antigen-binding protein or immunoconjugate, immunoconjugate, pharmaceutical composition, and, use of

专利摘要:
ANTIGEN OR IMMUNOCONJUGATE BINDING PROTEIN, IMMUNOCONJUGATE, PHARMACEUTICAL COMPOSITION, METHOD FOR TREATING A HUMAN PATIENT AFFECTED WITH AN INFLAMMATORY DISORDER OR DISEASE, AND, USE OF A COMPOSITION. The present invention relates to proteins that bind antigen and fragments thereof that specifically bind B Cell Maturation Antigen (BCMA), particularly human BCMA (hBCMA) and which inhibit the binding of BAFF and APRIL to the BCMA receptor. Pharmaceutical compositions, screening methods and pharmaceutical treatment are also described.
公开号:BR112013028779B1
申请号:R112013028779-9
申请日:2012-05-24
公开日:2021-01-05
发明作者:Paul Algate；Stephanie Jane Clegg；Jennifer L. Craigen；Paul Andrew Hamblin；Alan Peter Lewis；Radha Shah Parmar；Patrick Mayes；Trevor Anthony Kenneth Wattam
申请人:Glaxo Group Limited；
IPC主号:

专利说明:

Field of invention
[001] The present invention relates to proteins that bind antigen and fragments thereof that specifically bind B Cell Maturation Antigen (BCMA) and in particular human BCMA (hBCMA).
[002] The present invention also relates to methods of treating diseases or disorders with said antigen binding fragments, pharmaceutical compositions comprising said antigen binding fragments and methods of manufacture. Other embodiments of the present invention will be apparent from the description below. Fundamentals of the invention
[003] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily. It is a non-glycosylated integral membrane receptor for BAFF and APRIL ligands. BCMA ligands can also bind additional receptors: TACI (Transmembrane Activator and Calcium Modulator and Cyclophilin Ligand Interactor), which binds APRIL and BAFF; as well as BAFF-R (Receiver BAFF or BR3), which shows restricted but high affinity for BAFF. Together, these receptors and their corresponding ligands regulate different aspects of humoral immunity, B cell development and homeostasis.
[004] BCMA expression is typically restricted to the B cell lineage and is reported to increase in terminal B cell differentiation. BCMA is expressed by human plasma blasts, plasma cells of the tonsils, spleen and bone marrow, but also by B cells of tonsillar memory and cells of the germinal center, which have a low TACI-BAFFR phenotype (Darce et al, 2007). BCMA is virtually absent in naive and memory B cells (Novak et al., 2004a and b). The BCMA antigen is expressed on the cell surface so that it is accessible to the antibody, but it is also expressed in the Golgi complex. As suggested by its expression profile, BCMA signaling, typically linked with B cell survival and proliferation, is important in the late stages of B cell differentiation, as well as the survival of long-lived bone marrow plasma cells (O 'Connor et al., 2004) and plasmablasts (Avery et al., 2003). Furthermore, as BCMA binds APRIL with high affinity, the BCMA-APRIL signaling axis is suggested to predominate in the last stages of B cell differentiation, perhaps being the most physiologically relevant interaction.
[005] Multiple Myeloma (MM) is a clonal B cell malignancy that occurs at multiple sites within the bone marrow before it spreads into the circulation; again, or as a progression of monoclonal gammopathy of undetermined significance (MGUS). It is usually characterized by increases in paraprotein and osteoclast activity, as well as hypercalcemia, cytopenia, renal dysfunction, hyperviscosity and peripheral neuropathy. Decreases in both normal antibody levels and neutrophil numbers are also common, leading to a potentially lethal susceptibility to infection. BCMA has been implicated in the growth and survival of myeloma cell lines in vitro (Novak et al., 2004a and b; Moreaux et al., 2004).
[006] BCMA expression (both transcript and protein) is reported to correlate with disease progression in MM. Using Affymetrix microarrays, it was shown that the TACT and BCMA genes were overexpressed in Multiple Myeloma Cells (MMC) compared to their normal counterparts (Moreaux et al, 2004). Gene expression analysis was used to compare human myeloma cells with purified plasma cells from patients with MGUS and normal bone marrow as well as with primary tumor cells of leukemias of the B cell lineage (Bellucci et al, 2005). The BCMA gene was highly expressed in all myeloma samples. Although purified plasma cells from MGUS patients had lower BCMA expression, there was no significant difference when compared to the expression found in normal plasma cells or myeloma cells. In contrast, BCMA expression was significantly lower in B-cell Chronic Lymphocytic Leukemia (CLL), Pre-B Acute Lymphocytic Leukemia (ALL) and T-cell ALL (T-ALL). Mouse models that transgenically overexpress BAFF or APRIL have a significant increase in B cell lymphomas (Batten et al., 2004 - BAFF; Planelles et al., 2004 - APRIL). In humans, excess BAFF and APRIL have been detected in the sera and microenvironments of patients with various B cell malignancies, as well as other B cell disorders.
[007] All patent and literature references described within this specification are expressly and fully incorporated by reference. Brief Description of the Figures
[008] Figure 1: FMAT Binding Assay - Figure showing the results of the FMAT assay for the binding of CA8 antibody to HEK293 cells that express human and cyan BCMA. Human chimeric CA8 binds well to cells expressing human and cyan BCMA.
[009] Figure 2: ELISA Binding Assay - Figure showing the ELISA results for the binding of CA8 antibodies to recombinant human and cino BCMA proteins. This clearly shows that human chimeric CA8 antibodies bind to human BCMA and cino proteins equally.
[0010] Figure 3: BiaCore Binding Assay - Figure showing the binding of CA8 to BCMA-Fc, TACI-Fc and BAFF-R-Fc proteins in the Biacore experiment. The chimeric antibody CA8 does not bind to TALI or BAFF-R proteins.
[0011] Figure 4: Cell binding assay - Figure showing the binding of murine S307118G03, S3222110D07, S332121F02 and S332126E04 to multiple myeloma cells H929 and S3322110D07, S332121F02 and S332126E04 to the ARH77 transfected cells as determined by ARAC77 transfected.
[0012] BCMA H929 or ARH77-hBCMA 10B5 Multiple Myeloma cell line expressing transfectant cells were stained with anti murine BCMA antibodies (solid histogram) or murine IgG2a isotype control (open histograms). The cells were analyzed by FACS to detect antibody bound to the cells.
[0013] Figure 5: Cell binding assay - Figure showing the binding of chimeric CA8 to a panel of Multiple Myeloma cell lines as determined by FACS. Binding to H929, OPM-2, JJN-3 and U266 was tested by flow cytometry and measured mean fluorescence intensity (MFI) values to determine binding. Synagis was used as an irrelevant isotype control.
[0014] Figure 6: Cell binding assay - Figure showing the binding curves of humanized CA8 variants for ARH77 cells transfected with BCMA (A) and Multiple Myeloma H929 cells (B) as determined by FACS. The humanized variants J6M0, J6M1, J6M2, J9M0, J9M1 and J9M2 were tested by flow cytometry and the mean fluorescence intensity (MFI) values measured to determine the bond compared to the CA8 chimera.
[0015] Figure 7: Ligand neutralization assays - (A and B) Figure showing the ability of CA8 and J6M0 to neutralize the binding of recombinant BAFF or APRIL to recombinant BCMA coated on an ELISA plate. OD values were used to calculate antibody-mediated inhibition of the maximum signal obtained by binding only the relevant ligand to the recombinant BCMA. The data are reported as percent inhibition of the maximum signal. The tested antibodies were chimeric CA8 and humanized CA8 version J6M0 in both wild-type and afucosylated form (Potelligent). (A) Neutralization of the BAFF ligand binding; (B) Neutralization of APRIL ligand binding. (C) - Figure showing the ability of the J6M0 BCMA antibody to inhibit phosphorylation induced by BAFF or APRIL of NFCapaB in H929 cells. H-929 cells were washed 3 times to remove any sBCMA and resuspended in serum-free medium. J6M0 potelligent antibody was added to a 96 well plate to give a final reservoir concentration of up to 100 μg / ml along with BAFF or APRIL ligand to give a final reservoir concentration of 0.6 or 0.2 μg / ml respectively. The H-929 cells were then plated at 7.5 x 104 cells / reservoir in serum-free medium. 30 minutes later the cells were lysed and the levels of phosphorylated NFcapaB measured using an MSD pNFcapaB assay. The MSD reader 502819. That is, data from one of the independent experiments. Each data point is the average / sd of two replicates.
[0016] Figure 8: ADCC assay - Figure showing the ADCC activity of chimeric CA8 and defucosylated CA8 (enhanced with Fc) with target cells that express BCMA.
[0017] Human NK cells were incubated with targeting ARH77 10B5 cells transfected with BCMA labeled with europium in the presence of varying concentrations of the antibody. Europium release from target cells was measured and specific lysis calculated. (A) the ADCC dose response curves of chimeric CA8 compared to the isotype control. (B) the ADCC dose response curves for chimeric CA8 and defucosylated chimeric CA8 (enhanced with Fc) against the BCH-expressing ARH77 10B5 cell line.
[0018] Figure 9: ADCC assay - Figure showing ADCC assay on humanized antibodies with CA8 using target cells that express ARH77 BCMA.
[0019] Human PBMCs were incubated with target cells transfected with ARH77 BCMA labeled with europium in the presence of a range of concentrations from the J5, J6, J7, J8 or J9 series of humanized CA8 antibodies. Europium release from target cells was measured and specific lysis calculated. EC50 values are shown in μg / ml.
[0020] Figure 10: ADCC assay - Figure showing the chimeric ADCC activity, S332121F02 (A), S3322110D07 (B) S307118G03 (C) and S307118G03 H3L0 humanized (D) against ARH7710B5 target cells with NK cells purified as NK cells effector. Human NK target cells were incubated with target cells transfected with ARH77 10B5 BCMA labeled with europium in the presence of varying concentrations of the antibody. Europium release from target cells was measured and specific lysis calculated.
[0021] Figure 11: Dose response curves of the viability assay - Figure showing dose response curves in a cell viability assay for chimeric CA8 antibody, chimeric CA8-vcMMAE antibody conjugates and CA8-mcMMAF chimeric in human Multiple Myeloma cell lines (A) NCI-H929 (B) U266-B1 (C) JJN3 and (D) OPM2. The antibody was added to the cells and the number of viable cells after 96 hours measured using CelltiterGlo. The data points represent the average of CellTiterGlo measurements in triplicate. Error bars represent standard error.
[0022] Figure 12: Impact of chimeric antibody CA8 on the cell cycle. (A) Cell cycle histograms of NCI-H929 cells treated with unconjugated chimeric CA8, chimeric CA8vcMMAE ADC or chimeric CA8-mcMMAF 50 ng / ml for the indicated time points. Pactitaxel (100 nM) was used as a positive control for G2 / M cell cycle arrest and cell death. Control human IgG1 was used as a negative control. The cell cycle analysis was performed at the times shown in the graphs. (B) Quantification of the 4N DNA cell population indicative of G2 / M arrest and (C) sub-2N DNA cell population indicative of cell death for each of the indicated treatments. The cells were seeded in 12 well plates (2 x 105 cells per well in 1 ml of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding.
[0023] Figure 13: Impact of chimeric CA8 on phosphohistone H3.
[0024] Treatment with chimeric CA8 ADC results in increased phospho-Histone H3 staining of NCI-H929 cells. (A, B) Plots of cell dots stained with propidium iodide to measure the DNA content (FL3-H) x-axis and anti-phospho-Histone H3 antibody (Thr11) (FL1-H) y-axis after treatment with Control IgG (A) or chimeric CA8-mcMMAF (B). (C) Quantification of NCI-H929 positive cells in phospho-Histone H3 after a 48 hour treatment with the indicated concentrations of chimeric CA8 ADCs. Pactitaxel (100 nM) was used as a positive control for mitotic arrest and chimeric IgG1 control was used as a negative control. The cells were seeded in 12 well plates (2 x 105 cells per well in 1 ml of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell sowing.
[0025] Figure 14: Impact of chimeric CA8 on Annexin-V.
[0026] Treatment with chimeric CA8 ADC results in increased Annexin-V staining of NCI-H929 cells. (A) Anexin-V-FITC histograms (FL1-H; top panels) and staining with live cell propidium iodide (FL3-H; bottom panels) after treatment with increasing concentrations of chimeric CA8 ADCs (B) Quantification of Annexin-V positive NCI-H929 cells after a 96 hour treatment with the indicated concentrations of chimeric CA8 ADCs. Pactitaxel (100 nM) was used as a positive control for apoptosis and the control chimeric IgG1 was used as a negative control. The cells were seeded in 12 well plates (2 x 105 cells per well in 1 ml of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding.
[0027] Figure 15: Dose response curves of the viability assay - Figure showing dose response curves for unconjugated (naked) and vcMMAE antibody conjugates and humanized mcMMAF or J6M0 antibody drug conjugates. The antibody drug conjugates were tested against human Multiple Myeloma cell lines NCI-H929 and OPM2.
[0028] Figure 16: dose response curves of the viability assay - Figure showing dose response curves for unconjugated antibodies, conjugated anti-BMCA antibody drug vcMMAE and murine anti-BMCA antibodies S332121F02, S322110D07, S332126E04 and S307118G03 in human multiple myeloma cell lines NCI-H929 and U266-B1.
[0029] Figure 17: ADCC activity of ADC J6M0 molecules - Figure showing ADCC assay on J6M0 antibodies using target cells that express ARH77 BCMA. Human PBMCs were incubated with target cells transfected with ARH77 BCMA labeled with europium in the presence of a range of concentrations of J6M0 WT and potelligent BCMA antibodies conjugated to MMAE, MMAF, or not conjugated, the europium release was monitored in the Victor multi-label reader 2 1420.
[0030] Figure 18: ADCC dose response curves of CA8 J6M0 Potelligent against a panel of 5 strains of Multiple Myeloma - human PBMCs were incubated with multiple myeloma target cells in the presence of varying concentrations of CA8 J6M0 potelligent antibody at an E ratio : 50: 1 T for 18 hours. The percentage of target cells remaining in the effector plus the target mixture was then measured by FACS using a fluorescently labeled anti-CD138 antibody to detect the target cells and the calculated percentage cytotoxicity. A) Sample dose response curves for CA8 J6M0 potelligent against the five Multiple Myeloma cell lines tested. Each data point is of a sample value only.
[0031] Figure 19: Dose escalation effect of J6M0 and drug conjugate J6M0 on the growth and establishment of NCI-H929 cells in CB mice, 17 SCID Calculated tumor volumes of NCI-H929 tumors in CB17 SCID mice following the dosage intraperitoneal twice weekly from 50 or 100 μg of J6M0 anti-BCMA or non-conjugated IgG1 isotype control, or conjugated to MMAE or MMAF for 2 weeks. Data points represent mean tumor volume of n = 5 per group
[0032] Figure 20: Determination of serum soluble BCMA levels in healthy volunteers and myeloma patients. The serum samples that were collected from samples from a patient with MM were of a variety of stages (progressive disease, remission, relapse, newly diagnosed, and others). The samples shown in the figure are those of serum diluted 1/500 before the assay.
[0033] A human BCMA / TNFRSF17 interleaved ELISA kit from R & D Systems that measures levels of soluble human BCMA was used to detect BCMA following the standard protocol provided with the kit. Summary of the invention
[0034] The present invention provides antigen-binding proteins that bind to membrane-bound targets and in which the antigen-binding protein is capable of internalization. In another embodiment, an immunoconjugate is provided which comprises the antigen binding protein of the present invention and a cytotoxic agent. In another embodiment, the antigen binding protein has an ADCC effector function, for example, the antigen binding protein has an enhanced ADCC effector function.
The present invention provides antigen-binding proteins that specifically bind to BCMA, for example antibodies that specifically bind to BCMA and that inhibit the binding of BAFF and / or APRIL to the BCMA receptor. The present invention also provides antigen-binding proteins that specifically bind BCMA and that inhibit BAFF and / or APRIL binding to BCMA in which the antigen binding protein is capable of binding to FcyRIIIA or is capable of effector function mediated by FcyRIIIA.
The antigen-binding proteins of the present invention specifically bind to BCMA and inhibit the binding of BAFF and / or APRIL to BCMA in which the antigen-binding protein has enhanced FcyRIIIA binding or has enhanced FcyRIIIA-mediated effector function. In one embodiment, the antigen binding protein is capable of internalization.
[0037] In one aspect of the invention an antigen binding protein is provided which binds to membrane-bound BCMA, for example to serum BCMA.
[0038] In an embodiment of the present invention an immunoconjugate is provided which comprises the antigen binding protein of the present invention and a cytotoxic agent.
[0039] In another embodiment, proteins that bind antigen are conjugated to a toxin such as auristatin. In another embodiment, the drug conjugate is either vcMMAE or mcMMAF. In one embodiment, the immunoconjugate is also enhanced ADCC.
[0040] Antigen-binding proteins may be related to, or derived from, a CA8 murine monoclonal antibody. The amino acid sequence of the variable region of the murine CA8 heavy chain is provided as SEQ ID NO. 7 and the amino acid sequence of the CA8 murine light chain variable region is provided as SEQ ID NO. 9.
[0041] Antigen-binding proteins may be related to, or derived from, a murine monoclonal antibody 5336105A07. The amino acid sequence of the variable region of the murine heavy chain 5336105A07 is provided as SEQ ID NO. 140 and the amino acid sequence of the murine light chain variable region 5336105A07 is provided as SEQ ID NO. 144.
[0042] Other murine monoclonal antibodies from which the antigen-binding proteins of the present invention can also be derived are included in Table C.
[0043] The variable regions of the heavy chain (VH) of the antigen-binding proteins can comprise the CDRs or variants that follow from these CDR's (as defined by Kabat (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987)): A CDRH1 is provided as SEQ ID NO. 1 or SEQ ID NO. 182 CDRH2 is supplied as SEQ ID NO. 2 or SEQ ID NO. 183 CDRH3 is provided as SEQ ID NO. 3 or SEQ ID NO. 184
[0044] The light chain (VL) variable regions of the antigen-binding proteins can comprise the CDRs or variants that follow from these CDR's (as defined by Kabat (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987)): A CDRL1 is provided as SEQ ID NO. 4 or SEQ ID NO. 185 CDRL2 is supplied as SEQ ID NO. 5 or SEQ ID NO. 186 CDRL3 is supplied as SEQ ID NO. 6 or SEQ ID NO. 187
[0045] The invention also provides a polynucleotide sequence that encodes a variable region of the heavy chain of any of the proteins that bind antigen described herein, and a polynucleotide that encodes a variable region of the light chain of any of the proteins that bind antigen here described.
[0046] The invention also provides a polynucleotide sequence that encodes a heavy chain for any of the antigen binding proteins described herein, and a polynucleotide that encodes a light chain for any of the antigen binding proteins described herein.
[0047] Such polynucleotides represent the coding sequence that corresponds to the equivalent polypeptide sequences, however it will be understood that such polynucleotide sequences can be cloned into an expression vector together with a start codon, an appropriate signal sequence and a stop codon .
[0048] The invention also provides a transformed or transfected recombinant host cell that comprises one or more polynucleotides that encode a heavy chain and / or a light chain of any of the antigen binding proteins described herein.
[0049] The invention further provides a method for the production of any of the antigen-binding proteins described herein which method comprises the step of cultivating a host cell comprising a first and second vectors, said first vector comprising a polynucleotide that encodes a heavy chain of any of the antigen-binding proteins described herein and said second vector comprising a polynucleotide encoding a light chain of any of the antigen-binding proteins described herein, in a suitable culture medium, for example free culture medium of serum.
[0050] The invention further provides a pharmaceutical composition comprising an antigen binding protein as described herein and a pharmaceutically acceptable carrier.
[0051] In another aspect, the present invention provides a method of treatment or prophylaxis of a disease or disorder responsive to BCMA inhibition or blocking such as modulating the interaction between BCMA and its ligands, BAFF or APRIL which method comprises step of administering to said patient a therapeutically effective amount of the antigen binding protein thereof as described herein.
It is therefore an object of the present invention to provide a therapeutic method for the treatment of B cell-related disorders or diseases such as antibody-mediated or plasma-cell-mediated diseases or plasma cell malignancies such as for example Multiple Myeloma (MM ). In particular, it is an objective of the present invention to provide proteins that bind antigen, especially antibodies that specifically bind BCMA (e.g., hBCMA) and modulate (i.e., inhibit or block) the interaction between BCMA and its ligands such as BAFF and / or APRIL in the treatment of diseases and disorders responsive to the modulation of this interaction.
[0053] In another aspect of the present invention there is provided a method of treating a human patient afflicted with a B cell-related disorder or disease such as antibody-mediated or plasma-cell-mediated diseases or plasma cell malignancies such as for example Multiple Myeloma (MM) which method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.
[0054] In another aspect of the present invention there is provided a method of treating a human patient afflicted with Rheumatoid Arthritis, Psoriasis, Diabetes Mellitus Type 1 or Multiple Sclerosis which method comprises the step of administering to said patient a therapeutically effective amount of the protein of antigen binding as described herein. Detailed Description of the Invention
[0055] The present invention provides antigen-binding proteins that bind to membrane-bound targets and in which the antigen-binding protein is capable of internalization. In another embodiment, an immunoconjugate is provided which comprises the antigen binding protein of the present invention and a cytotoxic agent. In another embodiment, the antigen binding protein has an ADCC effector function, for example, the antigen binding protein has an enhanced ADCC effector function.
[0056] In such an embodiment proteins are provided that bind antigen or fragments thereof that specifically bind to BCMA, for example that specifically bind to human BCMA (hBCMA) and that inhibit the binding of BAFF and / or APRIL to the BCMA receptor.
[0057] In another embodiment, proteins that bind antigen or fragments specifically bind to BCMA and inhibit the binding of BAFF and / or APRIL to BCMA in which proteins that bind antigen or fragments thereof have the ability to bind to FcyRIIIA and mediate effector functions mediated by FcgRIIIA, or have enhanced effector functions mediated by FcgRIIIA. In an embodiment of the invention as provided herein, proteins that bind antigen are capable of internalization.
[0058] In one aspect of the invention, an antigen-binding protein according to the invention is provided as described herein that binds to membrane-bound BCMA, for example to serum BCMA.
[0059] In one aspect of the invention, an antigen binding protein is provided as described herein wherein the antigen binding protein comprises CDRH3 of SEQ ID NO. 3 or a variant of SEQ ID NO. 3.
[0060] In another aspect of the invention, an antigen binding protein is provided as described herein wherein the antigen binding protein further comprises one or more of: CDR H1 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.
[0061] In one aspect of the invention, an antigen binding protein is provided as described herein wherein the antigen binding protein comprises CDRH3 of SEQ ID NO. 184 or a variant of SEQ ID NO. 184.
