Methods for reducing alloantibody levels in subjects in need of solid organ transplantation

Abstract

A B cell maturation antigen (BCMA) is expressed on plasma cells. The present invention provides methods of desensitizing a patient in need of solid organ transplantation against HLA antibodies using bispecific antibodies that bind to both BCMA and CD3 and activate T cells by the CD3 complex in the presence of plasma cells expressing BCMA. In certain embodiments, the bispecific antibodies of the invention reduce alloantibody levels and / or reduce computational population reactive antibody levels to promote solid organ transplantation and / or improve long term graft survival and / or function.

Description

[0001] Reference to the Sequence Listing

[0002] This application incorporates by reference a computer-readable sequence listing in ST.26 XML format, named 11294WO01_Sequence, created on October 8, 2023, and containing 48,180 bytes. Field of the Invention

[0003] The present invention pertains to the medical field and relates to bispecific antibodies (and antigen-binding fragments thereof) that bind BCMA and CD3, as well as methods of using them, for example, to desensitize patients against HLA alloantibodies prior to solid organ transplantation. Background of the Invention

[0004] B-cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a type III transmembrane protein lacking a signal peptide and containing a cysteine-rich extracellular domain. BCMA, along with closely related proteins, promotes the survival of B cells at different developmental stages. BCMA is expressed only in B-cell lineage cells, particularly in the interfollicular region of germinal centers and on plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is essential for the optimal survival of long-lived plasma cells in the bone marrow.

[0005] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that is associated with the T-cell receptor complex (TCR) and is essential for T-cell activation. Functional CD3 is formed by the dimerization association of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to cause CD3 aggregation on T cells, thereby inducing T-cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T-cell activation. In addition, bispecific antibodies capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving targeting the T-cell immune response to tissues and cells expressing the target antigen.

[0006] Kidney transplantation is the preferred treatment for end-stage renal disease. Although hemodialysis saves lives in the short term, the 1-year and 5-year mortality rates are as high as 25% and 65% respectively, while the 5-year mortality rate of patients treated with kidney transplantation is as low as 3%. However, for patients with high levels of preformed anti-human leukocyte antigen (HLA) antibodies, organ transplantation is rarely performed due to the increased risk of antibody-mediated rejection (AMR) and shortened graft survival. Approximately 30% of the approximately 95,000 patients on the kidney transplantation waiting list in the United States are HLA-sensitized due to previous transplantation, blood transfusions, and pregnancy. Approximately half of these patients (about 13,000 patients) are considered highly sensitized (i.e., expected to react to ≥80% of donor HLA types). Calculating the panel reactive antibody (cPRA) score represents the probability that an organ transplantation candidate will encounter an incompatible donor and is used as a measure of the level of sensitization. Although the Kidney Allocation System (KAS) has generally increased the transplantation rate of highly sensitized individuals, 30% to 50% of patients with cPRA scores ≥90% remain on the kidney transplantation waiting list for more than 5 years, highlighting the unmet needs of this subset of sensitized patients. Among the approximately 11,000 patients with chronic kidney disease (CKD) and cPRA ≥90% on the kidney transplantation waiting list in the United States, the majority (about 7,000) have cPRA scores ≥99%. These patients have particularly high unmet needs, as evidenced by their more frequent removal from the waiting list and death due to medical comorbidities. In addition, despite the implementation of KAS in 2014, the average transplantation rate for the approximately 2,000 patients with cPRA ≥99.9% on the kidney transplantation waiting list is significantly lower than that of other HLA-sensitized patients. Therefore, desensitization strategies to facilitate successful transplantation in these patients are an urgent priority.

[0007] Individuals sensitive to multiple or common HLAs have limited transplant options because of the risk of AMR and subsequent graft failure if HLA-incompatible kidneys are transplanted. The pathogenesis of AMR involves antibodies against HLA produced by plasma cells and possibly B cells. Other effector molecules, including cytokines and complement, have also been shown to contribute to kidney pathology. Antibody-mediated rejection can manifest as hyperacute rejection, which occurs within minutes after vascular anastomosis and often leads to graft failure within hours. More commonly, AMR presents as acute / active and chronic forms that develop over months or years. Although hyperacute rejection rarely occurs due to improved HLA screening and crossmatching, the acute / active AMR rate in HLA-sensitized patients has been reported to be as high as 40% to 45% despite the use of plasmapheresis and intravenous immunoglobulin (IVIG) desensitization techniques. Although acute AMR can be controlled with short-term immunosuppression in many cases, it is a strong risk factor for the development of chronic AMR, which is the most common cause of graft failure in the United States. The DeKAF study showed that most patients with graft failure had evidence of chronic AMR, including deposition of split C4 complement component on biopsy (Matas et al., Am J Transplant, 18(5):1140-1150, 2018). In addition, the risk of chronic AMR in previous recipients of highly HLA-sensitized transplants increased four-fold even after waiting for HLA-compatible kidneys (Schinstock et al., Transplantation, 101(10):2429-2439, 2017). Sensitization and the presence of anti-HLA alloantibodies are also involved in the delayed transplantation of other solid organs, including heart (Kransdorf et al., Transplantation, 101(9):1971-1976, 2017) and lung (Barac et al., An Thorac Surg., 110(2):414-423, 2020) transplants. Therefore, there remains a far unmet need for more effective therapies to facilitate transplantation and prevent acute and chronic AMR after transplantation in individuals highly sensitive to HLA. SUMMARY OF THE INVENTION

[0008] The present disclosure generally relates to methods of using an anti-BCMA x anti-CD3 bispecific antibody (e.g., REGN5459 or REGN5458) to safely deplete plasma cells expressing B cell maturation antigen (BCMA) and reduce anti-HLA alloantibodies to facilitate solid organ transplantation (e.g., kidney transplantation). Plasma cell-targeted therapy (as described herein) will have a more durable HLA desensitization effect by eliminating the source of anti-HLA alloantibodies, thereby facilitating transplantation and reducing the risk of AMR post-transplantation. Anti-BCMA x CD3 antibodies (e.g., REGN5459 and REGN5458) target the surface protein BCMA selectively expressed on plasma cells. The bispecific antibody depletes plasma cells and newly activated BCMA-expressing B cells, including those that produce anti-HLA antibodies, without compromising the regulatory T cell response that promotes post-transplantation tolerance. The restricted pattern of BCMA expression in tissues makes it an attractive therapeutic target for preventing AMR.