[0062] In another aspect of the invention, an antigen binding protein is provided as described herein wherein the antigen binding protein further comprises one or more of: CDR H1 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.
[0063] In yet another aspect the antigen binding protein comprises CDR H3 of SEQ ID NO: 3: CDRH2: SEQ ID NO: 2: CDR H1 of SEQ ID NO: 1: CDRL1: SEQ ID NO: 4: CDRL2 : SEQ ID NO: 5 and CDRL3: SEQ ID NO: 6.
[0064] In yet another aspect the antigen binding protein comprises CDR H3 of SEQ ID NO: 184: CDRH2: SEQ ID NO: 183: CDR H1 of SEQ ID NO: 182: CDRL1: SEQ ID NO: 185: CDRL2 : SEQ ID NO: 186 and CDRL3: SEQ ID NO: 187.
[0065] In one aspect of the invention the antigen binding protein has an enhanced effector function. In another aspect, the antigen binding protein is conjugated to a cytotoxic agent. In yet another embodiment, the antigen binding protein both has an enhanced effector function and is conjugated to a cytotoxic agent.
[0066] The antigen-binding proteins of the present invention can comprise variable regions of the heavy chain and variable regions of the light chain of the invention that can be formatted into the structure of a natural antibody or functional fragment or equivalent thereof. An antigen-binding protein of the invention can therefore comprise the VH regions of the invention formatted into a life-size antibody, a (Fab ') 2 fragment, a Fab fragment, or equivalent thereof (such as scFV, bi-tri - or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain. The antibody can be an IgG1, IgG2, IgG3, or IgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain can be selected accordingly. The light chain constant domain can be a kappa or lambda constant domain. In addition, the antigen binding protein may comprise modifications of all classes, for example, IgG dimers, Fc mutants that no longer bind Fc receptors or mediate C1q binding. The antigen binding protein can also be a chimeric antibody of the type described in WO86 / 01533 which comprises an antigen binding region and a non-immunoglobulin region.
[0067] The constant region is selected according to any required functionality, for example, an IgG1 can demonstrate lytic capacity by binding to complement and / or will mediate ADCC (antibody-dependent cell cytotoxicity).
The antigen-binding proteins of the present invention are derived from the murine antibody that has the variable regions as described in SEQ ID NO: 7 and SEQ ID NO: 9 or non-murine equivalents thereof, such as from rat, human, chimeric or humanized variants thereof, for example they are derived from the antibody having the variable heavy chain sequences as described in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29 and / or the light chain variable sequences as described in SEQ ID NO: 31, SEQ ID NO: 33 and / or SEQ ID NO: 35.
[0069] In another embodiment the antigen-binding proteins of the present invention are derived from an antibody that has the variable sequences of the heavy chain as described in SEQ ID NO: 116 or SEQ ID NO: 118 and / or the variable sequences of the light chain as described in SEQ ID NO: 120, or SEQ ID NO: 122.
[0070] In another embodiment the antigen-binding proteins of the present invention are derived from an antibody that has the variable sequences of the heavy chain as described in SEQ ID NO: 140 and / or the variable sequences of the light chain as described in SEQ ID NO: 144.
[0071] In one aspect of the invention an antigen binding protein is provided which comprises an isolated heavy chain variable domain selected from any 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.
[0072] In another aspect of the invention an antigen binding protein is provided which comprises an isolated light chain variable domain selected from any of the following: SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35, SEQ ID NO: 120 or SEQ ID NO: 122.
[0073] In another aspect of the invention an antigen binding protein is provided which comprises an isolated heavy chain variable domain selected from any 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 a variable domain of isolated light chain selected from any of the following: SEQ ID NO: 31, SEQ ID NO: 33 and / or SEQ ID NO: 35.
[0074] In one aspect the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 23 and a light chain variable region encoded by SEQ ID NO: 31.
[0075] In one aspect the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 27 and a light chain variable region encoded by SEQ ID NO: 31. In one aspect the The antigen binding of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 29 and a light chain variable region encoded by SEQ ID NO: 31.
[0076] In one aspect the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 116 and a light chain variable region encoded by SEQ ID NO: 120
[0077] In one aspect the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 118 and a light chain variable region encoded by SEQ ID NO: 122
In one aspect, a polynucleotide encoding an isolated variable heavy chain is provided, said polynucleotide comprising SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO: 18, or SEQ ID NO: 20, or SEQ ID NO: 22, or SEQ ID NO: 24, or SEQ ID NO: 26, or SEQ ID NO: 28, or SEQ ID NO: 30 or SEQ ID NO: 117 or SEQ ID NO : 119 or SEQ ID NO: 141.
[0079] In one aspect, a polynucleotide encoding an isolated variable light chain is provided, said polynucleotide comprising SEQ ID NO: 32, or SEQ ID NO: 34, or SEQ ID NO: 36 or SEQ ID NO: 121 or SEQ ID NO: 123 or SEQ ID NO: 145.
In another aspect, a polynucleotide encoding an isolated variable heavy chain is provided, said polynucleotide comprising SEQ ID NO: 24, or SEQ ID NO: 28 or SEQ ID NO: 30 and a polynucleotide encoding a variable light chain said polynucleotide comprising SEQ ID NO: 32, or SEQ ID NO: 34 is isolated.
[0081] In yet another aspect, a polynucleotide encoding an isolated variable heavy chain is provided said polynucleotide comprising SEQ ID NO: 24 and a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ ID NO: 32.
[0082] In yet another aspect a polynucleotide encoding an isolated variable heavy chain is provided said polynucleotide comprising SEQ ID NO: 117 and a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ ID NO: 121.
[0083] In yet another aspect a polynucleotide encoding an isolated variable heavy chain is provided said polynucleotide comprising SEQ ID NO: 119 and a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ ID NO: 123.
[0084] In yet another aspect a polynucleotide encoding an isolated variable heavy chain is provided said polynucleotide comprising SEQ ID NO: 141 and a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ ID NO: 145.
[0085] In another aspect the antigen binding protein can comprise any of the variable heavy chains as described herein in combination with any of the light chains as described herein.
[0086] In one aspect the antigen-binding protein is an antibody or antigen-binding fragment thereof comprising one or more CDR's according to the invention described herein, or one or both of the variable domains of the heavy or light chain of according to the invention described herein. In one embodiment the antigen binding protein binds primate BCMA. In such an embodiment the antigen binding protein additionally binds non-human primate BCMA, for example cynomolgus monkey BCMA.
[0087] In another aspect the antigen binding protein is selected from the group consisting of a dAb, Fab, Fab ', F (ab') 2, Fv, diabody, tribody, tetrabody, minibody, and a minibody ,.
[0088] In one aspect of the present invention the antigen binding protein is a humanized or chimeric antibody, in another aspect the antibody is humanized.
[0089] In one aspect the antibody is a monoclonal antibody.
[0090] In one aspect of the present invention, an antibody with the heavy chain sequence as shown in SEQ ID NO: 55 or SEQ ID NO: 59 or SEQ ID NO: 61 is provided.
[0091] In one aspect of the present invention, an antibody with the light chain sequence as shown in SEQ ID NO: 63 or SEQ ID NO: 65 is provided.
[0092] In another aspect of the invention an antibody is provided with the heavy chain sequence of SEQ ID NO: 55 and a light chain sequence as shown in SEQ ID NO: 63.
[0093] In one embodiment, an antigen binding protein is provided that competes with an antigen binding protein of the invention as described herein. In such an embodiment, therefore, an antigen binding protein is provided that competes with an antigen binding protein comprising the heavy chain variable sequence of SEQ ID NO 23 and the light chain variable region of SEQ ID NO 31.
[0094] In another embodiment, therefore, an antigen binding protein is provided that competes with an antigen binding protein that comprises a heavy chain variable sequence selected from one of SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 116, SEQ ID NO 118 and SEQ ID NO 140 and a variable region of the light chain selected from one of SEQ ID NO 31, SEQ ID NO 120, SEQ ID NO 122 and SEQ ID NO 144.
[0095] In another aspect the antigen binding protein binds to human BCMA with high affinity for example when measured by Biacore the antigen binding protein binds to human BCMA with an affinity of 20 nM or less or an affinity of 15 nM or less or an affinity of 5 nM or less or an affinity of 1000 pM or less or an affinity of 500 pM or less or an affinity of 400 pM or less or 300 pM or less or for example about 120 pM . In another embodiment, the antigen binding protein binds to human BCMA when measured by Biacore between about 100 pM and about 500 pM or between about 100 pM and about 400 pM, or between about 100 pM and about 300 pM. In an embodiment of the present invention the antigen binding protein binds BCMA with an affinity of less than 150 pM.
[0096] In such an embodiment, this is measured by Biacore, for example as shown in Example 4.
[0097] In another aspect the antigen binding protein binds to human BCMA and neutralizes the binding of BAFF and / or APRIL ligands to the BCMA receptor in a cell neutralization assay in which the antigen binding protein has a 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 another embodiment of the present invention the antigen binding protein binds BCMA and neutralizes BCMA in a cell neutralization assay in which the antigen binding protein has an IC 50 of about 10 nM.
[0098] In such an embodiment, this is measured by a cell neutralization assay, for example as shown in Example 4.6.
[0099] Antigen-binding proteins, for example antibodies of the present invention can be produced by transfecting a host cell with an expression vector that comprises the coding sequence for the antigen-binding protein of the invention. A recombinant expression vector or plasmid is produced by placing these coding sequences for the antigen binding protein in operative association with conventional regulatory control sequences capable of controlling replication and expression in, and / or secretion from, a host cell . Regulatory sequences include promoter sequences, for example, CMV Promoter, and signal sequences that can be derived from other known antibodies. Similarly, a second expression vector can be produced that has a DNA sequence that encodes a complementary light or heavy chain antigen binding protein. In certain embodiments, this second expression vector is identical to the first except to the extent that the selected coding sequences and markers are involved, in order to ensure as much as possible that each polypeptide chain is functional expressed. Alternatively, the heavy and light chain coding sequences for the antigen binding protein can reside in a single vector.
A selected host cell is cotransfected by conventional techniques with either the first or second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both recombinant or synthetic light and heavy chains. . The transfected cell is then cultured by conventional techniques to produce the engineered antigen-binding protein of the invention. The antigen binding protein that includes the association of both recombinant heavy and light chain is screened from the culture by the appropriate assay, such as ELISA or RIA. Similar conventional techniques can be used to build other proteins that bind antigen.
[00101] Vectors suitable for the cloning and subcloning steps used in the methods and construction of the compositions of this invention can be selected by a person of skill in the art. for example, the conventional pUC series of cloning vectors can be used. One vector, pUC19, is commercially available from supplier firms, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden). In addition, any vector that is able to replicate easily, has an abundance of selectable cloning sites and genes (eg, antibiotic resistance), and is easily manipulated can be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.
[00102] Expression vectors can also be characterized by genes suitable for amplifying the expression of heterologous DNA sequences, for example, the mammalian dihydrofoliate reductase (DHFR) gene. Other vector sequences include a poly A signal sequence, such as bovine growth hormone (BGH) and the betaglobin (betaglopro) promoter sequence. The expression vectors useful here can be synthesized by techniques well known to those skilled in the art.
[00103] The components of such vectors, for example, replicates, selection genes, enhancers, promoters, signal sequences and the like, can be obtained from commercial or natural sources or synthesized by procedures known for use in targeting the expression and / or secretion of the recombinant DNA product in a selected host. Other appropriate expression vectors of which numerous types are known in the art for expression in mammal, bacteria, insect, yeast, and fungus can also be selected for this purpose.
[00104] The present invention also encompasses a cell line transfected with a recombinant plasmid that contains the coding sequences for the proteins that bind antigen of the present invention. Host cells useful for cloning and other manipulation of these cloning vectors are also conventional. However, cells from various strains of E. coli can be used for replication of cloning vectors and other steps in the construction of proteins that bind the antigen of this invention.
[00105] Host cells or cell lines suitable for the expression of the antigen binding proteins of the invention include mammalian cells such as NSO, Sp2 / 0, CHO (e.g. DG44), COS, HEK, a fibroblast cell ( for example, 3T3), and myeloma cells, for example, can be expressed in a CHO cell or a myeloma cell. Human cells can be used, thus allowing the molecule to be modified with human glycosylation patterns.
[00106] Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, for example, Sambrook et al., Cited above.
[00107] Bacterial cells may prove useful as host cells suitable for the expression of recombinant Fabs or other embodiments of the present invention (see, for example, Plückthun, A., Immunol. Rev., 130: 151-188 ( 1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial cell would have to be screened for retention of the antigen binding capacity. If the molecule expressed by the bacterial cell were produced in an appropriately folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule can express in the bacterial host and then be subsequently refolded. for example, several strains of E. coli used for expression are well known as host cells in the field of Biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli and the like can also be used in this method.
[00108] Where desired, cells from yeast strains known to those skilled in the art are also available as host cells, as well as insect cells, for example, Drosophila and Lepidoptera and viral expression systems. See, for example, Miller et al., Genetic Engineering, 8: 277-298, Plenum Press (1986) and references cited therein.
[00109] The general methods by which vectors can be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding protein of the invention from such a host cell can be all conventional techniques. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing serum-free cells in the suspension. Likewise, once produced, the antigen-binding proteins of the invention can be purified from cell culture contents according to standard procedures in the art, including precipitation with ammonium 16eroxidi, affinity columns, column chromatography, electrophoresis in gel and the like. Such techniques are within the skill of the art and do not limit this invention. for example, altered antibody preparations are described in WO 99/58679 and WO 96/16990.
[00110] Yet another method of expressing antigen-binding proteins can use expression in a transgenic animal, as described in U.S. Patent No. 4,873,316. This refers to an expression system that uses the animal casein promoter that when transgenically incorporated within a mammal allows the female to produce the desired recombinant protein in her milk.
[00111] In another embodiment of the invention there is provided a method of producing an antibody of the invention which method comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and / or heavy chain of the antibody of the invention and recover the antibody produced thereby.
[00112] According to the present invention there is provided a method of producing an anti-BCMA antibody of the present invention which binds to and neutralizes the activity of human BCMA which method comprises the steps of: providing a first vector encoding a heavy chain the antibody; providing a second vector that encodes an antibody light chain; transforming a mammalian host cell (e.g., CHO) with said first and second vectors; culturing the host cell of step (c) under conditions conducive to secretion of the antibody from said host cell within said culture medium; recover the antibody secreted from step (d).
[00113] Once expressed by the desired method, the antibody is then examined for activity in vitro using an appropriate assay. Presently conventional ELISA assay formats are used to assess the qualitative and quantitative binding of the antibody to BCMA. In addition, other in vitro assays can also be used to verify the effectiveness of neutralization prior to subsequent human clinical studies conducted to assess the persistence of the antibody in the body despite the usual clearance mechanisms.
[00114] The dose and duration of treatment refers to the relative duration of the molecules of the present invention in human circulation, and can be adjusted by a person of skill in the art depending on the condition being treated and the general health of the patient. It is considered that repeated dosing (for example, once a week or once every two weeks or once every 3 weeks) over an extended period of time (for example, four to six months) may be required to achieve maximum therapeutic efficacy.
[00115] In an embodiment of the present invention a transformed, transfected or transduced recombinant host cell is provided that comprises at least one expression cassette, for example where the expression cassette comprises a polynucleotide encoding a heavy chain of a protein of antigen binding according to the invention described herein and further comprising a polynucleotide encoding a light chain of an antigen binding protein according to the invention described here or where there are two expression cassettes and the 1st encodes the light chain and the second encodes the heavy chain. For example, in one embodiment, the first expression cassette comprises a polynucleotide that encodes a heavy chain of an antigen binding protein that comprises a constant region or antigen binding fragment thereof that is bound to a constant region according to the invention described herein and further comprising a second cassette comprising a polynucleotide encoding a light chain of an antigen binding protein comprising a constant region or antigen binding fragment thereof which is bound to a constant region according to invention described herein for example the first expression cassette comprises a polynucleotide that encodes a heavy chain selected from SEQ ID NO: 56, or SEQ ID NO: 60 or SEQ ID NO: 62 and a second expression cassette that comprises a polynucleotide that encodes a light chain selected from SEQ ID NO: 64 or SEQ ID NO: 66.
[00116] In another embodiment of the invention there is provided a stably transformed host cell comprising a vector comprising one or more expression cassettes encoding an antibody heavy chain and / or a light chain comprising a constant region or fragment antigen binding site that is bound to a constant region as described herein. For example, such host cells may comprise a first vector encoding the light chain and a second vector encoding the heavy chain, for example the first vector encoding a heavy chain selected from SEQ ID NO: 55, or SEQ ID NO: 59 or SEQ ID NO: 61 and a second vector encoding a light chain for example the light chain of SEQ ID NO: 63 or SEQ ID NO: 65. In such an example the first vector encodes a heavy chain selected from SEQ ID NO: 55 and a second vector encoding a light chain for example the light chain of SEQ ID NO: 63.
[00117] In another embodiment of the present invention, a host cell according to the invention described herein is provided in which the cell is eukaryotic, for example where the cell is a mammalian. Examples of such cell lines include CHO or NSO.
[00118] In another embodiment of the present invention, a method is provided for the production of an antibody comprising a constant region or antigen binding fragment thereof which is bound to a constant region according to the invention described herein this comprising the step of culturing a host cell in a culture medium, for example serum-free culture medium.
[00119] In another embodiment of the present invention, a method according to the invention described herein is provided in which said antibody is further purified to at least 95% or greater (for example, 98% or greater) with respect to said serum-free culture medium containing antibody.
[00120] In yet another embodiment, a pharmaceutical composition is provided which comprises an antigen binding protein and a pharmaceutically acceptable carrier.
[00121] In another embodiment of the present invention, a kit of parts is provided which comprises the composition according to the invention described herein together with instructions for use.
[00122] The mode of administration of the therapeutic agent of the invention can be any suitable route that releases the agent to the host. Antigen-binding proteins and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneous (s.c.), intrathecal, intraperitoneal, intramuscular (i.m.) or intravenously (i.v.). In such an embodiment, the antigen-binding proteins of the present invention are administered intravenously or subcutaneously.
[00123] Therapeutic agents of the invention can be prepared as pharmaceutical compositions that contain an effective amount of the antigen binding protein of the invention as an active ingredient in a pharmaceutically acceptable carrier. In one embodiment the prophylactic agent of the invention is an aqueous suspension or solution that contains 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 compositions for parenteral administration will comprise a solution of the antigen binding protein of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier. In one embodiment the carrier is an aqueous carrier. A variety of aqueous carriers can be used, for example, 0.9% saline, 0.3% glycine, and the like. These solutions can be made sterile and generally free of particulate matter. These solutions can be sterilized by conventional well-known sterilization techniques (for example, filtration). The compositions can contain pharmaceutically acceptable auxiliary substances as required to approach physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antigen binding protein of the invention in such a pharmaceutical formulation can vary widely, that is, from less than about 0.5%, usually to or at least about 1% to as much as about 15 or 20 % by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
[00124] Thus, a pharmaceutical composition of the invention for intravenous infusion can be completed to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 or 5 mg to about 25 mg of a binding protein of antigen of the invention per ml of Ringer's solution. The actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail, for example, in Remington’s Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. For the preparation of the intravenously administrable antigen-binding protein formulations of the invention see Lasmar U and Parkins D "The formulation of Biopharmaceutical products", Pharma. Sci.Tech.today, page 129-137, Vol. 3 (April 3, 2000); Wang, W "Instability, stabilization and formulation of liquid protein pharmaceutics", Int. J. Pharm 185 (1999) 129-188; Stability of Protein Pharmaceutical Part A and B and Ahern T. J., Manning M. C., New York, NY: Plenum Press (1992); Akers, M. J. "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; Izutsu, Kkojima, S. “Excipient crystalinity and its protein- structure-stabilizing effect during freeze-drying”, J Pharm. Pharmacol, 54 (2002) 10331039; Johnson, R, "Mannitol-sucrose mixtures-versatile formulation for protein peroxidise 19g19n", J. Pharm. Sci, 91 (2002) 914-922; and Ha, E Wang W, Wang Y.j. “Peroxide formation in polisorbate 80 and protein stability”, J. Pharm Sci, 91, 2252-2264, (2002) the entire contents of which are incorporated by reference and to which the reader is specifically directed.