[0009] In one aspect, the invention provides a method of reducing the level of alloantibodies in a subject in need of solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells, and a second antigen-binding domain that specifically binds to human CD3 on T cells.

[0010] In some embodiments, reducing the level of alloantibodies in the subject comprises reducing the level of alloantibodies to below a baseline alloantibody level measured prior to administering the bispecific antibody or an antigen-binding fragment thereof. In some cases, the method further comprises measuring the baseline alloantibody level in the subject prior to administering the bispecific antibody or an antigen-binding fragment thereof. In some cases, the alloantibody level and / or the baseline alloantibody level are measured by a single antigen bead assay, and the alloantibody level and / or the baseline alloantibody level correspond to the mean fluorescence intensity of the peak immunodominant anti-HLA antibody. In some cases, the reduction in the alloantibody level relative to the baseline alloantibody level corresponds to a reduction in the mean fluorescence intensity of the peak immunodominant anti-HLA antibody of ≥50%.

[0011] In some embodiments, the alloantibody level is measured by a single antigen bead assay, and the reduction in the alloantibody level corresponds to a reduction in the mean fluorescence intensity of the peak immunodominant anti-HLA antibody to <5000.

[0012] In one aspect, the present invention provides a method of reducing the calculated panel reactive antibody (cPRA) level in a subject in need of solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells, and a second antigen-binding domain that specifically binds to human CD3 on T cells.

[0013] In some embodiments, reducing the cPRA level in a subject comprises reducing the cPRA level in the subject to less than a baseline cPRA level measured prior to administering the bispecific antibody or antigen-binding fragment thereof. In some cases, the cPRA level in the subject is reduced to: <99%; <98%; <97%; <96%; <95%; <94%; <93%; <92%; <91%; <90%; <89%; <88%; <87%; <86%; <85%; <84%; <83%; <82%; <81%; or <80%.

[0014] In one aspect, the present invention provides a method of reducing a subject's sensitivity to anti-HLA antibodies prior to solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells and a second antigen-binding domain that specifically binds to human CD3 on T cells, wherein the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population having a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation.

[0015] In some embodiments, the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population having a pre-transplant cPRA level of 80%. In some embodiments, the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population having a pre-transplant cPRA level of 50% to <80%.

[0016] In one aspect, the present invention provides a method of reducing the risk of allograft rejection in a subject after solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells, and a second antigen-binding domain that specifically binds to human CD3 on T cells, wherein the risk of rejection in the subject during the post-transplantation interval is not greater than the risk of rejection in a control population having a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation.

[0017] In some embodiments, the risk of rejection in the subject during the post-transplantation interval is no greater than the risk of rejection in a control population with a pre-transplant cPRA level of 80%. In some embodiments, the risk of rejection in the subject during the post-transplantation interval is no greater than the risk of rejection in a control population with a pre-transplant cPRA level of 50% to <80%. In some cases, the post-transplantation interval is a 3-month interval starting one day after organ transplantation. In some cases, the post-transplantation interval is a 6-month interval starting one day after organ transplantation. In some cases, the post-transplantation interval is a 12-month interval starting one day after organ transplantation.

[0018] In some embodiments, the graft function in the subject remains at a functional level equal to or higher than the functional level of the control population during the post-transplantation interval.

[0019] In any one of the various embodiments of the methods discussed above or herein, the subject can be a human.

[0020] In any one of the various embodiments of the methods discussed above or herein, the solid organ can be selected from the group consisting of a kidney, a lung, a pancreas, or a heart. In some cases, the solid organ is a kidney. In some cases, the solid organ is a kidney and the subject has been on the kidney transplant waiting list for 5 years or longer. In some cases, the subject has chronic kidney disease. In some cases, the subject is undergoing hemodialysis. In some cases, the subject is highly sensitized and has end-stage renal failure requiring hemodialysis.

[0021] In any one of the various embodiments of the methods discussed above or herein, the bispecific antibody or its antigen-binding fragment can be as discussed below.

[0022] In some embodiments, the first antigen-binding domain comprises: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) that are contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 1; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) that are contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 5. In some instances, the first antigen-binding domain comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 2, an HCDR2 having the amino acid sequence of SEQ ID NO: 3, and an HCDR3 having the amino acid sequence of SEQ ID NO: 4. In some instances, the first antigen-binding domain comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8. In some instances, the first antigen-binding domain comprises an HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 5.

[0023] In some embodiments, the second antigen-binding domain comprises: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:13; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:5. In some cases, the second antigen-binding domain comprises: (a) an HCDR1 having the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:14; (b) an HCDR2 having the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:15; and (c) an HCDR3 having the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:16. In some cases, the second antigen-binding domain comprises an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:8. In some cases, the second antigen-binding domain comprises: (a) HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively; and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NO:6, 7, and 8, respectively; or (b) HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NO:14, 15, and 16, respectively; and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NO:6, 7, and 8, respectively. In some cases, the second antigen-binding domain comprises: (a) an HCVR having the amino acid sequence of SEQ ID NO:9 and an LCVR having the amino acid sequence of SEQ ID NO:5; or (b) an HCVR having the amino acid sequence of SEQ ID NO:13 and an LCVR having the amino acid sequence of SEQ ID NO:5.

[0024] In some embodiments, (a) the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, (a) the first antigen-binding domain comprises an HCVR consisting of the amino acid sequence of SEQ ID NO: 1 and an LCVR consisting of the amino acid sequence of SEQ ID NO: 5; and (b) the second antigen-binding domain comprises an HCVR consisting of the amino acid sequence of SEQ ID NO: 9 and an LCVR consisting of the amino acid sequence of SEQ ID NO: 5.

[0025] In some embodiments, (a) the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, (a) the first antigen-binding domain comprises an HCVR consisting of the amino acid sequence of SEQ ID NO: 1 and an LCVR consisting of the amino acid sequence of SEQ ID NO: 5; and (b) the second antigen-binding domain comprises an HCVR consisting of the amino acid sequence of SEQ ID NO: 13 and an LCVR consisting of the amino acid sequence of SEQ ID NO: 5.

[0026] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a human IgG heavy chain constant region. In some cases, the bispecific antibody comprises a heavy chain having a constant region comprising the amino acid sequence of SEQ ID NO:33. In some cases, the bispecific antibody comprises a heavy chain having a constant region comprising the amino acid sequence of SEQ ID NO:34. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4. In some embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to the wild-type hinge of the same isotype.

[0027] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain having the amino acid sequence of SEQ ID NO:29, a second heavy chain having the amino acid sequence of SEQ ID NO:30, and a common light chain having the amino acid sequence of SEQ ID NO:32.

[0028] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain having the amino acid sequence of SEQ ID NO:29, a second heavy chain having the amino acid sequence of SEQ ID NO:31, and a common light chain having the amino acid sequence of SEQ ID NO:32.

[0029] In any one of the various embodiments of the methods discussed above or herein, the bispecific antibody is administered according to a dosing regimen comprising a fractional initial dose. In some embodiments, the bispecific antibody is administered to a subject at a dose of 0.05 mg to 150 mg per week.

[0030] In one aspect, the present invention provides a dosing regimen for any one of the embodiments of the methods discussed above or herein, wherein the dosing regimen comprises administering a bispecific antibody to a subject at an initial dose in the first week of the dosing regimen, at a second dose in the second week of the dosing regimen, and at a third dose in the third week of the dosing regimen, wherein the third dose is equal to or greater than the second dose, and the second dose is greater than the initial dose.

[0031] In some embodiments, the initial dose is from 0.05 mg to 5 mg. In some embodiments, the second dose is from 0.15 mg to 25 mg. In some embodiments, the third dose is from 0.5 mg to 150 mg. In some cases, the initial dose is 0.05 mg, 0.15 mg, 0.5 mg, 1 mg, 1.5 mg, or 5 mg. In some cases, the second dose is 0.15 mg, 0.5 mg, 1.5 mg, 3 mg, 5 mg, 15 mg, or 25 mg. In some cases, the third dose is 0.5 mg, 1.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 40 mg, 50 mg, or 150 mg.

[0032] In various embodiments, any feature or component of the embodiments discussed above or herein can be combined, and such combinations are within the scope of the present disclosure. Any specified value discussed above or herein can be combined with another related value discussed above or herein to enumerate a range, and the values represent the upper and lower limits of the range, and such ranges are within the scope of the present disclosure.

[0033] Other embodiments will become apparent by referring to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Illustrates the reduction in serum IgG concentration after treatment of highly sensitive chronic kidney disease patients with REGN5459, as discussed in Example 2.

[0035] Figure 2A and 2B Shows the in vivo targeting of plasma cells by BCMA x CD3 bispecific antibody in BCMAhu / huCD3hu / hu mice ( Figure 2A ) and the quantification of BMPC ( Figure 2B ) experimental setup.

[0036] Figure 3A and 3B Shows the levels of IgA+ ( Figure 3A ) and IgM+ ( Figure 3B ) BMPC after administration of REGN5459 in BCMAhu / huCD3hu / hu mice.

[0037] Figure 4 Shows the quantification of B cells that experienced splenic antigen 7 days after administration of BCMA x CD3 bispecific antibody in BCMAhu / huCD3hu / hu mice.

[0038] Figure 5A 、 5BAnd 5C shows the quantification of serum IgA ( Figure 5A ), IgM ( Figure 5B ), and IgG1 ( Figure 5C ) levels in BCMAhu / huCD3hu / hu mice after administration of the BCMA x CD3 bispecific antibody.

[0039] Figure 6A , 6B And 6C depict a study design showing 15 weeks of continuous HDM exposure, where

[0040] continuous anti-IL-4Ra was initiated at week 12 and BCMAxCD3 ( Figure 6A ) was administered briefly at week 15; quantification of IgE+BMPC as a percentage of live cells ( Figure 6B ); and quantification of total BMPC ( Figure 6C ).

[0041] Figure 7A And 7B show a cynomolgus monkey study design ( Figure 7A ). Anti-IL-4Ra was administered weekly starting on day 1, and a single dose of BCMAxCD3 or an isotype control antibody was administered on day 22. Bone marrow aspirates were collected on days 15 and 43. ( Figure 7B ) Flow cytometric quantification of BMPC from bone marrow aspirates sampled at the indicated time points.

[0042] Figure 8A , 8B And 8C show the change over time in the mean concentration levels of serum IgG ( Figure 8B ) and IgE ( Figure 8C ) in multiple myeloma patients who received the BCMAxCD3 bispecific antibody ( Figure 8A ) at the indicated dose levels weekly. Detailed Description

[0043] Before describing the present invention, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0045] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are hereby incorporated by reference in their entirety.

[0046] Definitions

[0047] As used herein, the expression "CD3" refers to an antigen that is expressed on T cells as part of the multi-molecular T cell receptor (TCR) and consists of a homodimer or heterodimer formed by the binding of two of the four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-ε contains the amino acid sequence shown in SEQ ID NO:23; human CD3-δ contains the amino acid sequence shown in SEQ ID NO:24; human CD3-ζ contains the amino acid sequence shown in SEQ ID NO:25; and CD3-γ contains the amino acid sequence shown in SEQ ID NO:26. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human forms of the various proteins, polypeptides, or protein fragments, unless specifically indicated as being from a non-human species. Thus, unless indicated as being from a non-human species, e.g., "mouse CD3", "monkey CD3", etc., the expression "CD3" means human CD3.

[0048] As used herein, "an antibody that binds CD3" or "an anti-CD3 antibody" includes antibodies that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ) and their antigen-binding fragments, as well as antibodies that specifically recognize the dimer complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) and their antigen-binding fragments. The antibodies and antigen-binding fragments of the present invention can bind soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein as well as recombinant CD3 protein variants, such as monomeric and dimeric CD3 constructs that lack a transmembrane domain or are not associated with the cell membrane.

[0049] As used herein, the expression "cell surface-expressed CD3" refers to one or more CD3 proteins that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins that are within the functional T cell receptor on the cell membrane. The expression "cell surface-expressed CD3" includes CD3 proteins that are expressed as part of a homodimer or heterodimer on the cell surface (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The expression "cell surface-expressed CD3" also includes CD3 chains that are expressed on the cell surface by themselves (e.g., CD3-ε, CD3-δ, or CD3-γ) in the absence of other CD3 chain types. "Cell surface-expressed CD3" can include or consist of CD3 proteins that are expressed on the cell surface of cells that normally express CD3 proteins. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 proteins that are expressed on the cell surface of cells that do not normally express human CD3 on the surface but have been engineered to express CD3 on the surface.