[00125] In one embodiment the therapeutic agent of the invention, when in a pharmaceutical preparation, is present in unit dosage forms. The appropriate therapeutically effective dose will be easily determined by those skilled in the art. Suitable doses can be calculated for patients according to their weight, for example the appropriate doses can be in the range of about 0.1 to about 20 mg / kg, for example from about 1 to about 20 mg / kg , for example from about 10 to about 20 mg / kg or for example from about 1 to about 15 mg / kg, for example from about 10 to about 15 mg / kg or for example from 1 to 5 mg / kg. In one embodiment, the antibody is given from 1 to 5 mg / kg every 3 weeks. To effectively treat conditions such as Multiple Myeloma, SLE or IPT in a human, suitable doses can be within the range of about 0.1 to about 1000 mg, for example from about 0.1 to about 500 mg , for example about 500 mg, for example about 0.1 to about 100 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or from about 0.1 to about 40 mg, or for example from about 1 to about 100 mg, or from about 1 to about 50 mg, of an antigen binding protein of this invention, which can be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such a dose, if necessary, can be repeated at appropriate time intervals selected as appropriate by a physician.
[00126] The proteins that bind antigen described here can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective with conventional immunoglobulins and peroxidase known in the art and reconstitution techniques can be used.
[00127] In another aspect of the invention, an antigen-binding protein is provided as described herein for use in a drug.
[00128] In one aspect of the present invention an antigen-binding protein according to the invention is provided as described herein for use in the treatment of rheumatoid arthritis, Type 1 Diabetes Mellitus, Multiple Sclerosis or psoriasis wherein said method comprises step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.
[00129] In an embodiment of the present invention, methods are provided to treat cancer in a human which comprises administering to said human an antigen binding protein that specifically binds to BCMA. In some cases, the antigen-binding protein is part of an immunoconjugate.
[00130] In another aspect of the present invention there is provided an antigen binding protein according to the invention as described herein for use in the treatment of a B cell-mediated or plasma cell-mediated disease or antibody-mediated disease or disorder selected from Multiple Myeloma (MM), chronic lymphocytic leukemia (CLL), Non-secretory Multiple Myeloma, Multiple Myeloma Burning, monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (Bone, Extramedullary), Lymphoplasmacytic Lymphoma (LPL) Waldenstrom, plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, cryoglobulinemia Type I and II, light chain deposition disease, Goodpasture syndrome, purple idiopathic thrombocytopenic (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders, and acquired epidermolysis bullosa; or any B cell leukemia of Non-Hodgkin's lymphoma or Hodgkin's lymphoma (HL) with BCMA expression or any diseases in which patients develop neutralizing antibodies for recombinant protein replacement therapy in which said method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.
[00131] B cell disorders can be divided into defects in B cell development / immunoglobulin production (immunodeficiencies) and excessive / uncontrolled proliferation (lymphomas, leukemias). As used herein, B cell disorder refers to both types of disease, and methods are provided to treat B cell disorders with an antigen-binding protein.
[00132] In a particular aspect, the disease or disorder is selected from the group consisting of Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Solitary Plasmacytoma (Bone, Extramedullary), Waldenstrom's Macroglobulinemia.
[00133] In one aspect of the present invention the disease is Multiple Myeloma, Burning Multiple Myeloma (SMM) or Solitary Plasmacytoma (Bone, Extramedullary).
[00134] In one aspect of the present invention the disease is Multiple Myeloma.
[00135] In one aspect of the present invention the disease is systemic lupo erythematosus (SLE)
[00136] In one aspect of the present invention the disease is idiopathic thrombocytopenic purpura (ITP)
[00137] The use of the antigen binding protein as described herein in the manufacture of a drug for the treatment of diseases and disorders as described herein is also provided.
[00138] For example in one aspect of the invention the use of the antigen binding protein as described herein is provided for use in the treatment or prophylaxis of diseases and disorders responsive to modulation (such as inhibition or blocking) of the interaction between BCMA and the BAFF and APRIL ligands.
[00139] In another aspect of the invention the use of the antigen-binding protein as described herein is provided for use in the treatment or prophylaxis of an antibody-mediated or plasma cell-mediated disease or selected rheumatoid arthritis disorder, Type Diabetes Mellitus 1, Multiple sclerosis or psoriasis.
[00140] In another aspect of the invention the use of the antigen binding protein as described herein is provided for use in the treatment or prophylaxis of an antibody-mediated or plasma cell-mediated disease or selected Multiple Myeloma (MM) disorder, chronic lymphocytic leukemia (CLL), monoclonal gammopathy of undetermined significance (MGUS), Multiple Myeloma That Burns (SMM), Solitary Plasmacytoma (Bone, Extramedullary), Waldenstrom's Macroglobulinemia, Primary Amyloidosis (AL), Heavy Chain Disease, Lyst (SLE), POEMS syndrome / osteosclerotic myeloma, cryoglobulinemia Type I and II, light chain deposition disease, Goodpasture syndrome, Idiopathic thrombocytopenic purpura (ITP), Acute glomerulonephritis, Pemphigus and pemphigoid disorders and any acquired epidermolysis Not Hodgkin and BCMA-expressing leukemia or any disease in which patients develop neutralizing antibodies for replacement therapy that of recombinant protein wherein said method comprises the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.
[00141] In one aspect, the invention provides a pharmaceutical composition comprising an antigen binding protein of the present invention or a functional fragment thereof and a pharmaceutically acceptable carrier for the treatment or prophylaxis of rheumatoid arthritis, Type 1 Diabetes, Sclerosis Multiple or psoriasis or an antibody-mediated or plasma cell-mediated disease or selected disorder of Multiple Myeloma (MM), chronic lymphocytic leukemia (CLL), Monoclonal Gamopathy of undetermined significance (MGUS), Multiple Burning Myeloma (SMM), Solitary Plasmacytoma (Bone, Extramedullary), Waldenstrom's Macroglobulinemia, Primary Amyloidosis (AL), Heavy chain disease, Systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, cryoglobulinemia Type I and II, light chain deposition disease, syndrome of light chain Goodpasture, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders and bullous epidermolysis a acquired, any Non-Hodgkin's Lymphoma and BCMA-Expressed Leukemia or any disease in which patients develop neutralizing antibodies for recombinant protein replacement therapy in which said method comprises the step of administering to said patient a therapeutically effective amount of the protein of antigen binding as described herein.
[00142] In another embodiment of the present invention there is provided a method of treating a human patient afflicted with rheumatoid arthritis, Type 1 Diabetes Mellitus, Multiple Sclerosis or psoriasis or an antibody-mediated or plasma cell-mediated disorder or disease this method comprising the step of administering a therapeutically effective amount of the antigen binding protein according to the invention as described herein, for example a method of treating a human patient afflicted with an antibody-mediated or plasma cell-mediated disease or disorder is provided In another aspect of the present invention, an antigen-binding protein according to the invention is provided as described herein for use in the treatment of an antibody-mediated or plasma cell-mediated disease or selected Multiple Myeloma (MM) disorder , Chronic Lymphocytic Leukemia (CLL), Monoclonal gammopathy of undetermined significance (MGUS), Miel Burning Multiple Oma (SMM), Solitary Plasmacytoma (Bone, Extramedullary), Waldenstrom Macroglobulinemia, Primary Amyloidosis (AL), Heavy Chain Disease, Systemic Lupus Erythematosus (SLE), POEMS Syndrome / Osteosclerotic Myeloma, Cryoglobulin Type II , light chain deposition disease, Goodpasture syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders and acquired bullous epidermolysis, any Non-Hodgkin's lymphoma and leukemia with BCMA expression or any disease in which patients develop neutralizing antibodies for recombinant protein replacement therapy wherein said method comprises the step of administering a pharmaceutical composition comprising an antigen binding protein according to the invention herein in combination with a pharmaceutically acceptable carrier.
[00143] In another embodiment, a method of treating a human patient afflicted with Multiple Myeloma (MM) is provided.
[00144] Definitions
[00145] As used herein, the terms "cancer", "neoplasm", and "tumor" are used interchangeably and, in singular or plural form, refer to cells that have undergone a malignant transformation that make them pathological for the host organism . Primary cancer cells can be easily distinguished from non-cancer cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from an ancestral cancer cell. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When alluding to a type of cancer that normally manifests itself as a solid tumor, a “clinically detectable” tumor is one that is detectable based on the tumor mass; for example, by procedures such as computed tomography (CT) scanning, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and / or that is detectable because of the expression of one or more antigens specific cancers in a sample obtainable from a patient. Tumors can be hematopoietic cancer (or hematological or involved in blood or related to blood), for example, cancers derived from blood cells or immune cells, which can be referred to as "liquid tumors". Specific examples of clinical conditions based on hematological tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as Multiple Myeloma, MGUS and Waldenstrom's macroglobulinemia; lymphomas such as Non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.
[00146] Cancer can be any cancer in which an abnormal number of Blast cells or unwanted cell proliferation is present or which is diagnosed as a hematological cancer, which includes both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid leukemia (or myelocytic or myelogenous or myeloblastic) (undifferentiated or differentiated), acute promyeloid leukemia (or promyelocytic or promyelogenous or promyeloblastic), acute myelomonocytic leukemia (or myelomonoblastic), leukemia acute monocytic (or monoblastic), erythroleukemia and megakaryocytic (or megacarioblastic) leukemia. These leukemias can be alluded to together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and true polycythemia (PCV) . Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which can be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess transforming blasts (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.
[00147] Hematopoietic cancers also include lymphoid malignancies, which can affect lymph nodes, spleens, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, Non-Hodgkins B-cell lymphoma (B-NHLs). BNHLs can be indolent (or low grade), intermediate grade (or aggressive) or high grade (very aggressive). Indolent B cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) which includes nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and lymphoma of lymphoid tissue associated with the mucosa (MALT or extranodal marginal zone). Intermediate grade BNHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), large follicular cell lymphoma (or grade 3 or grade 3B), and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV-associated (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder or lymphoma (PTLD). B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom macroglobulinemia (WM), hair cell leukemia (HCL), large granular lymphocyte leukemia (LGL) , acute lymphoid leukemia (or lymphocytic or lymphoblastic), and Castleman's disease. NHL may also include Non-Hodgkin's T-cell lymphomas (T-NHLs), which include, but are not limited to otherwise unspecified T-cell Non-Hodgkin's lymphoma (NOS), peripheral T-cell lymphoma (PTCL), large anaplastic cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), natural nasal killer cell (NK) / T cell lymphoma, gamma / delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.
[00148] Hematopoietic cancers also include Hodgkin's lymphoma (or disease) which includes classic Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed-cell Hodgkin's lymphoma, predominant lymphocyte (LP) Hodgkin's lymphoma, LP Hodgkin's lymphoma , and lymphocyte depleted Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as Multiple Myeloma (MM) which includes burning MM, monoclonal gammopathy of undetermined (or unknown or uncertain) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL ), Waldenstrom's macroglobulinemia, plasma cell leukemia, and primary amyloidosis (LA). Hematopoietic cancers can also include other cancers of additional hematopoietic cells, which include polymorphonuclear (or neutrophil) leukocytes, basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues that include hematopoietic cells referred to herein as "hematopoietic cell tissues" include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as lymphoid tissues associated with the intestine), tonsils, Peyer plaques and appendix, and lymphoid tissues associated with another mucosa, for example, bronchial linings .
[00149] The term "antigen binding protein" as used herein refers to antibodies, antibody fragments and other protein constructs that are capable of binding and neutralizing human BCMA.
[00150] The terms Fv, Fc, Fd, Fab, or F (ab) 2 are used with their standard meanings (see, for example, Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)) .
[00151] The term "antibody" is used here in the broadest sense and specifically encompasses monoclonal antibodies (which includes monoclonal antibodies of natural size), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies)
[00152] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies that is, the individual antibodies that comprise the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific and are directed against a single antigen binding site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen.
[00153] A "chimeric antibody" refers to a type of engineered antibody in which a portion of the heavy chain and / or light chain is identical with the homologue that corresponds to the sequences in antibodies derived from a particular donor antibody class or subclass , while the remainder of the chains is identical to the homologues that correspond to the sequences in antibodies derived from another species or that belong to another class or subclass of antibody, as well as fragments of such antibodies, as long as they have the desired biological activity (Patent US No. 4, 816,567 and Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851-6855) (1984)).
[00154] A "humanized antibody" refers to a type of engineered antibody that has its CDR derivatives derived from a non-human donor immunoglobulin, the remaining immunoglobulin derived parts of the molecule being derived from one (or more) human immunoglobulins . In addition, structure support residues can be altered to preserve binding affinity (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 1002910032 (1989), Hodgson et al., Bio / Technology, 9: 421 (1991)). A suitable human receptor antibody can be selected from a conventional database, for example, the KABAT® database, Los Alamos database and Swiss Protein database, by homology to the nucleotide and amino acid sequence of the donor antibody. A human antibody characterized by homology to the donor antibody framework regions (on an amino acid basis) may be suitable to provide a heavy chain constant region and / or a matrix heavy chain variable region for insertion of the donor CDRs. A suitable receptor antibody capable of donating light chain constant or regions of variable structure can be selected in a similar manner. It should be noted that receptor heavy and light chain antibodies are not required to generate the same receptor antibody. The prior art describes several ways to produce such humanized antibodies - see, for example, EP-A-0239400 and EP-A-054951.
[00155] For nucleic acids, the term "substantial identity" indicates that two nucleic acids or projected sequences thereof, when optimally aligned and compared and, are identical, appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides at least about 90% to about 95%, or at least about 98% to about 99.5% of the nucleotides. Alternatively, substantial identity exists when the segments will hybridize under selective hybridization conditions, to the complement of the filament. “Identity” means, for polynucleotides and polypeptides, as the case may be, the comparison calculated using the algorithm provided in (1) and (2) below:
[00156] (1) The identity for polynucleotides is calculated by multiplying the total number of nucleotides in a given sequence by the integer that defines the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in said sequence, or: nn <xn - (xn • y), where nn is the number of nucleotide changes, xn is the total number of nucleotides in a given sequence, y is 0.95 by 95%, 0.97 by 97% or 1.00 per 100%, and • is the symbol for the multiplication operator, where any non-integer product of xn and y is rounded down to the nearest whole number before its subtraction from xn. Changes to a polynucleotide sequence that encode a polypeptide can create nonsense, wrong sense, or structural change in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide that follows such changes.
[00157] (2) The identity of the polypeptides is calculated by multiplying the total number of amino acids by the integer that defines the percent identity divided by 100 and then subtracting that product from said total number of amino acids, or: na <xa - (xa • y), where na is the number of amino acid changes, xa is the total number of amino acids in the sequence, y is 0.95 by 95%, 0.97 by 97% or 1.00 by 100%, and • is the symbol for the multiplication operator and in which any non-integer product of x and y is rounded down to the nearest whole number before its subtraction from x.
[00158] For the nucleotide and amino acid sequence, the term "identical" indicates the degree of identity between two nucleic acids or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
[00159] "Isolated" means altered "by the hand of man" from its natural state, has been changed or removed from its natural environment or both. for example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", which includes but is not limited to when such polynucleotides or polypeptides are introduced back into a cell, even if the cell is of the same species or type as the one from which the polynucleotide or polypeptide was separated.
[00160] Throughout this specification and the attached claims, the term "that comprises" and "comprises" incorporates "that consists of" and "consists of". That is, these words are intended to convey the possible inclusion of other elements or integers not specifically cited, where the context allows.
[00161] The term "specifically binds" as used throughout this specification in relation to the antigen-binding proteins of the invention means that the human BCMA-binding antigen binding protein (hBCMA) has no binding or negligible binding to other proteins human. The term, however, does not exclude the fact that proteins that bind the antigen of the invention can also be reactive with other forms of BCMA, for example, primate BCMA. For example, in one embodiment, the antigen binding protein does not bind to TACI or BAFF-R.
[00162] The term "inhibits" as used throughout this specification in relation to the antigen binding proteins of the invention means that the biological activity of BCMA is reduced in the presence of the antigen binding proteins of the present invention compared to the BCMA activity in the absence of such antigen-binding proteins. Inhibition may be due, but not limited to, one or more binding of the blocking ligand, preventing the ligand from activating the receptor and / or regulating BCMA. The inhibition can also refer to a BCMA antigen-binding protein that causes cell apoptosis or ADCC. The antibodies of the invention can neutralize the activity of BCMA BAFF ligands and / or APRIL binding to BCMA. Neutralization levels can be measured in several ways, for example, by using the assays as shown in the examples below, for example, in 4.4 in an H929 cell NFkB signaling assay. The BCMA BAFF and APRIL ligands are able to induce NFkB signaling and the downstream events that link to BCMA. BCMA neutralization in this assay is measured by estimating the ability of monoclonal antibodies to anti-BCMA antibodies to inhibit NFkB induction conducted by BAFF or APRIL.
[00163] Whether an antibody or antigen binding fragment thereof is capable of neutralization after this is indicative of inhibiting the interaction between human BAFF or APRIL and BCMA. Antibodies that are considered to have neutralizing activity against human BCMA must have an IC 50 of less than 30 micrograms / ml or less than 20 micrograms / ml, or less than 10 micrograms / ml or less than 5 micrograms / ml or less than than 1 microgram / ml or less than 0.1 microgram / ml in the H929 stimulus assay as shown in Example 4.4
[00164] The "CDRs" are defined as the region determining the complementary amino acid sequences of an antibody which are the hypervariable domains of immunoglobulin chains of light and heavy chains. There are CDRs of three heavy chains and three light chains (or regions of CDR regions) in the variable portion of an immunoglobulin. In this way, the "CDRs" as used herein can refer to all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain and all light chain CDRS, if appropriate).
[00165] CDRs provide the majority of residues and contact for the binding of the antibody to the antigen or epitope. The CDRs of interest in this invention are derived from heavy and light chain donor variable antibody sequences and include analogs of naturally occurring CDRs, whose analogs also divide or retain the same specificity and binding antigen and / or neutralizing capacity as the antibody donor from which they were derived.
[00166] CDR antibody sequences can be determined by the Kabat numbering system (Kabat et al; (Protein sequences of immunological interest NIH, 1987), alternatively these can be determined using the Chothia numbering system ( Al-Lazikani et al., (1997) JMB 273,927-948), the contact definition method (MacCallum RM, and Martem um.CR and Thornton JM, (1996), Journal of Molecular Biology, 262 (5), 732 -745) or any other method established for numbering the residues in an antibody and determining the CDRs known to the person skilled in the art
[00167] Other numbering conventions for CDR sequences available to a person skilled in the art include "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region using at least two of the methods of Kabat, Chothia, AbM and contact can be determined to provide the “minimum connection unit”. The minimum connection unit can be a sub-portion of a CDR.
[00168] Table A below represents a definition using each numbering convention for each CDR or link unit. The numbering scheme of is used in Table X for numbering the variable amino acid sequence domain. It should be noted that some of the CDR definitions may vary depending on the individual publication used. Table A