[0050] As used herein, the expression "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO:22 and can also be found in GenBank accession number NP_001183.2.

[0051] As used herein, "an antibody that binds BCMA" or "an anti-BCMA antibody" includes antibodies that specifically recognize BCMA and antigen-binding fragments thereof.

[0052] A "plasma cell" is a differentiated B lymphocyte capable of secreting antibodies, including isologous antibodies.

[0053] "Human leukocyte antigen" (HLA) is a cell surface protein that presents peptides to T lymphocytes as part of the immune recognition process, which is the basis of the adaptive immune response, and the genes encoding HLA are among the most polymorphic genes in the human genome. Approximately 30% of patients in need of solid organ transplantation become sensitized, i.e., there are anti-HLA antibodies. These isologous antibodies are produced after exposure to non-self HLA through pregnancy, blood transfusion, or a previous organ transplantation.

[0054] An "isologous antibody" is an antibody that is produced in response to exposure to incompatible blood group antigens and is directed against non-self HLA proteins.

[0055] The level of "calculated panel reactive antibody" (cPRA) is based on values of HLA antigens that are unacceptable for candidates for solid organ (e.g., kidney) transplantation. cPRA represents a method for determining the risk of organ rejection in a patient prior to transplantation and is an estimate of the percentage of donors that are incompatible with a particular recipient. A cPRA > 80% is considered highly sensitized.

[0056] As used herein, a "subject" refers to an individual (e.g., a human) in need of a solid organ transplantation (e.g., a kidney transplantation) who is at risk of antibody-mediated rejection of the transplanted organ due to the presence of alloantibodies. The subject can be an individual with chronic kidney disease or an individual with chronic kidney disease requiring hemodialysis.

[0057] As used herein, "chronic kidney disease" or "CKD" refers to a condition characterized by a decrease in renal function, which is due to kidney damage or a glomerular filtration rate (GFR) of less than 60 mL / min / 1.73 m 2 for at least 3 months. The different stages of CKD define a continuum and are divided into: stage 1 (kidney damage with normal GFR); stage 2 (mild decrease in GFR to 60 - 89 mL / min / 1.73 m 2 ); stage 3a (moderate decrease in GFR to 45 - 59 mL / min / 1.73 m 2 ); stage 3b (moderate decrease in GFR to 30 - 44 mL / min / 1.73 m 2 ); stage 4 (severe decrease in GFR to 15 - 29 mL / min / 1.73 m 2 ); and stage 5 (renal failure with GFR < 15 mL / min / 1.73 m 2 or dialysis).

[0058] As used herein, a "control group" refers to a group of individuals whose average calculated or measured parameter can be used as a comparative value. For example, a control group with a pre-transplant cPRA level of a particular value (e.g., 90%) refers to a group of individuals with an average cPRA level of a particular value, and the cPRA level of the control group can be used as a comparative value to evaluate the effect of an antibody therapy as discussed herein (e.g., reducing the level of alloantibodies or reducing the risk of antibody-mediated transplant rejection).

[0059] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0060] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that comprises at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., BCMA or CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains (two heavy chains (H) and two light chains (L)) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region comprises three domains: C H 1, C H 2, and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region comprises one domain (C L 1). The V H region and the V L region can be further subdivided into hypervariable regions known as complementarity determining regions (CDRs), which are interspersed with more conserved regions known as framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the invention, the FRs of an anti-BCMA antibody or an anti-CD3 antibody (or antigen-binding portion thereof) can be identical to the human germline sequence or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on a side-by-side analysis of two or more CDRs.

[0061] As used herein, the term "antibody" also includes antigen-binding fragments of whole antibody molecules. As used herein, terms such as "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody" etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biological techniques, for example, to arrange one or more variable domains and / or constant domains in a suitable configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

[0062] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. As used herein, other engineered molecules (such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains) are also encompassed within the expression "antigen-binding fragment".

[0063] Antigen-binding fragments of an antibody generally include at least one variable domain. A variable domain can have any size or amino acid composition and will generally include at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a V H domain associated with a V L domain, the V H and V L domains can be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimers and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or V L domain.

[0064] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains found within antigen-binding fragments of antibodies of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains can be directly linked to each other or can be joined by a full-length or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Additionally, antigen-binding fragments of antibodies of the invention can contain any one of the variable and constant domain configurations listed above with respect to each other and / or with one or more monomeric V H or VL Homodimers or heterodimers (or other multimers) in which the domains are non-covalently associated (e.g., via disulfide bonds).

[0065] As with whole antibody molecules, antigen-binding fragments can be multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will generally comprise at least two different variable domains, each of which is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of the antibodies of the invention.

[0066] The antibodies of the invention can function by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and thereby cause lysis of the target cell. Assays well-known and available in the art can be used to measure CDC and ADCC. (See, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether antibody-mediated cytotoxicity is desired.

[0067] In certain embodiments of the invention, the anti-BCMA x anti-CD3 bispecific antibodies of the invention are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), such as in the CDRs and particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto a human framework sequence.

[0068] In some embodiments, the antibodies of the invention can be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant human antibody combinatorial library (described further below), antibodies isolated from an animal transgenic with respect to human immunoglobulin genes (e.g., a mouse) (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, produced or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic with respect to human Ig sequences, to in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions are sequences that, although derived from and related to human germline V H and V L sequences, may not naturally occur within the human antibody germline repertoire.

[0069] Human antibodies can exist in two forms associated with hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, wherein the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds and the molecules of approximately 75-80 kDa are composed of covalently coupled light and heavy chains (half-antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0070] The occurrence frequency of the second form in the various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. The invention encompasses antibodies having one or more mutations in the hinge, C H 2 or C H 3 regions, which may be desirable, for example, in production to increase the yield of the desired antibody form.

[0071] The antibody of the present invention can be an isolated antibody. As used herein, "isolated antibody" means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, for the purposes of the present invention, an antibody that has been separated or removed from at least one component of an organism, or from the tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody". Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0072] Compared to the corresponding germline sequences from which the antibodies are derived, the anti-BCMA x anti-CD3 antibodies discussed herein can contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frameworks and / or CDRs are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence variations are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a variety of antibodies and antigen-binding fragments starting from the heavy and light chain variable region sequences disclosed herein, which contain one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / 或VLAll framework and / or CDR residues within the domain are mutated back to the residues in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., mutated residues that are only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues that are only within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Additionally, the antibodies of the invention can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residues of a particular germline sequence while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. After obtaining antibodies and antigen-binding fragments containing one or more germline mutations, one or more desired properties of the antibodies and antigen-binding fragments, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity, etc., can be readily tested. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0073] The invention also includes anti-BCMA x anti-CD3 antibodies that comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes anti-BCMA x anti-CD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences shown herein or the full-length heavy and light chain sequences described herein.

[0074] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (termed a paratope) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope can include a sugar, phosphoryl group, or sulfonyl group moiety on an antigen.

[0075] When referring to a nucleic acid or a fragment thereof, the terms "substantially identical" or "substantially the same" indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap as discussed below, at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases exhibit nucleotide sequence identity. In some cases, a nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. In some cases, a bispecific antibody or an antigen-binding fragment thereof is 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences discussed herein (e.g., CDR, HCVR, LCVR, heavy chain or light chain sequences).

[0076] When applied to polypeptides, the terms "substantially similar" or "substantially similarity" mean that when two peptide sequences are optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, they have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the differences in the non-identical residue positions consist of conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In cases where the conservative substitutions in two or more amino acid sequences differ from each other, the percentage of sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitutions. The means for making such an adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains, namely cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445 (incorporated herein by reference). A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0077] Sequence analysis software is typically used to measure the sequence similarity of polypeptides, which is also referred to as sequence identity. Protein analysis software uses similarity metrics assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, the GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant proteins. See, e.g., version 6.1 of GCG. The polypeptide sequences can also be compared using FASTA (a program in GCG 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions between the query and the search sequence and the percentage of sequence identity (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST with default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0078] As used herein, the term "binding" in the context of an antibody, immunoglobulin, antibody binding fragment, or Fc-containing protein binding to, for example, a predetermined antigen, such as a cell surface protein, or a fragment thereof, generally refers to an interaction or association between two entities or molecular structures, such as an antibody-antigen interaction.

[0079] For example, when measured using an antigen as a ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as an analyte (or anti-ligand) in a BIAcore 3000 instrument by, for example, surface plasmon resonance (SPR) technology, the binding affinity typically corresponds to a K D value of about 10 -7 M or less, such as about 10 -8 M or less, such as about 10 -9M or less. Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also frequently used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA et al., J Immunol Methods. 2005, 302(1-2):68-77).

[0080] Thus, the antibody or antigen-binding protein of the present invention binds to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to a K D value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, an affinity of an antibody corresponding to a K D value equal to or less than ten-fold that of a non-specific antigen can be considered undetectable binding, yet such an antibody can be paired with a second antigen-binding arm for generating the bispecific antibody of the present invention.

[0081] The term "K D " (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between K D and binding affinity, so the smaller the K D value, the higher the affinity, i.e., the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, so the K D value is smaller, and conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, so the K D value is larger. In some cases, compared with the binding affinity of a molecule ( 例如 antibody) for another interacting partner molecule (e.g., antigen Y), a higher binding affinity (or KD) of a specific molecule (e.g., antibody) for its interacting partner molecule (e.g., antigen X) can be expressed as a binding ratio determined by dividing the larger K D value (lower or weaker affinity) by the smaller K D (higher or stronger affinity), e.g., expressed as 5-fold or 10-fold greater binding affinity, as appropriate.

[0082] The term "k d " (seconds-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is also referred to as the k off value.

[0083] The term "k a”(M-1·sec-1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or an antibody-binding fragment.

[0084] The term “K A ”(M-1 or 1 / M) refers to the binding equilibrium constant of a specific antibody-antigen interaction, or the binding equilibrium constant of an antibody or an antibody-binding fragment. The binding equilibrium constant is obtained by dividing k a by k d .

[0085] The term “EC50” or “EC 50 ” refers to the half-maximal effective concentration, which includes the antibody concentration that induces half of the response between baseline and maximum after a specific exposure time. EC 50 essentially represents the antibody concentration at which 50% of the maximum effect is observed. In certain embodiments, the EC 50 value is equal to the concentration at which the antibody of the present invention produces half-maximal binding to cells expressing CD3 or BCMA, as determined by, for example, FACS binding assays. Thus, as the EC 50 or half-maximal effect concentration value increases, a decrease or attenuation in binding is observed.

[0086] In one embodiment, the decrease in binding can be defined as an increase in the EC 50 antibody concentration capable of binding to half of the maximum amount of target cells.

[0087] In another embodiment, the EC 50 value represents the concentration of the antibody of the present invention that causes half-maximal depletion of target cells through T cell cytotoxic activity. Thus, as the EC50 or half-maximal effect concentration value decreases, an increase in cytotoxic activity ( 例如 , T cell-mediated plasma cell killing) is observed.

[0088] A method for desensitizing a patient against HLA alloantibodies to facilitate a successful solid organ transplantation

[0089] The present disclosure provides methods of using an anti-BCMA x anti-CD3 bispecific antibody (e.g., REGN5459 or REGN5458) to safely deplete cells expressing B cell maturation antigen (BCMA) (e.g., plasma cells) and reduce anti-HLA alloantibodies to facilitate solid organ transplantation (e.g., kidney transplantation). Plasma cell-targeted therapies (as described herein) will have a more durable HLA desensitization effect by eliminating the source of anti-HLA alloantibodies, thereby facilitating transplantation and reducing the risk of antibody-mediated rejection (AMR) post-transplant. Anti-BCMA x CD3 antibodies (e.g., REGN5459 and REGN5458) target the surface protein BCMA that is selectively expressed on plasma cells. The bispecific antibodies deplete plasma cells and newly activated BCMA-expressing B cells, including those that produce anti-HLA antibodies, without compromising regulatory T cell responses that promote tolerance post-transplant. Desensitization of a patient to anti-HLA alloantibodies can be determined, for example, by reducing the level of alloantibodies in a subject, by reducing the level of calculated panel reactive antibody (cPRA) in a subject, or by comparing the risk of rejection of a subject to a control population at the time of transplantation or within a specific time period post-transplant.