[00169] Through this specification, the amino acid residues in the antibody sequences are numbered according to the Kabat scheme. similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" follow the Kabat numbering system as presented in Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987.
[00170] The terms "variants" refer to at least one, two or three amino acid bands in the sequence. These amino acid changes can be deletion, substitution or addition but are, preferably, substitution. In such an embodiment, substitutions are conservative substitutions.
[00171] In an alternative embodiment, the sequence variant sequence contains at least one substitution while the canon of the antigen binding protein is retained.
[00172] The complementarity determining regions (CDRs) L1, L2, L3, H1 and H2 tend to structurally present one of a finite number of main chain conformations. The particular canonical structure of a CDR is defined by both the length of the CDR and the arc packaging, determined by residues located at key positions in both the CDRs and the structural regions (structural determination of residues or SDRs). Martin and Thornton (1996; J Mol Biol 263: 800-815) generated an automatic method to define the canonical standards "key residue". Group analysis is used to define canonical classes for the CDR series and canonical patterns are then identified by analyzing isolated hydrophobics, hydrogen bond residues and, for example, conserved glycines. The CDRs of the antibody sequences can be assigned to canonical classes by comparing the sequences with the standard key residues and recording each standard using identity or similarity matrices.
[00173] The terms "VH" and "VL" are used here to refer to a heavy chain variable domain and a light chain variable domain respectively of an antibody.
[00174] As used herein the term "domain" refers to a folded protein structure having a tertiary structure independent of the rest of the protein. In general, domains are responsible for the distinct functional properties of proteins and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and / or the domain. A "single antibody variable domain" is a folded polypeptide domain that comprises sequences characteristic of antibody variable domains. Therefore, this includes complete antibody variable domains and modified variable domains, for example, where one or more arcs have been replaced by sequences that are not characteristic of antibody variable domains or antibody variable domains that have been truncated or comprise N-terminal extensions or C-terminal, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the natural-sized domain.
[00175] The phrase "single immunoglobulin variable domain" refers to an antibody variable domain (VH, VHH, VL) that specifically binds an antigen or epitope regardless of a different V region or domain. A single variable immunoglobulin domain may be evident in one format (for example, homo- or hetero-multimer) with another, variable regions or different variable domains in the other regions or domains that are required for antigen binding by the immunoglobulin variable domain unique (i.e., where the single immunoglobulin variable domain binds antigen regardless of the additional variable domains). An "antibody domain" or "dAb" is the same as an "unique immunoglobulin variable domain" that is capable of binding to an antigen as the term is used herein. A single immunoglobulin variable domain can be a human antibody variable domain, but it also includes single antibody variable domains from other species, such as rodent (for example, as described in WO 00/29004), sand shark and camelid VHH dAbs . Camelid VHH are single variable domain immunoglobulin polypeptides that are derived from species including camel, llama, alpaca, dromedary and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains can be humanized according to standard techniques available in the art and such domains are still considered to be "domain antibodies" according to the invention. As used herein “VH includes camelid VHH domains. NARV are another type of unique variable immunoglobulin domain that has been identified in cartilaginous fish that include the sand shark. These domains are also known as a new receptor and antigen variable region (usually abbreviated to V (NAR) or NARV). For additional details see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.
[00176] The term "epitope binding domain" refers to a domain that specifically binds an antigen or epitope regardless of a different V region or domain, this may be an antibody domain (dAb), for example, a simple human, camelid or shark immunoglobulin variable domain or this may be a domain that is a derivative of a structure selected from the group consisting of CTLA-4 (Evicorpo); lipocalin; protein A-derived molecules, such as protein A Z domain (Affibody, SpA), A-domain (Avimer / Maxibody); heat shock proteins, such as GroEl and GroES; 29eroxidise29g (trans-body); ancirin repeat protein (DARPin); peptide aptamer; lectin-like domain C (Tetranectin); y- human crystalline and human ubiquitin (afilins); PDZ domains; scorpion toxin kunitz toxins from human protease inhibitors and fibronectin (adnectin); that were subjected to the protein project in order to obtain binding to a ligand other than the natural ligand.
[00177] CTLA-4 (antigen 4 associated with cytotoxic T lymphocyte) is a receptor of the CD28 family mainly on CD4 + T cells. Its extracellular domain has an Ig fold similar to the variable domain. The arcs corresponding to the CDRs of the antibodies can be replaced with a heterologous sequence to confer different binding properties. CTLA-4 molecules designed to have different binding specificities are also known as Evibodies. For additional details see Journal of Immunological Methods 248 (1-2), 31-45 (2001)
[00178] Lipocalins are a family of extracellular proteins that carry small hydrophobic molecules, such as steroids, bilins, retinoids and lipids. These have a rigid 8-blade secondary structure with several arches at the open end of the conical structure that can be designed to bind to different target antigens. Anticalins are between 160 and 180 amino acids in size and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633.
[00179] An antibody is a structure derived from Protein A from Staphilococcus aureus that can be designed to bind to the antigen. The domain consists of a bundle of three helices of approximately 58 amino acids. The libraries were generated by randomizing surface residues. For additional details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1
[00180] Avimers are multi-domain proteins derived from the A domain structure family. The natural domains of approximately 35 amino acids adopt a defined disulfide linked structure. Diversity is generated by changing the variation presented by the A family of domains. For further details see Nature Biotechnology 23 (12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16 (6), 909917 (June 2007)
[00181] A transferrin is a monomeric serum transport glycoprotein. Transferrins can be designed to bind to different target antigens by inserting peptide sequences into a permissive surface arc. Examples of designed transferrin structures include the Transbody. For additional details, see J. Biol. Chem 274, 24066-24073 (1999).
[00182] Planned ancirin repeat proteins (DARPins) are derived from Ancirin which is a family of proteins that mediate the binding of integral membrane proteins to the cytoskeleton. A single ancirin repeat is a 33-residue motif consisting of two α-propellers and a β loop. These can be designed to bind different target antigens by randomizing residues in the first a-helix and a β loop of each repetition. Its connection interface can be increased by increasing the number of modules (an affinity maturation method). For additional details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.
[00183] Fibronectin is a structure that can be designed to be linked to an antigen. Adnectins consist of a main chain of the natural amino acid sequence of the 10th domain of the 15 type III human fibronectin (FN3) repeat units. Three arcs at one end of the e-sandwich can be designed to allow an adnectin to specifically recognize a therapeutic target of interest. For additional details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, W02005056764 and US6818418B1.
[00184] Peptide aptamers are combinatorial recognition molecules that consist of a protein of constant structure, typically thioredoxin (TrxA) that contains a restricted variable peptide arc inserted in the active site. For additional details, see Expert Opin. Biol. The R. 5, 783-797 (2005).
[00185] Microbodies are derived from naturally occurring microproteins 25 to 50 amino acids in length that contain 3 to 4 cysteine bridges - examples of microproteins include KalataB1 and conotoxin and knotins. Microproteins have an arc that can be designed to include up to 25 amino acids without affecting the total microprotein fold. For additional details on projected knotin domains, see WO2008098796.
[00186] Other epitope binding domains include proteins that have been used as a framework to design different target antigen binding properties include human y-crystalline and human ubiquitin and human ubiquitin (afilines), kunitz-like domains of human protease inhibitors, PDZ domains of the Ras AF-6 binding protein, scorpion toxins (caribdotoxin), lectin-like domain C (tetranectins) are reviewed in Chapter 7 - Non-Antibodies Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel ) and Protein Science 15: 14-27 (2006). The epitope-binding domains of the present invention can be derived from any of these alternative protein domains.
[00187] As used herein, the term "antigen binding site" refers to a site on a protein that is capable of specifically binding to the antigen, this may be a single domain, for example, a binding domain epitope or it can be paired VH / VL domains as seen in a standard antibody. In some embodiments of the single chain invention, the Fv domains (ScFv) can provide antigen binding sites.
[00188] The terms "mAbdAb" and dAbmAb "are used herein to refer to proteins that bind antigen of the present invention. The two terms can be used interchangeably, and are intended to have the same meaning as used here.
[00189] The term "antigen binding protein" as used herein refers to antibodies, antibody fragments for example, an antibody domain (dAb), ScFv, Fab, Fab2, and other protein constructs. The antigen binding molecules can comprise at least one variable domain of Ig, for example, antibodies, antibody domain (dAbs), Fab, Fab ', F (ab') 2, Fv, ScFv, diabodies, mAbdAbs, antibodies, antibodies heteroconjugates or bispecific antibodies. In one embodiment, the binding antigen molecule is an antibody. In another embodiment, the binding antigen molecule is a dAb, that is, a unique immunoglobulin variable domain such as a VH, VHH or VL that specifically binds to an antigen or epitope regardless of a region or domain V different. The binding antigen binding molecules may be capable of binding to two targets, i.e., these may be dual targeting proteins. The binding antigen molecules can be a combination of the antibodies and antigen binding fragments, such as for example, one or more domain antibodies and / or one or more ScFvs bound to a monoclonal antibody. The binding antigen binding molecules can also comprise a non-Ig domain, for example, a domain that is a derivative of a structure selected from the group consisting of CTLA-4 (Evibody); lipocalin; protein A-derived molecules, such as Protein A domain Z (Affibody, SpA), domain A (Avimer / Maxibody); Heat shock proteins such as GroEl and GroES; 31eroxidise31g (trans-body); ancirin repeat protein (DARPin); peptide aptamer; lectin-like domain C (Tetranectin); y- human crystalline and human ubiquitin (afilines); PDZ domains; kunitz-like domains of scorpion toxin from human protease inhibitors and fibronectin (adnectin); who were subjected to the protein design in order to obtain the OSM binding. As used herein "antigen binding protein" will be able to antagonize and / or neutralize human OSM. In addition, an antigen binding protein can inhibit and / or block OSM activity by binding to OSM and prevent a natural ligand from binding and / or activate the gp130 receptor.
[00190] The term "Effect or Function" as used herein is understood to refer to one or more of antibody-dependent cell cytotoxic activity (ADCC), complement-dependent activity (CDC) -mediated cytotoxic responses, Fc-mediated phagocytosis and antibody recycling via the FcRn receptor. For IgG antibodies, the effect or functionality that includes ADCC and ADCP is mediated by the interaction of the heavy chain constant region with a family of Fcy receptors present on the surface of immune cells. In humans, these include FcyR1 (CD64), FcyR11 (CD32) and FcyR111 (CD16). The interaction between the antigen-binding protein bound to the antigen and the formation of the Fc / Fcy complex induces a range of effects that include cytotoxicity, immune cell activation, phagocytosis and inflammatory cytokine release and cytokines.
[00191] The interaction between the constant region of an antigen-binding protein and several Fc receptors (FcR) is believed to measure the effect or functions of the antigen-binding protein. The significant biological effects may be a consequence of effect or functionality, in particular, antibody-dependent cellular cytotoxicity (ADCC), complement fixation (complement-dependent cytotoxicity or CDC), and antigen binding protein half life / release. Usually, the ability to mediate the effect or function requires the binding of the antigen-binding protein to an antigen and not all proteins that bind the antigen will mediate each effect or function.
[00192] Effect or function can be measured in several ways which include, for example, by binding FcyR111 to natural killer cells or by using FcyR1 to monocytes / macrophages for measurement as to the ADCC actuating function. for example, an antigen-binding protein of the present invention can be estimated for the effective function of ADCC in a natural killer cell assay. Examples of such assays can be seen in Shields et al, 2001 The Journal of Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of Biological Chemistry, Vol 268, p25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.
[00193] Examples of assays to determine CDC function include those described in 1995 J Imm Meth 184: 29-38.
[00194] Some isotypes of human constant regions, in particular IgG4 and IgG2 isotypes, essentially need the functions of a) complement activation by the classical path; and b) antibody-dependent cell cytotoxicity. Various modifications to the constant region of the heavy chain of proteins that bind antigen can be carried out depending on the desired effect or property. IgG1 region constants that contain specific mutations have been separately described to reduce binding to Fc receptors and therefore reduce ADCC and CDC (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. Imunol 1991, 147; 26572662; 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).
[00195] In an embodiment of the present invention an antigen binding protein is provided that comprises a constant region such that the antigen binding protein has reduced ADCC and / or complement or functionality activation. In such an embodiment the heavy chain constant region may comprise a naturally invalid IgG2 or IgG4 isotype constant region or a mutated IgG1 constant region. Examples of suitable modifications in EP0307434. An example comprises replacing alanine residues at positions 235 and 237 (numbering the EU index).
[00196] Human IgG1 region constants containing specific mutations or altered glycosylation at the Asn297 residue have been described to enhance binding to Fc receptors. In some cases, mutations have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103; 40054010; Shields et al. J Biol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Imunol, 2007, 44; 1815-1817).
[00197] In one embodiment of the present invention, such mutations are, at one or more of the selected positions of 239, 332 and 330 (IgG1) or equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D and 1332E and A330L. In one embodiment, the antigen binding protein of the invention described herein is mutated at positions 239 and 332, for example, S239D and 1332E or in another embodiment it is mutated at three or more selected positions of 239 and 332 and 330, for example, S239D and 1332E and A330L. (EU index number).
[00198] In an alternative embodiment of the present invention, an antigen binding protein is provided that comprises a heavy chain constant region with an altered glycosylation profile such that the antigen binding protein has enhanced actuating function. For example, where the antigen binding protein has enhanced ADCC or enhanced CDC or where it has as much effect as enhanced ADCC and CDC function. Examples of suitable methodologies for producing proteins that bind the antigen with an altered glycosylation profile are described in WO2003011878, WO2006014679 and EP1229125, all of which can be applied as proteins that bind the antigen of the present invention.
[00199] The present invention also provides a method for producing an antigen binding protein according to the invention comprising the steps of: a) culturing a recombinant host cell that comprises an expression vector comprising the isolated nucleic acid as described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase has been inactivated in the recombinant host cell; and b) recovering the antigen binding protein.
[00200] Such methods for producing antigen-binding proteins can be performed, for example, using the POTELLIGENTTM technology system available from BioWa, Inc. (Princeton, NJ) in which CHOK1SV cells that need a copy The functional function of the FUT8 gene produces monoclonal antibodies that have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that is increased over an identical monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects of the POTELLIGENTTM technology system are described in US7214775, US6946292, W00061739 and W00231240 all of which are incorporated herein by reference. That person skilled in the art will also recognize other appropriate systems.
[00201] In one embodiment of the present invention, an antigen binding protein is provided which comprises a chimeric heavy chain constant region, for example, an antigen binding protein which comprises a chimeric heavy chain constant region with at least an Ig2 CH2 domain such that the antigen binding protein has enhanced effector function, for example, in which it has enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. In such an embodiment, the antigen binding protein can comprise an IgG3 CH2 domain or both CH2 domains can be IgG3.
[00202] Also provided is a method of producing an antigen binding protein according to the invention comprising the steps of: a) culturing a recombinant host cell that comprises an expression vector that comprises an isolated nucleic acid as described herein in that the expression vector comprises a nucleic acid sequence encoding an Fc domain that has both the amino acid residues of the IgG1 and IgG3 Fc domain; and b) recovering the antigen binding protein.
[00203] Such methods for producing antigen-binding proteins can be performed, for example, using the COMPLEGENTTM technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kogyo (now Kyowa Hakko Kirin Co., Ltd.) Co., Ltd. In which a recombinant host cell comprising an expression vector in which a nucleic acid sequence encoding a chimeric Fc domain that has both the IgG1 and IgG3 Fc amino acid residues is expressed to produce an antigen-binding protein that has enhanced complement-dependent cytotoxicity (CDC) activity that is increased relative to an identical antigen-binding protein that otherwise lacks such a chimeric Fc domain. Aspects of the COMPLEGENTTM technology system are described in W02007011041 and U520070148165 each of which are incorporated here by reference. In an alternative embodiment, CDC activity can be increased to introduce specific sequence mutations into the Fc region of an IgG chain. That person skilled in the art will also recognize other appropriate systems.
[00204] It is evident to those skilled in the art that such modifications can not only be alone, but can be used in combination with each other in order to intensify the additional effective function.
[00205] In such an embodiment of the present invention an antigen binding protein is provided which comprises a heavy chain constant region comprising a chimeric and mutated heavy chain constant region for example, wherein an antigen binding protein comprising at least one IgG3 CH2 domain and an IgG1 CH2 domain, where the IgG1 CH2 domain has one or more mutations at the selected positions 239 and 332 and 330 (for example, the mutations can be selected from 5239D and 1332E and A330L) such that the antigen binding protein has enhanced effector function, for example, in which it has one or more of the following functions, enhanced ADCC or enhanced CDC, for example, in which it has enhanced ADCC and enhanced CDC. In one embodiment the IgG1 CH2 domain has mutations 5239D and 1332E.
[00206] In an alternative embodiment of the present invention an antigen binding protein is provided which comprises a constant chimeric region of the heavy chain and which has an altered glycosylation profile. In such an embodiment the heavy chain constant region comprises at least one IgG3 CH2 domain and one IgG1 CH2 domain and has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8: 3 or less, for example, in which the antigen-binding protein is defucosylated so that said antigen-binding protein has an enhanced effector function compared to an equivalent of the antigen-binding protein with a heavy chain immunoglobulin constant region that lacks said mutations and altered glycosylation profile, for example, in which it has one or more of the following functions, enhanced ADCC or enhanced CDC, for example, in which it has enhanced ADCC and enhanced CDC. In an alternative embodiment the antigen binding protein has at least one IgG3 CH2 domain and at least one constant domain of the IgG1 heavy chain in which so many IgG CH2 domains are mutated according to the limitations described herein.
[00207] In one aspect of the invention there is provided a method of producing an antigen binding protein according to the invention described herein comprising the steps of: a) culturing a recombinant host cell that contains an expression vector that contains an acid isolated nucleic acid as described herein, said expression vector even though it comprises an Fc nucleic acid sequence encoding a chimeric Fc domain that has both the amino acid residues of the IgG1 and IgG3 Fc domains, and wherein the FUT8 gene encoding alpha- 1,6-fucosyltransferase was inactivated in the recombinant host cell; and b) Recovering an antigen-binding protein
[00208] Such methods for producing antigen-binding proteins can be performed, for example, using the ACCRETAMABTM technology system available from BioWa, Inc. (Princeton, NJ) that combines the POTELLIGENTTM and COMPLEGENTTM technology systems to produce a protein antigen binding agent that has both enhanced ADCC and CDC activity that is increased relative to an identical monoclonal antibody otherwise lacking a chimeric Fc domain and having fucose in the oligosaccharide.
[00209] In yet another embodiment of the present invention an antigen binding protein is provided which comprises a mutated and chimeric constant region of the heavy chain in which said antigen binding protein has an altered glycosylation profile such that antigen-binding protein has enhanced effector function, for example, in which it has one or more of the following functions, enhanced ADCC or enhanced CDC. In one embodiment the mutations are selected from positions 239 and 332 and 330, for example, the mutations are selected from S239D and 1332E and A330L. In another embodiment the heavy chain constant region comprises at least one IgG3 CH2 domain and one IgG1 Ch2 domain. In one embodiment the constant region of the heavy chain has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8: 3 or less for example, the antigen binding protein is defucosylated, so that said antigen binding protein has an enhanced effector function compared to an equivalent non-chimeric antigen binding protein or an immunoglobulin heavy chain constant region that lacks said mutations and altered glycosylation profile. Immunoconjugates
[00210] Also provided is an immunoconjugate (interchangeably referred to as "antibody-drug conjugate", or "ADCs") comprising an antigen-binding protein according to the invention as described herein that includes, but is not limited to, one antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a development inhibitor, a toxin (for example, a protein toxin, an enzymatically active toxin of animal, plant, fungal or bacterial origin, or fragments of the same), or a radioactive isotope (that is, a radioconjugate).
[00211] Immunoconjugates were used for the local release of cytotoxic agents, that is, drugs that kill or inhibit the development or proliferation of cells, 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; US Pat. No. 4,975,278). Immunoconjugates allow for targeted delivery of a portion of drug to a tumor, and intracellular accumulation in it, where systematic administration of unconjugated drugs can result in unacceptable levels of toxicity to normal cells as well as tumor cells seek 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 antibodies and monoclonal antibodies have been listed as useful in these strategies (Rowland et al., (1986) Cancer Imunol. Imunother. 21: 183-87). The drugs in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins used in antibody toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, lesser molecule toxins r such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92 (19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13: 786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58: 2928; Hi nMan et al (1993) Cancer Res. 53: 3336-3342).
[00212] In one embodiment, the present invention includes immunoconjugates that have the following general structure: ABP - ((Ligand) n - Ctx) m
[00213] Where ABP is an antigen binding protein The linker is absent or any of the cleavable or non-cleavable linker described here Ctx is any cytotoxic agent described here n is 0, 1, 2, or 3 and is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[00214] Examples of antibodies linked by an MC linker with auristatins such as MMAE and MMAF are described in the following structures:

[00215] In certain embodiments, an immunoconjugate comprises an antigen binding protein, which includes but is not limited to, an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful in the generation of immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A-chain diphtheria, active fragments of diphtheria toxin unbinding, A-chain exotoxia (from Pseudomonas aeruginosa), rice A-chain, abrin A-chain, A-chain modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcine, crotin, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin , phenomycin, enomycin, and trichothecenes. See, for example, WO 93/21232 published October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 211A1 212Bi 131I 131I 90Y 186R,,, na,, and e.
[00216] The antigen-binding proteins of the present invention can also be conjugated to one or more toxins, which include, but are not limited to, a calicheamicin, maytansinoids, dolastatin, aurostatin, a trichothecene, and CC1065, and derivatives of these toxins that has toxin activity. Suitable cytotoxic agents include, but are not limited to, an auristatin which includes dovaline-valine-dolaisoleunin-dolaproine-phenylalanine (MMAF) and monomethyl auristatin E (MMAE) as well as ester forms of MMAE, a minor groove bonding agent DNA, a lower DNA slot alkylating agent, an enedino, a lexitropsin, a duocarmicin, a taxane, which includes paclitaxel and docetaxel, a puromycin, a dolastatin, a maytansinoid, and a vinca alkanoid. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chaliqueamicin, maytansine, DM-1, DM-4, netropsin. Suitable cytotoxic agents include anti-tubulin agents, such as auristatin, vinca alkanoid, podophyllotoxin, taxane, baccatin derivative, cryptophysin, maytansinoid, combretastatin, or dolastatin. The antitubulin agent includes dimethylvaline-valinadolaisoleuin-dolaproin-phenylalanine-p-phenylene-diamine (AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, doclxoxone, paclitaxel, doclotaxel, paclitaxel, epletaxel, paclitaxel, docletaxel, paclitaxel, epothilone B, nocodazole, colloquine, colcimid, estramustine, cemadotine, discodermolide, maytansine, DM-1, DM-4 or eleuterobin.
[00217] Antibody drug conjugates were produced by conjugating the smaller molecule anti-tubulin agent of monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) to antibodies. In the case of MMAE the linker consists of a thiol reactive maleimide, a caproil spacer, the dipeptide valinacitrulin, and p-aminobenzyloxycarbonyl, a group of its own immolative fragment. In the case of MMAF a protease resistant maleimidocaproyl ligand is used. The conjugation process leads to heterogeneity in the binding of the drug antibody, varying in both numbers of drugs bound in each antibody molecule (mol ratio [MR]), and the binding site. The most prevalent species is the material with an MR = 4; less prevalent are materials with MR of 0, 2, 6, and 8. The average total MR drug-to-antibody is approximately 4. Immunoconjugate production
[00218] The binding points are cysteines produced by the mild reduction of antibody interchain disulfides that is carried out while the antibodies are immobilized on the protein G affinity resin (thus able to use the wide reagent excesses without intermediate purifications). While immobilized, a large excess of TCEP will totally reduce the interchain disulfides but have no impact on the binding of the antibody to the resin.
[00219] The number of thiols per antibody generated by this procedure depends on the source and isotype of the antibodies. For example, human IgG1s (and chimeric mouse-human) have 4 reducible disulfides, and thus generate 8 thiols in total reduction, whereas murine IgG1s have 5 reducible disulfides and produce 10 thiols. If ADCs with maximum drug loading (for example, 10 drugs per antibody for murine IgG1s) are desired, then the maleimido-drug-binder can simply be added to the immobilized antibodies in sufficient excess to ensure complete conjugation. However, ADCs with few drugs per antibody can also be prepared from fully reduced antibodies by which they include a biologically inert coating agent such as N-ethyl maleimide (NEM) that occupies some of the thiols available in the antibody. When maleimido-drug-ligand and coating agent are added simultaneously to the totally reduced antibody and in the large excess (at least 3 times), the two maleimide electrophiles compete for the limiting number of available thiols. In this way, drug loading is determined by the relative thiol reaction rates of the drug-binder and coating agent, and thus can be considered to be under kinetic control. The relative reaction rates of maleimido-drug-binders vary significantly, and therefore the molar ratio of the drug-NEM binder present in a reaction mixture must be determined empirically to achieve on a panel of ADCs with a desired level of loading of damn it. The mol fraction of the drug ligands SGD-1006 (vcMMAE) and SGD-1269 (mcMMAF) in the NEM mixtures that produce ADCs with approximately 4 drugs by the antibody are summarized in Table 2 by the common human and murine IgG isotypes. Auristatinas and Dolastatinas
[00220] In some embodiments, the immunoconjugate comprises an antigen-binding protein or conjugated analogue antibody and peptide derivatives of dolastatin or dolostatin, auristatin (U.S. Patent No. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and cell division and nuclear (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584) and have anticancer (US Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). The drug portion of dolastatin or auristatin (which are derived from dolastatin pentapeptide) can be linked to the antibody via the N (amino) terminal or the C (carboxyl) terminal of the peptide drug portion (WO 02/088172).
Exemplary embodiments of auristatins include the N DE and DF terminally linked monomethyluristatin drug moieties, described in "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent No. 7,498,298, the description of which is expressly incorporated by reference in its entirety. As used herein, the abbreviation "MMAE" refers to monomethyl auristatin E. As used here the abbreviation "MMAF" refers to dovaline-valine-dolaisoleuine-dolaproin-phenylalanine.
[00222] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more fragments of amino acids and / or peptides. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry. Auristatin / dolastatin drug portions can be prepared according to the methods of: Pat. U.S. No. 5,635,483; Pat. U.S. 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, G. R., et al. Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15: 859-863. See also Doronina (2003) Nat Biotechnol 21 (7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent No. 7,498,298, filed on November 5, 2004, incorporated herein by reference in its entirety (described, for example, binders and methods of preparing monomethylvaline compounds such as conjugated to MMAE and MMAF ligands). Biologically active organic compounds that act as cytotoxic agents, specifically pentapeptides, are described in U.S. Patent No. 6,884,869; 7,498,298; 7,098,308; 7,256,257; and 7,423,116. Monoclonal antibodies bound with MMAE adn MMAF as well as various derivatives of auristatins and methods of making these are described in U.S. Patent No. 7,964,566.
[00223] Examples of auristatins include MMAE and MMAF the structures of which are shown below:
Maytansine and Maytansinoids
[00224] Maytansinoids are mitototic inhibitors that act by inhibiting the tubulin polymerization. Maytansine was first isolated from the western-made Maytenus serrata shrub (U.S. Pat. No. 3,896,111). Subsequently, it was recovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Highly cytotoxic maytansinoid drug drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. Methods for isolating ansamitocins are described in U.S. Patent No. 6,573,074. Synthetic maytansinol and derivatives and analogues thereof are described, for example, in U.S. Patent No. 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; 4,309,428; 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.
[00225] Antibody-Maytansinoid conjugates are prepared by chemically binding an antibody to a maytansinoid molecule without significantly decreasing the biological activity of the antibody or the maytansinoid molecule. See, for example, Pat. U.S. No. 5,208,020. An average of 3-4 Maytansinoid molecules conjugated by the antibody molecule has been shown to be effective in enhancing the cytotoxicity of the target cells without negatively affecting the function or solubility of the antibody, although still a toxin / antibody molecule should be expected to enhance cytotoxicity in use of the naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are described, for example, in U.S. Patent No. 5,208,020 and the other Non-Patent Patents and Publications refer to the previously mentioned. Maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or in other positions of the maytansinol molecule, such as various maytansinol esters. Methods for preparing matansinoids by binding with antibodies are described in U.S. Patent No. 6,570,024 and 6,884,874. Calicheamicin
[00226] The calicheamicin family of antibiotics is capable of producing double stranded DNA breaks in subpicomolar concentrations. For the preparation of the calicheamicin family conjugates, see U.S. Patent No. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all in the American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, .gama, 1l, .alpha, 21, .alpha, 31, N-acetyl-.gama, 11, PSAG and .teta.l1 (Hi nMan et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and the US Patents previously mentioned to American Cyanamid). Another antitumor drug that the antibody can be conjugated to is FFQ which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, the cellular absorption of these agents through the antibody mediated by internalization greatly enhances their cytotoxic effects. Other cytotoxic agents
[00227] Other anti-tumor agents that can be conjugated to antibodies include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively by the LL-E33288 complex described in U.S. Patent No. 5,053,394, 5,770,710, as well as speramicins (U.S. Pat. No. 5,877,296).
[00228] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, diphtheria toxin non-binding fragments, A-chain exotoxia (from Pseudomonas aeruginosa), A-chain rice, A-abrin chain , modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcine, crotine, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, WO 93/21232 published on October 28, 1993.
[00229] The present invention further considers an immunoconjugate formed between an antibody and a compound with nucleolytic activity (for example, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
[00230] For selective tumor destruction, the antibody may comprise a relatively radioactive atom. A variety of radioactive isotopes are available for the production of antibodies to radioconjugates. Examples include At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it can comprise a radioactive atom by scintigraphic studies, for example, tc99m or 1123, or a rotation label for nuclear magnetic resonance (NMR) imaging (also known as resonance imaging) magnetic, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[00231] Radio- or other labels can be incorporated into the conjugate in known ways. For example, the peptide can be biosynthesized or it can be synthesized by chemical amino acid synthesis using the appropriate amino acid precursors involving, for example, fluorine-19 instead of hydrogen. Labels such as tc99m or 1123, Re186, Re188 and 1n111 can be linked via a cysteine residue in the peptide. Yttrium-90 can be linked via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail. Preparation of ADCs
[00232] In drugs conjugated by the antibody, the antibody can be conjugated directly to the cytotoxic agent or through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a linker peptide cleavable by an intracellular protease, such as a lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine linker (phe-lys). Other suitable binders include hydrolyzable binders at a pH of less than 5.5, such as a hydrazone binder. Additional suitable cleavable linkers include disulfide linkers.
[00233] Bristol-Myers Squibb has described cleavable conjugate drugs of particular lysosomal enzyme. See, for example, Pat. U.S. No. 6,214,345. Seattle Genetics has published Applications in U.S. Patent Applications 2003/0096743 and U.S. Patent Applications 2003/0130189, which describes p-aminobenzylethers in drug release agents. The binders described in these applications are limited to the aminobenzyl ether compositions.
[00234] Antigen-binding protein and cytotoxic agent conjugates can be made using a variety of bifunctional protein-binding agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidil-4- ( N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as NCI dimethyl adipimidate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), compounds of bis-azido (such as bis (pazidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and fluorine bis compounds -active (such as 1,5-difluoro-2,4-dinitrobenzene).
[00235] Additionally, the binder can be composed of one or more binder components. Exemplary binding components include 6-maleimidocaproil (“MC”), maleimidopropanoil (“MP”), valine-citrulline (“valcit”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), 4 - N-Succinimidyl (2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 (“SMCC”), and N- (4-iodo-acetyl) aminobenzoate Succinimidyl ("STAB"). Additional binding components are known in the art and some are described herein. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Patent No. US 7,498,298, deposited on November 5, 2004, the contents of which are therefore incorporated by reference in their entirety.
[00236] The linkers can also comprise amino acids and / or amino acid analogs. The amino acid-binding components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citruline (vc or val-cit), 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 comprise an amino acid binding component include those of naturally occurring as well as minor amino acids and analogues of non-naturally occurring amino acids, such as citrulline. The amino acid-binding components can be designed and optimized in their selectivity by enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
[00237] Proteins that bind antigen and antibodies can be made reactive by conjugation with binding reagents. Nucleophilic groups in antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) secondary chain amine groups, for example, lysine, (iii) secondary chain thiol groups, for example, cysteine, and (iv) amino or hydroxyl sugar groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups in the linking moieties and binding reagents that include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamide groups; (iii) aldehydes, ketones, carboxyl, and maleimide. Certain antibodies have reducible interchain bisulfides, that is, cysteine bridges. Antibodies can be made reactive by conjugation with binding reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge in this way will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibodies by reacting lysines with 2-iminothiolane (Traut reagent) resulting in the conversion of an amine to a thiol. Reactive thiol groups can be introduced into the antibody (or fragment thereof) by introducing one, two, three, four or more cysteine residues (for example, preparing mutant antibodies that comprise one or more unnatural cysteine amino acid residues) .
[00238] Proteins that bind antigen and antibodies can also be modified to introduce electrophilic moieties, which can react with nucleophilic substituents on the binding reagent or drug. The sugars of the glycosylated antibodies can be oxidized, for example, with periodate oxidizing the reagents, to form groups of aldehyde or ketone that can react in the amine group of the binding reagents or drug moieties. The resulting Schiff imine base groups can form a stable bond, or can be reduced, for example, by the borohydride reagents to form the stable amine bonds. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with glactose oxidase or sodium meta-periodate can produce carbonyl groups (aldehyde and ketone) in the protein that can react with the appropriate groups in the drug (Hermanson, Bioconjugate Techniques) . In another embodiment, proteins containing residues of N-terminal serine or threonine can react with sodium meta-periodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3 : 138-146; US Pat. No. 5,362,852). Such aldehydes can be reacted with a drug or nucleophile linker moiety.
[00239] Nucleophilic groups in a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups in the binding moieties and binding reagents that include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehyde, ketone, carboxyl, and maleimide groups.
[00240] In some embodiments, the ligand is cleavable by a cleavage agent that is present in the intracellular environment (for example, within a lysosome or endosome or caveolea). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, which includes, but is not limited to, a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two long amino acids or at least three long amino acids. Cleavage agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze derivatives of the dipeptide drug resulting in the release of the active drug into target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). Peptidyl ligands can be cleavable by enzymes that are present in cells, for example, a peptidyl ligand that is cleavable by thiol-dependent cathepsin-B protease, which is highly expressed in cancerous tissue, can be used (for example, a Phe-Leu linker or a Gly-Phe-Leu-Gly (SEQ ID NO: 50)). Other such binders are described, for example, in U.S. Pat. U.S. No. 6,214,345. In specific embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, US Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit ligand). An advantage of using the intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
[00241] In the other embodiments, the cleavable binder is sensitive to pH, that is, sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive hydrolyzable binder under acidic conditions, for example, an unstable acidic binder that is hydrolyzable in the lysosome (for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or others) to be used. (See, for example, US Patent No. 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 ligands are relatively stable under conditions of neutral pH, such as those in the blood, but are unstable below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone link (see, for example, U.S. Pat. No. 5,622,929)).
[00242] In still other embodiments, the linker is cleavable under reducing conditions (for example, a disulfide linker). A variety of disulfide binders are known in the art, which include, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridylditium) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alfamethyl-alpha- (2-pyridyl-dithio) toluene) -, SPDB and SMPT (See, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immuoconjugates: Conjugates Antibody in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also US Pat. . 4,880,935.)
[00243] In still other specific embodiments, the ligand is a malonate ligand (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl ligand (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).
[00244] Typically, the ligand is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment", in the context of a ligand, means that no more than about 20%, typically no more than about 15%, more typically no more than about 10% , and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the binders, in a sample of ADC or ADC derivative, are cleaved when the ADC or derivative ADC present in an extracellular environment (for example, in plasma). Whether a ligand is not substantially sensitive to the extracellular environment can be determined, for example, by independent incubation with plasma both (A) the ADC and ADC derivative (the “sample ADC”) and (b) an equal molar amount of unbound antibody or therapeutic agent (the “control sample”) for a predetermined period of time (for example, 2, 4, 8, 16, or 24 hours) and then comparing an amount of unconjugated antibody or therapeutic agent present in the sample ADC with that present in the control sample, as measured, for example, by high performance liquid chromatography.
[00245] In other non-mutually exclusive embodiments, the ligand promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (i.e., in the environment of the ADC therapeutic agent linker-agent or ADC derivative as described herein). In still other embodiments, the linker promotes cell internalization when conjugated by both the therapeutic agent and the antigen or antibody-binding protein or derivative thereof (i.e., in the ADC environment or ADC derivative as described herein).
[00246] A variety of binders that can be used with the present compositions and methods are described in WO 2004010957 entitled "Drug Conjugates and Their Use for treating Cancer, An Autoimmune disease or an Infectious disease" deposited on July 31, 2003, and US Provisional Order No. 60 / 400,403, entitled “Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease”, filed on July 31, 2002 (the description of which is incorporated by reference here).
[00247] Alternatively, a fusion protein comprising the antigen-binding protein and cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The length of DNA can comprise respective regions that encode two portions of the conjugate adjacent to one another or separated by a region that encodes a peptide linker that does not destroy the desired properties of the conjugate.
[00248] In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) by use in pre-targeting the tumor in which the antibody-receptor conjugate is administered to the patient, followed by removal of the unbound conjugate by circulation using a clarifying agent and then administration of a "ligand" (for example, avidin) that is conjugated to a cytotoxic agent (for example, a radionucleotide).
[00249] The term "non-human antibody or antibody fragment thereof" as used herein means referring to antibodies or fragments thereof that originate from any species other than humans in which human includes chimeric antibodies.
[00250] The term "donor antibody" refers to an antibody (monoclonal, and / or recombinant) that contributes the amino acid sequence of its variable domains, CDRs, or other functional fragments or analogues thereof to a first immunoglobulin associate , so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralization of the donor antibody activity characteristic.
[00251] The term "recipient antibody" refers to an antibody (monoclonal and / or recombinant) heterologous to the donor antibody, which contributes to all (or any portion, but preferably all) of the amino acid sequence encoding its structure regions heavy chain and / or light chain and / or its constant regions from the light and / or heavy chain to the first associated with immunoglobulin. The human antibody is the recipient antibody. The term "human receptor sequence" as used herein means to refer to an antibody structure or antibody fragment thereof which comprises the amino acid sequence of a VH or VL structure derived from a human antibody or antibody fragment thereof or a human consensus sequence structure in CDR's from non-human species can be incorporated.
[00252] The term "incorporation" of CDR’s or hypervariable regions as used herein covers any medium by non-human CDR’s are situated with the human receptor structure. It will be estimated that it can be obtained in several ways, for example, nucleic acids encoding the desired amino acid sequence can be generated by mutating the nucleic acids encoding the non-human variable domain sequence so that the structure residues therefrom are switched to human receptor structure residues, or by mutating the nucleic acid encoding the human variable domain sequence so that CDR's are switched to non-human residues, or to synthesize the nucleic acids encoding the desired sequence. In one embodiment the final sequence is generated in silico.
[00253] The present invention is now described by way of example only. The attached claims may include a generalization of one of the more of the following examples. Examples Example 1 Monoclonal Antibody Generation and Selection 1.1 Immunization Strategies
[00254] CA8 precursor of murine anti-human BCMA mAb was identified from hybridomas derived from mice immunized with human-sized human BCMA. One BALB / c mouse was immunized i.p. with 25 μg of recombinant protein (rBCMA) combined with CFA. The mouse was boosted three times at one-month intervals with 25 μg of natural-sized rBCMA protein + 10 μg of stable emulsion of monophosphoryl lipid A (MPL-SE) (Corixa Corporation, Seattle, WA) and given a prefusion boost 30 μg of rBCMA iv protein 3 days before fusion. Hybridomas were generated and cloned using the ClonaCell-HY hybridoma cloning kit (StemCell Technologies, Vancouver, BC) or using a conventional method. In the conventional method, the B cells of the spleens of the immunized animal were fused with Sp2 / 0 myeloma cells in the presence of PEG (Sigma-Aldrich, St. Louis, MO). After overnight recovery, the fused cells were plated at the limiting dilution in 96 well plates and subjected to hypoxanthine-aminopterin-thymidine selection. Hybridoma culture supernatants were examined for the presence of anti-BCMA antibodies by ELISA and flow cytometry.
[00255] The anti-human BCMA murine precursor mAb 5307118G03 was identified from hybridomas derived from SJL mice immunized with the recombinant human BCMA / TNFRSF17-Fc (R&D 193-Fc) chimera using the RIMMS method (multiple sites of rapid immunization ). On Day 0, 5 μg of protein per mouse were emulsified in AS02a adjuvant at 2 sites on the dorsum (over the hips and over the shoulders) and underlying the main lymph nodes at 4 sites in the front. On day 6 and day 11 2.5 μg of protein per mouse in RIBI adjuvant was injected underlying the main lymph nodes at 4 sites in the front. On day 14 the animals were sacrificed. Lymph nodes and spleen were excised, disrupted and a somatic cell fusion induced by PEG1500 performed using a 3: 1 ratio with X63 AG8 653.GFP.BcI-2,11 mouse myeloma cells (BioCat 112754; R17209 / 58) . The fusion was plated on 10 x 96 well plates and screened directly from them.
[00256] The anti-human BCMA murine precursor mAb 5336105A07 has been identified from hybridomas derived from identical immunizations. Lymph nodes and spleen were excised on day 14, ruptured, and a Cytopulse electrofusion was performed using a 1: 1 ratio with X63 AG8 653.GFP.BcI-2.11 mouse myeloma cells (BioCat 112754; R17209 / 58). The fusion was plated on omnitrays containing semi-solid medium before selecting 10 x 96 well plates and was screened directly from these 5 days later.
[00257] The murine anti-human BCMA precursor mAbs S332121F02 and S332126E04 were identified from hybridomas derived from SJL mice immunized with recombinant Fc fusion of the human BCMA (4-53) BCMA extracellular domain using the RIMMS (Rapid Immunization) method. On Day 0, 5 μg of protein per mouse was emulsified in AS02a adjuvant at 2 sites on the back (over the hips and over the shoulders) and underlying the main lymph nodes at 4 sites in the front. On day 6 5 μg of recombinant cyan BCMA-Fc protein per mouse in RIBI adjuvant was injected underlying the main lymph nodes at 4 sites in the front. On day 11 2.5 μg of recombinant human BCMA-Fc and 2.5 μg of recombinant cino BCMA-Fc per mouse in RIBI adjuvant was injected underlying the main lymph nodes at 4 sites in the front. On day 14 the animals were sacrificed and the cells treated as for S307118G03.
The murine anti-human BCMA precursor mAb S322110D07 was identified from hybridomas derived from SJL mice immunized with recombinant Fc fusion of the human BCMA extracellular domain (453) in complex with recombinant human April (R&D 5860-AP / CF) pre-mixed in a 1: 1 molar ratio. The mice were immunized i.p. with 5 μg of April / BCMA-Fc de Cino complex in PBS, suspended in RIBI adjuvant, 100 μl dose per mouse and reinforced 3 times at 3 to 4 week intervals with 2.5 μg of April / BCMA complex -Fino de Cino in PBS, suspended in RIBI adjuvant, 100 μl dose per mouse injected through the intraperitoneal route and given a pre-fusion boost of the same immunogen 1 day before fusion and treated as for S307118G03.
The murine anti-human BCMA precursor mAbs S335115G01 and 5335122F05 were identified from hybridomas derived from SJL mice immunized with a recombinant Fc fusion mixture from the human BCMA extracellular domain (4-53) and recombinant Fc fusion from the domain extracellular cyan BCMA (4-52) using the RIMMS method (multiple sites for rapid immunization). On Day 0, 2, 5 μg of each protein per mouse were emulsified in adjuvant AS02a and injected at 2 sites on the back (over the hips and over the shoulders) and underlying the main lymph nodes at 4 sites in the front. On day 6 and day 11 2.5 μg of each protein per mouse in RIBI adjuvant was injected underlying the main lymph nodes at 4 sites in the front. On day 14 the animals were sacrificed. The lymph nodes and spleen were excised, ruptured and a Cytopulse electrofusion was performed using a 1: 1 ratio with the X63 AG8 653.GFP.BcI-2,11 mouse myeloma cells (BioCat 112754; R17209 / 58). The fusion was plated into omnitrays containing semi-solid medium before choosing 32 x 96 well plates and was screened directly from these 5 days later. Example 2 Humanization. 2.1 Cloning of CA8 Hybridoma Variable Regions
[00260] The total RNA was extracted from CA8 hybridoma cells, the heavy and light variable domain cDNA sequence was then generated by reverse transcription and polymerase chain reaction (RT-PCR). The advanced primer for RT-PCR was a mixture of specific primers degenerate for leader sequences of the murine immunoglobulin gene and the reverse primer was specific for antibody constant regions. Reverse primers specific for IgG1, IgG2a and IgG2b were used in this case since the isotype was unknown. To design the primers, multiple DNA sequence alignments of the leader sequences of the mouse VH and Vk genes were generated. 2.2 Cloning of chimeric CA8
[00261] DNA expression constructs encoding the chimeric antibody have been prepared again by constructing overlapping oligonucleotides that include restriction sites for cloning into mammalian expression vectors as well as a human signal sequence. The HindIII and SpeI restriction sites were introduced into the structure of the VH domain that contains the signal sequence for cloning into mammalian expression vectors that contain the human y constant region. The HindIII and BsiWI restriction sites were introduced into the structure of the VL domain that contains the signal sequence for cloning into mammalian expression vectors that contain the human kappa constant region. 2.3 Cloning of humanized CA8 variants
[00262] DNA expression constructs encoding humanized antibody variants have been prepared again by the construction of overlapping oligonucleotides that include restriction sites for cloning into mammalian expression vectors as well as a human signal sequence. The HindIII and SpeI restriction sites were introduced into the structure of the VH domain containing the signal sequence for cloning into mammalian expression vectors containing the human y constant region. The HindIII and BsiWI restriction sites were introduced into the structure of the VL domain that contains the signal sequence for cloning into the mammalian expression vector containing the human kappa constant region. 2.4 Expression of recombinant CA8 antibodies (which includes antibody quantification)
[00263] The expression plasmids encoding the heavy and light chains respectively were transiently cotransfected into HEK 293 6E cells and expressed on a small scale to produce antibody. Antibodies were quantified by ELISA. ELISA plates were coated with anti-human IgG (Sigma 3382) at 1 mg / ml and blocked with blocking solution (4% BSA in Tris-buffered saline). Various dilutions of the tissue culture supernatants were added and the plate was incubated for 1 hour at room temperature. Dilutions of a known standard antibody were also added to the plate. The plate was washed in TBST and the binding was detected by the addition of a peroxidysis labeled anti-human light chain antibody (Sigma A764) to a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temperature before washing in TBST. The plate was developed by adding OPD substrate (Sigma P987) and color development was stopped by adding 2M H2SO4. Absorbance was measured at 490 nM and a standard curve plotted using data for known standard dilutions. The standard curve was used to estimate the concentration of the antibody in tissue culture supernatants. Larger scale antibody preparations were purified using protein A and concentrations were measured using a Nanodrop (Thermo Scientific). Table i. Planning of humanized heavy and light variants of variable CA8
2.5 Defucosylated antibody production
[00264] To generate defucosylated antibodies the heavy and light chains respectively were cotransfected into CHO DG44 MS705 BioWas cells and expressed in scale to produce antibody. In summary, 30 μg of DNA was linearized overnight with NatI, the DNA was precipitated with ethanol and redissolved in TE buffer. From the culture, 2.4 X 107 DG44 BioWa cells were obtained and washed in 14 ml of heated PBS-sucrose. The cells were spun and the pellet resuspended in 1.6 ml of PBS-sucrose. half (0.8 ml) of the cells previously mentioned, suspended in PBS-sucrose, were added to a BioRad cell with 30 μg of linearized DNA (in 50 μl of TE buffer). A GenePulser BioRad was programmed at 380 V with a capacitance of 25 μF and the cuvette was introduced for electroporation. The 850 μl resulting from electroporated cells and DNA were added to heated (80 ml) SFM512 medium (which includes phenol red, 2XHT (nucleosides), glutamax and Gibco supplement 4). Finally, the 80 ml resulting from cell suspension was transferred (150 μl / reservoir) to each reservoir in one of the 4 X 96-well plates. After 48 hours, the medium was changed to nucleoside free by appropriately removing 130 μl of conditioning and replacing with 150 μl of fresh SFM512 selection medium (which includes phenol red and glutamax). Every 3 to 4 days, 130 to 150 μl of conditioned medium was removed and replaced with fresh selection medium. The reservoirs were monitored for color change and tested for IgG concentration as discussed earlier. 2.6 Additional Antibodies - Cloning of Hybridoma Variable Regions
[00265] Total RNA was extracted from hybridoma cells S307118G03, S332121 F02, S332126E04, S322110D07, 5336105A07, S335115G01 and 5335122F05. The cDNA sequence of the heavy and light variable domain was then generated by reverse transcription and polymerase chain reaction (RT-PCR). The advanced primer for RT-PCR was a mixture of degenerate primers specific for the leading sequences of the murine immunoglobulin gene and the reverse primer was specific for antibody constant regions, in this case isotype IgG2a. The initiators were designed based on a strategy described by Jones and Bendig (Bio / Technology 9: 88, 1991). RT-PCR was performed for both V region sequences to allow subsequent verification of the correct V region sequences. DNA sequence data were obtained for products from region V generated by RT-PCR. 2.7 Additional antibodies - Cloning of chimeras
[00266] The DNA expression constructs encoding the chimeric antibodies have been prepared again by the advantage PCR cloning (Clonetech) infusion of the V gene PCR products into mammalian expression vectors. This cloning method allowed the fusion of the murine variable regions to the human IgG1 H chain and L chain constant regions. 2.8 S307118G03 - Cloning of humanized variants
[00267] The cloning was carried out as for paragraph 2.3. 2.9 S307118G03 Expression of recombinant antibodies
[00268] The expression plasmids encoding the relevant heavy and light chains (listed in Table 8 below) were transiently cotransfected into HEK 293 6E cells and expressed on a small scale to produce antibody. The antibodies were purified with Protein A from the supernatants and quantified using the Nanodrop spectrophotometer.
[00269] 8 below) were transiently cotransfected into HEK 293 6E cells and expressed on a small scale to produce antibody. The antibodies were purified with Protein A from the supernatants and quantified using the Nanodrop spectrophotometer. Example 3 Conjugation of antibodies to vcMMAE and mcMMAF to form antibody drug conjugates (ADC) Table B Chemical structures of drug ligands

[00270] Gammabind Plus Protein G Sepharose resin paste (GE Healthcare) (75 μl) was added to each reservoir of a deep reservoir filter plate (2 ml capacity). The antibodies to be conjugated were grouped by species and isotype and up to 0.5 mg of each antibody transferred to each reservoir on the plate. Each antibody was transferred to two separate reservoirs to facilitate the preparation of two conjugates, with the drug ligands SGD-1006 and SGD-1269. The filter plate was then shaken at 1200 RPM for 2 hours at 5 ° C to bind the antibodies to the resin. The filter plate was then centrifuged at 500x g for 3 minutes to ensure complete deposition of all fluids and resin for the fluid in each reservoir.
[00271] The bound antibodies were then reduced by adding 500 μL of 10 mM TCEP in 100 mM KPO4, 150 mM NaCI, pH 7, 1 mM EDTA and stirring for 30 minutes at 22 ° C. Following reduction, the plate was again centrifuged to remove the TCEP solution and subsequently washed with PBS + 1 mM EDTA, 1 ml per well. The wash solution was removed by centrifugation and the process repeated 3 times for a total of 4 washes. The bound and reduced antibodies were then conjugated using a mixture of NEM and drug linker prepared according to the molar fractions indicated in Table 2. Table 2.
* also for murine / human chimeric IgG1
[00272] Separate mixtures of NEM and drug ligand were thus prepared for each antibody species / isotype using 10 mM stock solutions of SGD-1006, SGD-1269 DMSO (See Table B) and NEM. When mixed in the appropriate ratio, the total maleimide concentration was therefore still 10 mM, and this value was used to calculate the volume of maleimide solution to be added to each reservoir. for example for a murine IgG1 with 5 reducible disulfides (10 thiols available when reduced) 0.5 mg of the antibody at 150 kD is 3.33 nMol which corresponds to 33.3 nMol of thiol. A 3-fold excess is therefore 100 nMol of total maleimide or 10 μl of the mixture at 10 mM of the drug / NEM binder. For the SGD-1269 conjugate this mixture would then be prepared with 5.86 μl of SGD-1269 and 4.14 μl of NEM. The maleimide mixture would then be diluted in 500 μl of PBS before addition to the immobilized reduced antibody. In practice, since multiple antibodies of each isotype were simultaneously conjugated, a single mixed solution of SGD-1269 / NEM for each isotype was prepared by multiplying the number of reservoirs containing this isotype by 10 μl per reservoir after diluting in a volume of PBS equal to 500 μl times the number of reservoirs. In the same way a total of eight mixtures of drug binder / NEM were prepared - four with SGD-1006 and four with SGD-1269 - and diluted in PBS. These mixtures were then added to the reduced antibodies (500 μl per reservoir) and the plate was shaken for 30 minutes at 22 ° C. The plate was then centrifuged as above to remove the excess reaction solution, and subsequently washed 4 times with PBS as before.
[00273] The bound ADCs were then eluted by adding 200 μl of 50 mM glycine pH 2.5 to each reservoir and shaking the plate for 3 minutes at 1200 RPM. Under stirring, 20 μl of neutralization buffer (1 M potassium phosphate, pH 7.4, 500 mM NaCl, 0.2% Tween-20) was added to each reservoir of a 1 ml collection plate. The ADCs were then eluted on the collection plate by rotating at 1500x g for 6 minutes. The collection plate was then slightly agitated to ensure complete mixing of the neutralization buffer.
[00274] The concentration of each ADC was then determined with an absorbance plate reader by transferring the solutions on a UV assay plate (Costar model 3635, Corning) and measuring the optical density at 280 nM. An average IgG extinction coefficient of 1.45 ml mg-1 cm-1 was used to provide an adequate estimate of ADC concentration through the panel. To confirm successful conjugation, a reverse phase protein HPLC method (described below) was used to estimate the drug load of isotype controls. For the plate containing the CA8 humanization variants, this method was used to estimate the load of all ADCs directly.
[00275] The reverse phase protein chromatography method to determine drug loading uses the polymeric stationary phase of PLRPS (Agilent Technologies). Since the antibodies were totally reduced during the conjugation process, all antibody subunits elute from the column as unique polypeptide chains that allow subpopulations of light and heavy chain species with varying levels of drug loading to be assessed separately. Thus, the analysis of these data allows the calculation of the average light chain drug load and the average heavy chain drug load as independent factors that can then be combined to determine the average antibody drug load with the basic knowledge that each antibody it is comprised of two light and two heavy chains. The chromatographic conditions were as follows: A PRLP-S column, 1000 Ã…, 50 x 2.1 mm, 8 μm particle size (Agilent Technologies) with water + 0.05% TFA as mobile phase A and acetonitrile + 0 , 01% TFA as mobile phase B; elution with a linear gradient of 27% B to 42% B in 12.5 minutes.
[00276] Anti-BCMA antibodies were conjugated to SGD-1006 and SGD-1269 in three separate batches over a period of seven months. In the first batch a total of 29 antibodies were conjugated (resulting in 58 ADCs). The drug load of each isotype control determined by PLRP chromatography and the data are summarized in Table 3. Table 3.