[0090] Desensitization can be achieved, for example, by a method of reducing the level of alloantibodies in a subject in need of solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on a cell (e.g., plasma cell), and a second antigen-binding domain that specifically binds to human CD3 on a T cell. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in conjunction with this method are discussed in more detail herein.

[0091] In some embodiments, reducing the level of alloantibodies in a subject comprises reducing the alloantibody level to below the baseline alloantibody level measured prior to administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the method further comprises measuring the baseline alloantibody level in the subject prior to administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the alloantibody level and / or the baseline alloantibody level are measured by a single antigen bead assay, and the alloantibody level and / or the baseline alloantibody level correspond to the mean fluorescence intensity of the peak immunodominant anti-HLA antibody. In some cases, the reduction in the alloantibody level relative to the baseline alloantibody level corresponds to a reduction in the mean fluorescence intensity (MFI) of the peak immunodominant anti-HLA antibody of ≥50%. In some cases, the reduction in the alloantibody level corresponds to a reduction or a reduction of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% in the MFI of the peak immunodominant anti-HLA antibody.

[0092] In some embodiments, the alloantibody level is measured by a single antigen bead assay, and a decrease in the alloantibody level corresponds to a decrease in the mean fluorescence intensity of the peak immunodominant anti-HLA antibody to <5000. In some cases, the alloantibody level is measured by a single antigen bead assay, and a decrease in the alloantibody level corresponds to a decrease in the mean fluorescence intensity of the peak immunodominant anti-HLA antibody to <10000, <9500, <9000, <8500, <8000, <7500, <7000, <6500, <6000, <5500, <5000, <4900, <4800, <4700, <4600, <4500, <4400, <4300, <4200, <4100, <4000, <3900, <3800, <3700, <3600, <3500, <3400, <3300, <3200, <3100, <3000, <2900, <2800, <2700, <2600, <2500, <2400, <2300, <2200, <2100, <2000, <1900, <1800, <1700, <1600, <1500, <1400, <1300, <1200, <1100, <1000, <900, <800, <700, <600, <500, <490, <480, <470, <460 or <450.

[0093] Desensitization can be achieved, for example, by a method of reducing the calculated panel reactive antibody (cPRA) level in a subject in need of a solid organ transplant, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on a cell (e.g., a plasma cell), and a second antigen-binding domain that specifically binds to human CD3 on a T cell. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in conjunction with this method are discussed in more detail herein.

[0094] In some embodiments, reducing the cPRA level of a subject comprises reducing the cPRA level of the subject to below the baseline cPRA level measured prior to administration of the bispecific antibody or its antigen-binding fragment. In some cases, the cPRA level of the subject is reduced to: <99%; <98%; <97%; <96%; <95%; <94%; <93%; <92%; <91%; <90%; <89%; <88%; <87%; <86%; <85%; <84%; <83%; <82%; <81%; or <80%. In some cases, the cPRA level of the subject is reduced to or reduced to less than the following: 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89.5%, 89%, 88.5%, 88%, 87.5%, 87%, 86.5%, 86%, 85.5%, 85%, 84.5%, 84%, 83.5%, 83%, 82.5%, 82%, 81.5%, 81%, 80.5%, 80%, 79.5%, 79%, 78.5%, 78%, 77.5%, 77%, 76.5%, 76%, 75.5%, 75%, 74.5%, 74%, 73.5%, 73%, 72.5%, 72%, 71.5%, 71%, 70.5%, 70%, 69.5%, 69%, 68.5%, 68%, 67.5%, 67%, 66.5%, 66%, 65.5%, 65%, 64.5%, 64%, 63.5%, 63%, 62.5%, 62%, 61.5%, 61%, 60.5%, 60%, 59.5%, 59%, 58.5%, 58%, 57.5%, 57%, 56.5%, 56%, 55.5%, 55%, 54.5%, 54%, 53.5%, 53%, 52.5%, 52%, 51.5%, 51%, 50.5%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%.

[0095] The present disclosure also provides a method of reducing the sensitivity of a subject to anti-HLA antibodies prior to solid organ transplantation, wherein the method comprises administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on a cell (e.g., a plasma cell) and a second antigen-binding domain that specifically binds to human CD3 on a T cell, wherein the risk of rejection in the subject at the time of organ transplantation is no greater than the risk of rejection in a control population having a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in conjunction with this method are discussed in more detail herein.

[0096] In some embodiments, the risk of rejection in the subject at the time of organ transplantation is no greater than the risk of rejection in a control population having a cPRA level of 80% prior to transplantation. In some embodiments, the risk of rejection in the subject at the time of organ transplantation is no greater than the risk of rejection in a control population having a cPRA level of 50% to <80% prior to transplantation. In some embodiments, the risk of rejection in the subject at the time of organ transplantation is no greater than the risk of rejection in a control population having a cPRA level of 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89.5%, 89%, 88.5%, 88%, 87.5%, 87%, 86.5%, 86%, 85.5%, 85%, 84.5%, 84%, 83.5%, 83%, 82.5%, 82%, 81.5%, 81%, 80.5%, 80%, 79.5%, 79%, 78.5%, 78%, 77.5%, 77%, 76.5%, 76%, 75.5%, 75%, 74.5%, 74%, 73.5%, 73%, 72.5%, 72%, 71.5%, 71%, 70.5%, 70%, 69.5%, 69%, 68.5%, 68%, 67.5%, 67%, 66.5%, 66%, 65.5%, 65%, 64.5%, 64%, 63.5%, 63%, 62.5%, 62%, 61.5%, 61%, 60.5%, 60%, 59.5%, 59%, 58.5%, 58%, 57.5%, 57%, 56.5%, 56%, 55.5%, 55%, 54.5%, 54%, 53.5%, 53%, 52.5%, 52%, 51.5%, 51%, 50.5%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% prior to transplantation.

[0097] The present disclosure also provides a method of reducing the risk of allograft rejection in a subject after solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on a cell (e.g., a plasma cell), and a second antigen-binding domain that specifically binds to human CD3 on a T cell, wherein the risk of rejection in the subject during the post-transplantation interval is no greater than the risk of rejection in a control population having a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in conjunction with this method are discussed in more detail herein.

[0098] In some embodiments, the risk of rejection during the post-transplantation interval for the subject is not greater than the risk of rejection for a control population with a pre-transplant cPRA level of 80%. In some embodiments, the risk of rejection during the post-transplantation interval for the subject is not greater than the risk of rejection for a control population with a pre-transplant cPRA level of 50% to <80%. In some embodiments, the risk of rejection during the post-transplantation interval for the subject is not greater than the risk of rejection for a control population with a pre-transplant cPRA level of 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89.5%, 89%, 88.5%, 88%, 87.5%, 87%, 86.5%, 86%, 85.5%, 85%, 84.5%, 84%, 83.5%, 83%, 82.5%, 82%, 81.5%, 81%, 80.5%, 80%, 79.5%, 79%, 78.5%, 78%, 77.5%, 77%, 76.5%, 76%, 75.5%, 75%, 74.5%, 74%, 73.5%, 73%, 72.5%, 72%, 71.5%, 71%, 70.5%, 70%, 69.5%, 69%, 68.5%, 68%, 67.5%, 67%, 66.5%, 66%, 65.5%, 65%, 64.5%, 64%, 63.5%, 63%, 62.5%, 62%, 61.5%, 61%, 60.5%, 60%, 59.5%, 59%, 58.5%, 58%, 57.5%, 57%, 56.5%, 56%, 55.5%, 55%, 54.5%, 54%, 53.5%, 53%, 52.5%, 52%, 51.5%, 51%, 50.5%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%. In some cases, the post-transplantation interval is a 3-month interval starting one day after organ transplantation. In some cases, the post-transplantation interval is a 6-month interval starting one day after organ transplantation. In some cases, the post-transplantation interval is a 12-month interval starting one day after organ transplantation.In various embodiments, the post-transplantation interval is a time period that begins one day after organ transplantation and ends at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months after transplantation.

[0099] In some embodiments, the graft function in the subject remains at a functional level that is equal to or higher than the functional level of a control population during the post-transplantation interval.

[0100] In any one of the various embodiments of the methods discussed above or herein, the subject can be a human.

[0101] In any one of the various embodiments of the methods discussed above or herein, the solid organ can be selected from the group consisting of a kidney, a lung, a pancreas, a heart, a colon or a part thereof, a liver or a part thereof, or skin. In some cases, the solid organ is a kidney.

[0102] The methods discussed herein address a highly unmet need for facilitating solid organ transplantation (e.g., kidney transplantation) in patients who are highly sensitive to HLA. Taking kidney transplantation as an example, fewer than 10% of highly sensitive patients in need of a kidney will receive a kidney transplant due to the difficulty in finding a compatible organ donor. Patients who are highly sensitive to HLA are generally defined as having a cPRA ≥ 80%. The cPRA score represents the probability that an organ transplant candidate will encounter an incompatible donor and is used as a measure of the level of sensitivity. Although the kidney allocation system has generally increased the transplantation rate for highly sensitive individuals, 30% to 50% of patients with a cPRA score ≥ 90% remain on the kidney transplant waiting list for more than 5 years, highlighting the unmet need for this subset of sensitive patients. Among the approximately 11,000 patients with chronic kidney disease and a cPRA ≥ 90% on the US kidney transplant waiting list, the majority (approximately 7,000 patients) have a cPRA score > 98%. These patients have a particularly high unmet need, as evidenced by their more frequent removal from the waiting list and death due to medical comorbidities. In addition, despite the implementation of the kidney allocation system in 2014, the average transplantation rate for approximately 2,000 patients with a cPRA ≥ 99.9% on the kidney transplant waiting list is significantly lower than that of other HLA-sensitive patients. Patients who are highly sensitive to HLA rely on hemodialysis for several years, which is associated with poor quality of life, high morbidity, and a mortality rate of up to 20% to 25% while on the kidney transplant waiting list; therefore, treatments that increase the likelihood of kidney transplantation are beneficial. In some cases, the methods discussed herein can be applied to subjects seeking solid organ transplantation who have been on a waiting list (e.g., a kidney transplant waiting list) for at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years or longer. Although desensitization according to the methods discussed herein does not necessarily result in an organ transplant due to limited availability, a reduction in the number of unacceptable HLA matches will increase the likelihood of transplantation for each patient. In addition, patients who receive an organ during the study period and have an acceptable crossmatch can receive an organ (e.g., a kidney) transplant.

[0103] Plasma cells are promising targets for treating antibody-mediated autoimmune and alloimmune diseases, including desensitizing individuals highly sensitive to HLA, which prevents them from safely receiving transplants. Due to eliminating the source of anti-HLA alloantibodies, plasma cell-targeted therapies may have a more durable HLA desensitization effect than plasma exchange and other antibody-reducing treatments. Bispecific anti-BCMA x anti-CD3 antibodies (including REGN5459 and REGN5458) can be used to bind BCMA on plasma cells and CD3 on T cells. Although both REGN5459 and REGN5458 contain the same BCMA-binding arm, the difference between these two molecules is their respective affinity for CD3, where REGN5459 binds CD3 on T cells with a lower affinity than REGN5458. In vitro, each of these bispecific antibodies triggers the activation of Jurkat T lymphocytes only in the presence of cells expressing human BCMA. REGN5459 and REGN5458 induce T cell activation and cytotoxicity against tumor cells with a wide range of BCMA protein expression levels; the cell surface expression level of BCMA on target cells does not affect the potency of REGN5459 or REGN5458. REGN5459 and REGN5458 can mediate the killing of multiple cell lines and primary cells with a range of BCMA cell surface expression (including normal plasma cells in bone marrow and secondary lymphoid organs), as demonstrated in healthy cynomolgus monkeys. Due to the lower affinity of REGN5459 for CD3, REGN5459 is hypothesized to have a lower likelihood of causing CRS and is similar in efficacy in plasma cell depletion.

[0104] Clinically relevant metrics for sensitive patients in need of solid organ (e.g., kidney) transplantation include the reactivity range of donor-specific antibodies (DSA), particularly the reactivity range of anti-HLA alloantibodies measured in Luminex-based single antigen bead (SAB) assays. The SAB assay measures the reactivity of a patient's serum or plasma to a panel of HLA-coated beads, expressed as mean fluorescence intensity (MFI). By using center-specific cut-off values and algorithms, HLA proteins can be identified on a per-patient basis where an individual has an unacceptable preformed alloantibody threshold level. The patient's own HLA genotype is considered to distinguish true allo-reactivity from background fluorescence signals. Based on the number of unacceptable HLA matches and the estimated HLA allele frequencies in the relevant population, a cPRA level (range: 0% to 100%) can be calculated and used as a measure of sensitivity level, with the cPRA level representing the probability of encountering an organ transplant-incompatible donor.