[00277] For the second batch an additional 25 antibodies were conjugated (resulting in 50 ADCs). The drug load of each isotype control was again determined by PLRP chromatography and the data are summarized in Table 4. Table 4.

[00278] In the third batch 30 antibodies were conjugated (resulting in 60 ADCs), which includes 13 humanized variants of CA8. In this final batch, drug loads for all ADCs have been determined and are summarized in the following two plate maps. (Table 5 & 6) Table 5
Table 6

[00279] Average drug load and% CV are indicated for each series of isotype in the background. An uncharacteristically large variability in drug load was observed for SGD-1269 ADCs prepared with mIgG2b antibodies; the reason for this is uncertain. Also, CA8 antibodies enhanced with Fc produced somewhat lower drug load levels than other human CA8 variants; to treat this, CA8 enhanced with additional Fc was conjugated in a solution phase reaction to better match the drug load obtained for the other antibodies. Example 4 - Binding Data 4.1 FMAT binding assay to show the binding of Chimeric CA8 to cells expressing human or cyan BCMA.
[00280] Human cryopreserved transfected human HEK293 cells, transfected with cytokine and simulated BCMA were recovered from LN2 storage. The test reservoirs were prepared with human chimeric CA8 antibody, in a range of different concentrations, mixed with HEK293 cells transfected with human BCMA, cino and simulated BCMA respectively. Secondary anti-human IgG conjugate FMAT Blue was added for the detection of human chimeric CA8. The assay plates were left for a minimum of 90 minutes before the result was read on the AB18200 (FMAT) plate reader.
[00281] This showed that the chimeric CA8 antibody binds well to both human and cyan BCMA proteins expressed in HEK293 cells.
[00282] Results are shown in Figure 1. 4.2 ELISA experiment showing the binding of chimeric CA8 to recombinant BCMA protein
[00283] The chimeric CA8 antibodies were tested for binding to human BCMA and cino BCMA expressed as Fc fusions. Human BCMA-Fc and BCMA of cino-Fc were coated on the ELISA plates and the plates were blocked using BSA to reduce non-specific binding. Chimeric CA8 antibodies were added in a concentration range of 5 μg / ml to 0.1 μg / ml to ELISA plates coated with human and cino BCMA. Any bound human chimeric CA8 antibody was detected using secondary antibody conjugated to anti-human IgG HRP as appropriate. The HRP substrate (TMB) was added to develop the ELISA. This showed that the CA8 antibody binds to recombinant human and cino BCMA in an ELISA assay.
[00284] The results are shown in Figure 2. 4.3 Biacore experiment to show binding of CA8 antibody to BCMA and TACI proteins to determine cross-reactivity with the TACI protein.
[00285] The chimeric CA8 antibody was injected and captured in protein A. (A sensor chip derived from protein A was used). Residual protein A binding was blocked with an injection of a high concentration of human IgG solution. BCMA-Fc, TACI-Fc or BAFF-R-Fc solutions were then tested for antibody binding. The 3 proteins were injected in the sequence and binding events were measured. The surface was regenerated between the injection of each protein.
[00286] Sensograms were analyzed in the Biaevaluation program. The double reference subtraction was done to remove the noise from the instrument and any non-specific connection of the sensogram curves.
[00287] This showed that CA8 was specific for binding to BCMA binding and not to TACI and BAFFR.
[00288] The binding of the CA8 antibody to BCMA-Fc, TACI-Fc and BAFF-R-Fc was plotted as shown in Figure 3. 4.4 Cell binding and neutralization data 4.4.1 Binding of murine anti BCMA antibodies to cells of multiple myeloma and cells that express BCMA
[00289] The Multiple Myeloma cell line H929 and ARH77-hBCMA 10B5 BCMA expressing transfectant cells were stained with murine S332211D07, S3332121F02 or S332126E04 or murine isotype control at 5 μg / ml. The H929 Multiple Myeloma cell line was stained with murine S307118G03. The cells were incubated for 20 mins at room temperature (RT) and then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. The cells were incubated with a secondary PE labeled anti-mouse IgG antibody for 15 minutes at RT and then washed with FACS buffer to remove unbound antibody. The cells were analyzed by FACS to detect the antibody bound to the cells.
[00290] The results (Figure 4) showed that all 4 murine antibodies linked to the Multiple Myeloma cell line H929 and the three antibodies tested on the transfected ARH77 BCMA cells linked to them. 4.4.2 Binding curve of chimeric CA8 to multiple myeloma cells as determined by FACS
[00291] A multiple myeloma cell line panel was used to determine the binding of chimeric CA8. Cell lines H929, OPM-2, JJN-3 and U266 were stained with chimeric CA8 or irrelevant antibody (Synagis) in varying concentrations for 20 minutes at room temperature. The cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. The cells were incubated with an anti-human IgG antibody labeled with secondary PE for 15 minutes at room temperature and then washed with FACS buffer to remove unbound antibody. The cells were analyzed by FACS and the mean fluorescence intensity (MFI) values measured to determine binding.
[00292] The results showed that chimeric CA8 bound to the Multiple Myeloma cell lines H929, OPM-2, JJN-3 & U266 in a dose dependent manner (Figure 5). 4.4.3 Binding of humanized CA8 to transfected BCMA cells as determined by FACS
[00293] ARH77-hBCMA 10B5 BCMA expressing transfectant cells or H929 cells were stained with chimeric CA8 or humanized variants of CA8 designated J6M0, J6M1, J6M2, J9M0, J9M1, J9M2 in varying concentrations for 20 minutes at room temperature. The cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. The cells were incubated with an anti-human IgG antibody labeled with secondary PE for 15 minutes at room temperature and then washed with FACS buffer to remove unbound antibody. The cells were analyzed by FACS and the mean fluorescence intensity (MFI) values measured to determine binding.
[00294] The results showed that chimeric CA8 and all antibodies tested apart from J9M2 bound to ARH77hBCMA 10B5 BCMA that expresses transfectant cells and H929 cells in a dose dependent manner (Figure 6). 4.5 Demonstration of the ability of CA8 and the humanized version J6M0 to neutralize the binding of BAFF or APRIL to the recombinant BCMA.
[00295] The purpose of this assay was to evaluate the ability of the CA8 antibody, and humanized version of J6M0 in both wild and afucosylated (Potelligent) form, in various concentrations, to neutralize the ligand binding capacity of BCMA, BAFF or APRIL .
[00296] The 96 well flat-bottom plates were coated overnight with 1 μg / ml of human BCMA Fc 4-53 recombinant solution in PBS. Following a washing step using 0.05% TWEEN20, the plates were blocked with a 2% solution of bovine serum albumin in PBS for 1 hour at room temperature. The plates were washed as before and 40 μl of each antibody (murine IgG, murine CA8, and chimeric CA8), leaving at 10 μg / ml, titrated 1 in 2 in duplicate was added to the relevant reservoirs and incubated for 1 hour at room temperature. 40 μl of 2% BSA was added to the relevant control reservoirs. 10 μl of recombinant human BAFF (2149BF / CF, R&D Systems) or recombinant human APRIL (5860-AP / CF, R&D Systems) were added at 30 ng / ml and 750 ng / ml respectively, giving a final concentration of 6 ng / ml and 150 ng / ml respectively in each reservoir. The equivalent volume of 2% BSA has been added to the relevant control reservoirs. The plates were allowed to incubate for 2 hours at room temperature, after which they were washed as before. Biotinylated anti-human ligand (BAFF BAF124 or APRIL BAF884, R&D Systems) was added to the relevant reservoirs at 50 ng / ml and incubated for 1 hour. Following a washing step, 50 μl of a 1: 4000 dilution of Streptavidin-HRP (Amersham RPN4401) was added to each well and incubated for 30 minutes at room temperature. The washing process was repeated once again followed by the addition of 100 μl of Tetramethylbenzidine substrate solution (T8665, Sigma) in each reservoir. The plates were incubated for 20 to 25 minutes at room temperature, wrapped in foil. The reaction was stopped with the addition of 100 μl of 1 M H2SO4. The optical density was determined at 450 nM using a Spectromax reader. See Figure 7A and B.
[00297] In a plate-based assay for neutralizing the binding of BAFF or APRIL to BCMA, the EC50 values calculated 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 the J6M0 potelligent version were 0.748 and 0.616 ng / ml respectively.
[00298] 4.6 Effect of chimerized BC8 antibody and humanized J6M0 on BAFF or APRIL-induced phosphorylation of NFkB in H929 cells.
[00299] In a set of experiments, H-929 cells were plated at 75,000 cells / well in a 96 well plate in serum-free medium. The chimeric CA8 antibody was added 24 hours later to give the final reservoir concentration up to 200 μg / ml. Ten minutes later, BAFF or APRIL ligands were added to the cells to give the final reservoir concentration of 0.6 or 0.3 μg / ml respectively. After 30 minutes the cells were lysed and phosphorylated, NfcapaB levels measured using an MSD pNFcapaB assay.
The chimeric BCMA CA8 antibody neutralized NfcapaB cell signaling induced by both BAFF and APRIL in H-929 cells. It was particularly potent in neutralizing BAFF-induced NfcapaB cell signaling in this cell type with an average IC50 of 10 nM, compared to 257 nM for APRIL-induced NfcapaB cell signaling.
[00301] Data averaged over 2 experiments
[00302] IC50s were 10 nM for BAFF-induced NfcapaB neutralization and 257 nM for APRIL-induced NfcapaB neutralization (mean of 2 independent experiments) are shown in Table 7. Table 7
Another set of experiments was carried out to try to understand why there was such a discrepancy between the power in neutralizing APRIL and BAFF in the cell-based system. Following the discovery of the soluble form of BCMA the experimental design was changed to include a step where H929 cells were washed before the assay to reduce the interference of antibody binding to soluble BCMA. H-929 cells were washed 3 times to remove any sBCMA and resuspended in serum-free medium. The J6M0 potelligent antibody was added to a 96 well plate to give a final reservoir concentration of up to 100 μg / ml along with BAFF or APRIL ligands to give a final reservoir concentration of 0.6 or 0.2 μg / ml respectively. The H-929 cells were then plated at 7.5 x 104 cells / reservoir in serum-free medium. 30 minutes later the cells were lysed and the levels of phosphorylated NFcapaB measured using an MSD pNFcapaB assay. These are data from an experiment. Each data point is the average / sd of two duplicates. The data for this experiment is shown in Figure 7c. The IC50s for the inhibition of BAFF and APRIL signaling were determined to be 0.91 μg / ml and 2.43 μg / ml respectively. 4.7 Protein analysis of anti-BCMA chimeric and humanized CA8 constructs
[00303] Initial screening of chimeric and humanized CA8 variants was performed on ProteOn XPR36 (Biorad). The method was as follows; Protein A was immobilized on a GLC chip (Biorad, Cat No: 176-5011) by primary amine binding, CA8 variants were then captured on this surface and recombinant human BCMA materials (internal or commercial US Biological, B0410) (round only) 2)) passed at 256, 64, 16, 4, 1 nM with a 0 nM injection (ie, buffer only) used for double reference binding curves, the buffer used is the HBS-EP buffer. 50 mM NaOH was used to regenerate the capture surface. The data were adjusted to the 1: 1 model using the analysis software inherent to ProteOn XPR36. Round 1 corresponds to the first screening of humanized CA8 variants (series J0 to J5) and round 2 to the second screening of humanized CA8 variants (series J5 to J9). Both rounds were performed at 25 ° C.
[00304] The data obtained from round 1 are shown in Table 8 and the data from round 2 are shown in Table 9. Several molecules in Round 2 (Table 09) failed to give measurable affinity values for ProteOn, this was due to the rate dissociation that is beyond the sensitivity of the machine in this assay, this however indicates that all of these molecules have firmly bound to recombinant human BCMA. As of Round 1, data indicates that some constructs showed no link to recombinant cino BCMA. Table 8: Round 1 - Kinetic analysis of anti-BCMA molecules against recombinant human BCMA
Table 9. Round 2 - Kinetic analyzes of anti-BCMA molecules against recombinant human BCMA for antibodies J8M0, J9M0, J8M1, J9M2, J7M2, J5M0, J7M1, J7M0, J8M2, J9M1, J5M2, J5M1 the dissociation rate was beyond the assay sensitivity therefore no data is shown.
4.8 BIAcore analysis of chimeric and humanized CA8 anti-BCMA constructs (series J7 to J9)
[00305] Protein A was immobilized on a CM5 chip (GE Healthcare, Cat No: BR-1005-30) by the primary amine bond and this surface was then used to capture the antibody molecules. Recombinant human BCMA (US Biological, B0410) was used as an analyte at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. The capture surface was regenerated using 50 mM NaOH. All binding curves were double reference with a buffer injection (ie, 0 nM) and the data were adjusted using the 1: 1 model inherent in the T100 evaluation software. The run was carried out at 37 ° C, using HBS-EP as the conduction buffer.
[00306] The results showed the tested molecules with the exception of J9M2 that binds to recombinant human BCMA, with similar affinity as the chimeric molecule. The data generated from this experiment are presented in table 10. Table 10: Analysis of kinetics of humanized anti-BCMA molecules against recombinant human BCMA
4.9 BIAcore analysis of anti-BCMA chimeric and humanized CA8 constructs J6M0 and J9M0
[00307] Protein A was immobilized on a CM5 chip (GE Healthcare, Cat No: BR-1005-30) by the primary amine bond and its surface was then used to capture the antibody molecules. Recombinant human BCMA (US Biological, B0410) was used as an analyte at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. The regeneration of the capture surface was carried out using 50 mM NaOH. All connection curves were double reference with a buffer injection (ie, 0 nM) and the data were adjusted for the use of the 1: 1 model inherent in the T100 evaluation software. The round was carried out at 25 ° C and 37 ° C for experiment 1 and only 37 ° C for experiment 2 using HBS-EP as the conduction buffer.
[00308] Both rounds identified J9M0 as the best molecule in terms of global affinity for human BCMA. The data generated from this experiment are shown in table 11. Table 11 Kinetic analysis of humanized anti-BCMA molecules against human BCMA
4.10. PrateOn analysis of new anti-BCMA chimeric constructs
[00309] The initial screening of the new chimeric variants of the second batch of hybridomas was carried out in PrateOn XPR36 (Biorad). The method was as follows; Protein A was immobilized on a GLM chip (Biorad, Cat No: 176-5012) by primary amine binding, anti-BCMA variants were then captured on this surface and recombinant human BCMA (internal material) passed at 256, 64, 16, 4 , 1 nM with a 0 nM injection (ie, buffer only) used for double reference binding curves, the buffer used is the HBS-EP buffer. The regeneration of the capture surface was carried out using 50 mM NaOH. The data were adjusted to the 1: 1 model using the analysis software inherent to the PrateOn XPR36. The round was carried out at 25 ° C.
[00310] The data generated from this experiment are shown in table 12. Table 12: Kinetic analysis of humanized anti-BCMA molecules against human BCMA
Example 5 Cell Death Assays. 5.1 The ADCC potencies of chimeric CA8 and chimeric CA8 defucosylated versions in ARH77 cells that express BCMA
[00311] Human natural killer cells (NK) were incubated with transfected target cells ARH77 BCMA (10B5) labeled with europium in the presence of varying concentrations of the antibody in an E: T ratio of 5: 1 for 2 hours. The europium release from the target cells was measured and the specific lysis calculated.
[00312] Result: Chimeric CA8 and defucosylated chimeric CA8 killed target cells expressing BCMA via ADCC. The defucosylated chimeric antibody showed more potent ADCC activity, as measured by a higher percent lysis obtained with all tested target cells and a ten-fold lower EC50 in the target cell line expressing high BCMA 10B5, compared to the precursor chimeric antibody. . See Figures 8A and 8B. 5.2 ADCC activity of humanized CA8 antibodies using target cells that express ARH77 BCMA express and PBMC as effectors
[00313] Human PBMCs were incubated with transfected cells targeting ARH77 BCMA (10B5) with europium in the presence of varying concentrations of humanized versions of CA8 antibody (5 μg / ml to 0.005 μg / ml) in an E: T ratio of 5: 1 for 2 hours. The europium release from the target cells was measured and the specific lysis calculated. Result:
[00314] Result: All J5, J6, J7 J8 & J9 series of humanized CA8 variants showed ADCC activity against cell line 10B5 expressing BCMA high ARH77 in a dose dependent manner. ADCC was at a similar level as that found in experiments using a chimeric CA8 molecule. See Figure 9. 5.3 ADCC potencies of 5322110F02, 5322110D07 and chimeric 5307118G03 and 5307118G03 humanized H3L0 against ARH77 10B5 cells expressing BCMA with purified NK cells as effector cells
[00315] Natural Human Exterminating Cells (NK) Humans were incubated with transfected target cells ARH77 BCMA labeled with europium in the presence of varying concentrations of the antibody in an E: T ratio of 5: 1 for 2 hours. Europium release from target cells was measured and specific lysis calculated. Result: all of the 4 antibodies tested showed ADCC activity against ARH77 10B5 cells. See Figure 10. 5.4 Antibody-conjugated drug (ADC) activity of chimeric CA8 ADCs.
[00316] Measure the chimeric CA8 antibody ADC activity, CA8-mcMMAF chimeric antibody drug conjugates and CA8-vcMMAE chimeric antibody drug conjugates against human Multiple Myeloma cell lines. Multiple Myeloma cell lines were treated with a chimeric CA8 antibody drug conjugate to determine the ADC concentrations required for growth and death inhibition.
[00317] The tested antibody drug conjugates were added to reservoirs containing multiple myeloma cells in concentrations ranging from 1 μg / ml to 5 ng / ml. The plates were incubated at 37 ° C for 96 hours at which point viable cells were quantified using Titer Glo cell. The unconjugated chimeric CA8 antibody did not show any significant growth inhibitory activity at the antibody concentrations that were tested. The chimeric CA8-mcMMAF antibody conjugate showed greater growth inhibitory activity than the chimeric CA8-vcMMAE antibody-drug conjugate in all 4 of the Multiple Myeloma cell lines that were tested. See Figure 11 and Table 13 Table 13 IC50 values represented in ng / ml for the chimeric drug-antibody CA8-vcMMAE and chimeric CA8-mcMMAF conjugates in 4 different Multiple Myeloma cell lines
5.5 Measure the stop activity of the chimeric CA8 antibody cell cycle, chimeric CA8-mcMMAF antibody conjugate and chimeric CA8-vcMMAE antibody conjugate against human Multiple Myeloma H929 cell line.
[00318] To determine by which mechanism the chimeric CA8 antibody-drug conjugate (ADC's) causes growth inhibition in multiple myeloma cells, the cell cycle of NCI-H929 cells was monitored by measuring cell DNA content by staining with fixed cell propidium iodide at multiple time points following treatment with chimeric CA8 antibody and chimeric CA8 ADC.
[00319] At the concentration of the chimeric CA8 ADC tested (50 ng / ml), the chimeric CA8-mcMMAF ADC caused a significant G2 / M cell cycle arrest (4N DNA content) that peaked within 48 hours. In the last 48, 72 and 96 hours, treatment with the chimeric CA8-mcMMAF ADC resulted in the accumulation of a cell population with sub-2N DNA content, which is representative of cell death. At the concentration of 50 ng / ml tested, the chimeric CA8-vcMMAE ADC had no significant effect on the G2 / M cell cycle arrest or sub-G1 accumulation. See Figure 12. 5.6 Dyeing with Phospho-Histone-H3 (Thr11) as a marker for mitotic arrest induced by the chimeric CA8 antibody conjugate mcMMAF-drug and chimeric CA8 antibody conjugate mcMMAF-drug.
[00320] To determine whether the accumulation of cells with 4N DNA content is a specific result of mitotic arrest induced by the chimeric CA8 ADCs, NCI-H929 cells were stained with an anti-phospho-Histone H3 antibody that follows treatment with increasing concentrations of Unconjugated chimeric CA8, chimeric CA8-vcMMAE or chimeric CA8-mcMMAF for 48 hours.
[00321] Treatment with chimeric CA8 ADCs resulted in a dose-dependent accumulation of NCI-H929 cells that stained positive for 65eroxidi-Histone H3 (Thr11), a specific mitotic cell marker. The chimeric AD8 CA8-mcMMAF caused accumulation of positive cells in 65eroxidi-Histone H3 at lower concentrations than the chimeric AD8 CA8-vcMMAE. See Figure 13. 5.7 Measuring apoptosis in NCI-H929 cells in response to chimeric CA8 ADCs by Annexin V staining.
[00322] To determine whether the accumulation of cells with sub-2N DNA content is a specific result of apoptosis induced by the chimeric CA8 ADCs, the NCI-H929 cells were stained with an anti-Annexin-V antibody that follows treatment with concentrations crescents of unconjugated chimeric CA8, chimeric CA8-vcMMAE or chimeric CA8-mcMMAF for 48 hours. Treatment with chimeric CA8 ADCs resulted in a dose-dependent accumulation of NCI-H929 cells that stained positive for Annexin-V, a specific apoptosis marker. The chimeric CA8-mcMMAF ADC caused accumulation of positive cells in Annexin-V at lower concentrations than the chimeric CA8-vcMMAE ADC. See Figure 14. 5.8 Antibody-drug conjugate (ADC) activity of humanized anti-BCMA-drug CA8 antibody conjugate variants.
[00323] Cells were plated on 96 well plates (4,000 cells per well in 100 μl RPMI + 10% FBS)
[00324] Naked antibody or ADC were added 6 hours after cell seeding and the plates were incubated for 144 hours. Growth inhibition in the presence of antibodies or ADCs was measured at 144 hours using Cell Titre glo. The data points represent the average of CellTiterGlo measurements in triplicate. Error bars represent standard error.
[00325] Multiple Myeloma cell lines NCI-H929 and OPM2 were treated with humanized anti-BCMA-CA8 antibody conjugate to determine the ADC concentrations required for growth and death inhibition. The mcMMAF and vcMMAE-drug antibody conjugate forms of these antibodies showed significant growth inhibitory activity comparable to that found with the CA8 chimera. The J6M0 variant showed higher potency than the chimera and the data are shown in figure 15 in H929 cells and OPM2 cells. The mcMMAF-drug antibody conjugate showed greater growth inhibitory activity than the vcMMAE-drug antibody conjugate for all antibodies in both cell lines tested. The results for all humanized variants are shown in Table 14. Table 14. IC50 values represented in ng / ml for the anti-BCMA-drug antibody conjugate in NCI-H929 and U266-B1 cells
5.9 Antibody-Drug Conjugate (ADC) activity of another murine-drug anti-BCMA antibody conjugate.
[00326] The cells were plated in a 96 well plate (4,000 cells per well in 100 μl of RPMI + 10% FBS)
[00327] Antibody or ADC 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 Cell Titre glo. The average of CellTiterGlo triplicate measurements is shown. Tables 15a and 15b are from experiments performed at different times on different series of antibodies. Multiple Myeloma cell lines NCI-H929 and U266-B1 were used for antibodies in Table 15a.
[00328] The mcMMAF and vcMMAE antibody murine antibody conjugate forms S322110D07, S332121F02 and S332136E04 showed significant growth inhibitory activity. The mcMMAF-drug antibody conjugate showed greater growth inhibitory activity than the vcMMAE-drug antibody conjugate in all of the murine anti-BCMA antibodies tested where activity was observed. IC50 figures are shown in Table 15a. See Figure 16 for dose response curves for these three antibodies and also S107118G03. Error bars represent standard error. The NCI-H929, U266-B1, JJN3 and OPM2 cells for the antibodies in Table 15b were treated with a different series of anti-murine anti-BCMA antibody conjugate to determine the ADC concentrations required for growth and death inhibition. IC50 figures are shown in Table 15b. All 5 antibodies shown in the table had significant ADC activity. Table 15a. IC50 values represented in ng / ml for the anti-BCMA-drug antibody conjugate in NCI-H929 and U266-B1 cells.
5.10 ADCC power of J6M0 conjugated, afucosylated (Potelligent)
[00329] J6M0 afucosylated conjugated to MMAE or MMAF was tested in ADCC assays using BCMA transfectants to ensure that their ADCC activity was not compromised by the conjugation. Europium-labeled ARH77-10B5 cells were incubated with several J6M0 WT and BCMA Potelligent antibodies in concentrations up to 10,000 ng / ml for 30 minutes before adding PBMCs (PBMC: 50: 1 target cell ratio). Two hours later an aliquot of cell medium was sampled and mixed with enhancement solution. After 30 minutes on a plate shaker, europium release was monitored on the Victor 2 1420 multi-label reader. Data points represent mean values in triplicate. These data are representative of 2 experiments.
[00330] There was no significant difference in ADCC potency between the unconjugated and ADC forms of J6M0 Potelligent. In the same experiment a wild-type version of J6M0 was included to show how the power compares with the afucosylated version. As expected, defucosylation resulted in a lower EC50 and higher maximum lysis. No lysis was observed with the F6 deficient form of J6M0. (Figure 17). 5.11 ADCC power of J6M0 afucosylated in MM cell lines
[00331] Human PBMCs were incubated with target multiple myeloma cells at a 50: 1 E: T ratio in the presence of varying concentrations of afucosylated J6M0 (Potelligent). The percentage of target cells remaining in the effector + target cell mixture after 18 hours was measured by FACS using a fluorescently labeled anti-CD138 antibody to detect the target cells and the calculated percent lysis. This is representative of several experiments.
[00332] J6M0 Potelligent antibodies showed ADCC activity against all five target multiple myeloma cell lines tested. This was important to test since studies earlier were performed using transfected cells. The results are shown in Figure 18. The complete data set with multiple donors is shown in Table 16. The potencies were all in a similar range as that found with transfectants. ADCC activity was not directly related to BCMA surface expression in these cell lines. Table 16 EC50 values generated in 13 independent assays using 11 donors (designated A to K) across the five Multiple Myeloma cell lines.
Example 6. Xenograft data 6.1 Murine xenografts from human MM cell lines were tested to ensure that the antibody potency detected in vitro could also be demonstrated in vivo.
[00333] The cell line selected for the xenograft studies was NCI-H929 which is sensitive to death by ADC and ADCC in vitro. The studies were performed in CB, 17 immunocompromised SLID mice that lack T and B cells but maintain NK cells to allow ADCC activity. However, it should be mentioned that although human IgG1 can join murine Fc receptors, enhancement with Potelligent does not improve affinity as it does with human Fc receptors. 6.2 Impact of J6M0 unconjugated and conjugated with MMAE or MMAF on the growth of the NCI-H929 tumor.
[00334] In order to independently analyze the activities of both ADCC and ADC of J6M0 we tested antibody J6M0 in the presence and absence of conjugation with MMAF or MMAE. By testing the unconjugated J6M0, any of the antitumor effects could be attributed to some combination of ADDC and functional inhibitory activity.
[00335] Mice with NCI-H929 tumors that reached a volume of 200 mm3 on average were treated with a human IgG1 control or the J6M0 antibody (unconjugated, MMAE or MMAF) twice weekly at a dose of 50 μg or 100 μg, for 2 weeks. The results of this study show that a dose of 100 μg of the J6MO-MMAF conjugate resulted in the elimination of tumors in these mice that completed the dosage. The J6MO-MMAF mice were maintained for 40 days after the last dose without any tumor recurrence. These results from this experiment demonstrate that the MMAF conjugation had increased antitumor activity in both the unconjugated J6M0 antibody and the J6MO-MMAE conjugate See Figure 19. Example 7 Assessment of Soluble BCMA Levels from MM Patient Serum 7.1 It is currently unknown whether BCMA is present extracellularly and can be detected in the blood.
[00336] In this work, we determine the serum level of human BCMA from patients with MM. Serum samples from 54 patients with MM and plasma cell dyscrasia and 20 normal control samples were analyzed by ELISA. Individual human approval was obtained from the Western Institutional Review Board. 7.2 Assessment of serum human BCMA levels
[00337] Blood, from patients and normal controls at the clinic, was collected in serum collection tubes. MM patient samples were of a variety of stages (progressive disease, remission, relapse, newly diagnosed, and others). The blood samples were spun at 10,000 rpm for 10 minutes and the serum was transferred into sterile microcentrifuge plastic tubes.
[00338] A human BCMA / TNFRSF17 ELISA kit from R & D Systems (catalog # DY193E) that measures levels of soluble human BCMA was used to detect BCMA following the standard protocol provided with the kit.
[00339] In summary, 96 well microplates were coated with 100 μl per well to capture antibody and incubated overnight at 4 ° C. The plates were 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 at room temperature for 2 hours. The plates were washed three times with wash buffer. 100 μl of serum or standard sample was added to each reservoir and incubated for 2 hours at room temperature. The plates were washed three times with wash buffer and then 100 μl of the detection antibody was added to each well and incubated for 2 hours at room temperature. 100 μl of Streptavidin-HRP was added to each well after washing the plates three times and incubated in a dark environment for 20 minutes. The plates were washed three times and added with 50 μl of stop solution and then determined by the microplate reader with a wavelength of 570 nM.
[00340] A series of tests were performed in order to determine the appropriate serum dilution factor for the levels of BCMA that were present. A dilution factor of 1: 500 was found to be suitable for most samples and is the dilution factor used in the data shown in Figure 20. The complete data set is shown in Table 17.
[00341] The serum samples of patient and normal control diluted and conducted in triplicates had the levels of BCMA determined. Serum BCMA levels were significantly elevated in the sera of patients with MM compared to the normal controls in this study. When the disease subset was further divided, there was a tendency for elevated serum BCMA levels in the sera of patients with MM progressing compared to those in remission. This is the first report that identifies serum BCMA in any human disease and suggests that these levels may be a new biomarker for monitoring disease status and therapeutic responses in patients with MM and for other patients with plasma cell-mediated diseases. Table 17. The figures represent the serum concentration of BCMA soluble in ng / ml calculated from samples diluted to 1/50, 1/500 and 1/5000. The P values were calculated using the one-tailed t-test and the 95% significance values are below in the table.
P-Values (One-Tail T Test, 95% Significance) ~ 1-500 Single Normal vs Progressive: p = 0.0010 * Progressive vs Remission: p = 0.0146 * ~ 1-500 Normal vs Progressive Triplicate: p = 0.0004 * Progressive vs Remission: p = 0.0091 * ~ 1-50 Test 1 Normal vs Progressive: p = 0.0171 * Progressive vs Remission: p = 0.0777 ~ 1-50 Test 2 Normal vs Progressive: p = 0.0184 * Progressive vs Remission: p = 0.0876 * shows significance Summary of Sequence (Table C)

权利要求:
Claims (25)
[0001]
1. Antigen binding protein, characterized by the fact that it specifically binds to BCMA and that inhibits BAFF and / or APRIL binding to BCMA, in which the antigen binding protein is capable of binding to FcyRIIIA or is capable of effector function mediated by FcyRIIIA and in which the antigen binding protein is capable of internalization and comprises: i) CDRH3 as presented in SEQ ID NO. 3, variant N99D; ii) CDRH1 as presented in SEQ ID NO. 1; iii) CDRH2 as presented in SEQ ID NO. two; iv) CDRL1 as presented in SEQ ID NO. 4; v) CDRL2 as presented in SEQ ID NO. 5; and vi) CDRL3 as presented in SEQ ID NO. 6.
[0002]
2. Antigen-binding protein according to claim 1, characterized in that the antigen-binding protein has enhanced FcyRIIIA binding or has enhanced FcgRIIIA-mediated effector functions.
[0003]
3. Antigen-binding protein according to claim 2, characterized by the fact that the antigen-binding fragment has enhanced ADCC effector function.
[0004]
Antigen-binding protein according to any one of claims 1 to 3, characterized in that the antigen-binding protein is defucosylated.
[0005]
Antigen-binding protein according to any of claims 1 to 4, characterized in that the antigen-binding fragment does not bind to Taci.
[0006]
Antigen-binding protein according to any one of claims 1 to 5, characterized in that it comprises a variable region of the heavy chain encoded by any of SEQ ID NO: 23 or SEQ ID NO: 27 or SEQ ID NO : 29.
[0007]
Antigen-binding protein according to any one of claims 1 to 6, characterized in that it comprises a variable region of the light chain encoded by any of SEQ ID NO: 31 or SEQ ID NO: 33.
[0008]
Antigen-binding protein according to claims 1 to 7, characterized in that the antigen-binding protein comprises a variable region of the heavy chain encoded by SEQ ID NO: 23 and a variable region of the encoded light chain by SEQ ID NO: 31.
[0009]
Antigen-binding protein according to claims 1 to 7, characterized in that the antigen-binding protein comprises a heavy chain encoded by SEQ ID NO: 27 and a light chain encoded by SEQ ID NO: 31 .
[0010]
Antigen-binding protein according to any one of claims 1 to 9, characterized in that the antigen-binding protein is a humanized monoclonal antibody.
[0011]
Antigen-binding protein according to claim 10, characterized in that the antibody is an IgG1 isotype.
[0012]
Antigen-binding protein according to any one of the preceding claims, characterized in that the antigen-binding protein is a fragment that is a Fab, Fab ', F (ab') 2, Fv, diabody, tribody , tetrabody, minibody, minibody, isolated VH or isolated VL.
[0013]
13. Immunoconjugate, characterized by the fact that it comprises the antigen binding protein as defined in any of claims 1 to 12 and a cytotoxic agent.
[0014]
Immunoconjugate according to claim 13, characterized in that the antigen binding protein is linked to the cytotoxic agent via a linker.
[0015]
Immunoconjugate according to claims 13 or 14, characterized in that the cytotoxic agent is an auristatin or a dolostatin.
[0016]
16. Immunoconjugate according to any one of claims 13 to 15, characterized by the fact that the cytotoxic agent is selected from MMAE and MMAF.
[0017]
17. Immunoconjugate according to any one of claims 13 to 16, characterized by the fact that the cytotoxic agent is covalently linked to said antigen binding protein.
[0018]
18. Immunoconjugate according to claim 13, characterized in that the antigen-binding protein is linked to the cytotoxic agent by means of a cleavable linker.
[0019]
19. Immunoconjugate according to any one of claims 14 to 17, characterized in that said ligand is a non-cleavable ligand.
[0020]
20. Immunoconjugate according to any one of claims 14 to 19, characterized by the fact that the ligand is selected from 6-maleimidocaproíla (MC), maleimidopropanoíla (MP), valine-citrulline (val-cit), alanine-phenylalanine ( ala-phe), N-succinimidyl p-amino-benzyloxycarbonyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 (SMCC), and (4-iodo-acetyl) N-succinimidyl aminobenzoate (SIAB).
[0021]
21. Immunoconjugate according to any one of claims 13 to 20, characterized in that said immunoconjugate is engulfed by a tumor cell when contacted with a tumor cell.
[0022]
22. Pharmaceutical composition, characterized in that it comprises an antigen-binding protein or immunoconjugate as defined in any one of claims 1 to 21 and a pharmaceutically acceptable carrier.
[0023]
23. Use of a composition as defined in claim 22, characterized in that it is in the manufacture of a medicament to treat a human patient afflicted with an inflammatory disorder or disease.
[0024]
24. Use of a composition as defined in claim 22, characterized in that it is in the manufacture of a medicament for the treatment of a human patient afflicted with B-cell lymphoma such as Multiple Myeloma (MM) or Chronic Lymphocytic Leukemia (CLL) .
[0025]
25. Antigen-binding protein or immunoconjugate according to any of claims 1 to 21, characterized in that they are for use in the treatment of a human patient afflicted with a B-cell lymphoma such as Multiple Myeloma (MM) or Chronic Lymphocytic Leukemia (CLL).

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/28 (2006.01), A61K 47/00 (2006.01) |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-05| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-04-20| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2609 DE 05/01/2021 QUANTO A PRIORIDADE UNIONISTA. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161490732P| true| 2011-05-27|2011-05-27|

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US61/490,732|2011-05-27|

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US201261647196P| true| 2012-05-15|2012-05-15|

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US61/647,192|2012-05-15|

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PCT/EP2012/059762|WO2012163805A1|2011-05-27|2012-05-24|Bcma-binding proteins|

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