[0105] Because anti-HLA alloantibodies are well-characterized prognostic biomarkers and can predict clinical outcomes of solid organ (e.g., kidney) transplantation, anti-HLA monitoring is part of the consensus guidelines established by the Transplantation Society Antibody Consensus Panel. In the presence of anti-HLA alloantibodies against donor-specific HLA above a certain threshold (expressed as MFI), the risk of AMR and graft failure is significantly increased. Peak anti-HLA MFI > 3,000 is associated with a significant increase in AMR and reduced 1-year, 3-year, and 5-year graft survival, while patients with anti-HLA MFI of 465 to approximately 3,000 showed similar AMR rates and graft survival to controls with little to no anti-HLA (highest MFI ＜ 465). Other studies have also found that the risk of post-transplant AMR is significantly increased in patients with peak anti-HLA MFI > 3,000 or > 8,000 before transplantation. Thus, there is a clear correlation between high peak anti-HLA MFI, AMR, and reduced graft survival; anti-HLA MFI > 3,000 to 5,000 is generally considered high-risk, and MFI > 10,000 is almost universally considered unacceptable for transplantation in HLA-matched (non-desensitized) patients. In addition, there are other less standardized DSA detection techniques, including measuring the titer of anti-HLA alloantibodies by serial dilution of serum samples used for SAB assays; specifically measuring C1q-binding anti-HLA alloantibodies using a similar Luminex platform; and cell-based physical crossmatch assays for T cells (representing class I HLA), B cells (representing class II HLA), and endothelial progenitor cells (for testing reactivity against non-HLA antigens). Cell-based crossmatch assays can be flow cytometry-based assays and cytotoxicity-based assays.

[0106] Although plasma cells are the target cells of anti-BCMA x anti-CD3 antibodies (e.g., REGN5459 and REGN5458), the rarity of these cells in the circulation may limit reliable quantification. Accordingly, the concentrations of Ig subclasses (IgA, IgM, IgE, IgG, IgG1, IgG2, and IgG3), as well as pathogen-specific antibody titers, can be monitored as potential surrogates for the level of antibody-producing plasma cells. Sensitive patients treated according to the methods discussed herein and patients who have received solid organ (e.g., kidney) transplants can be followed for a period of time (e.g., 12 months) after transplantation (when bispecific anti-BCMA x CD3 antibodies are no longer administered) to assess post-transplant outcomes. Graft function after transplantation (e.g., kidney transplantation) will be the primary measure of transplant success, and the incidence of graft failure and delayed graft function can be determined. The incidence of allograft rejection, the histological form of rejection (cell-mediated vs. antibody-mediated vs. mixed), and the Banff classification of kidney allograft pathology can also be determined by kidney biopsy, which is obtained as part of the standard of care or to obtain a cause when rejection is suspected (e.g., in the setting of elevated serum creatinine, worsening hypertension, or increased proteinuria). Anti-HLA MFI and cPRA can also be determined because these biomarker surrogates are highly correlated with relevant clinical outcomes, including post-transplant AMR and graft survival. The incidence of CMV, EBV, and BKV reactivation and infection will also be evaluated because these viruses are common after transplantation and the effect of plasma cell depletion therapy on their incidence is unclear.

[0107] In the case of kidney transplantation, the incidence of delayed graft function (defined as the need for dialysis within 7 days after transplantation) can be evaluated because delayed graft function is associated with a higher rate of acute rejection and is one of the strongest risk factors for chronic allograft nephropathy, corresponding to a 40% reduction in long-term graft survival. The incidence and classification of biopsy-proven TCMR and AMR can be monitored because both types of rejection alter graft histology and have a negative impact on graft survival. In addition, since 25% to 45% of highly sensitized patients experience AMR after transplantation and 80% of these AMR episodes occur in the first month after transplantation, the initial incidence of AMR in sensitized transplant recipients previously treated with REGN5459 or REGN5458 can be evaluated. Finally, the change in estimated glomerular filtration rate (eGFR) over time can be monitored as a key functional indicator of graft function after transplantation.

[0108] Combination therapy

[0109] The bispecific anti-BCMA x anti-CD3 antibodies and antigen-binding fragments thereof discussed herein can be combined with other therapies to desensitize patients against anti-HLA alloantibodies. In addition to the bispecific antibody, such combinations can include the use of IVIG and / or plasmapheresis. A regimen combining plasmapheresis with low-dose IVIG (100 mg / kg) has demonstrated a transient decrease in anti-HLA levels and, in some cases, facilitated transplantation upon repeated administration, depending on the response to anti-HLA alloantibodies. In addition to the bispecific antibody, other combinations can include rituximab, antibody blockers of interleukin (IL)-6 / IL-6 receptor (e.g., clazakizumab / tocilizumab), proteasome inhibitors (e.g., carfilzomib, bortezomib), other plasma cell-targeted therapies (e.g., daratumumab), and imlifidase / IgG-degrading enzyme (IdeS) from Streptococcus pyogenes.

[0110] The present invention provides methods that include administering a pharmaceutical composition comprising any one of the exemplary bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. The additional therapeutic active ingredient(s) can be administered before, simultaneously with, or immediately after the administration of the bispecific antigen-binding molecule of the present invention; (for the purposes of this disclosure, such an administration regimen is considered to be an "combined" administration of the bispecific antigen-binding molecule with the additional therapeutic active ingredient(s)).

[0111] The present invention includes pharmaceutical compositions in which the bispecific antigen-binding molecules of the present invention are co-formulated with one or more additional therapeutic active ingredients as discussed herein.

[0112] Bispecific antibodies and antigen-binding fragments thereof

[0113] The present invention includes bispecific antigen-binding molecules that specifically bind CD3 and BCMA. Such molecules can be referred to herein as, for example, "anti-BCMA x anti-CD3" or "anti-CD3 / anti-BCMA" or "anti-CD3xBCMA" or "CD3xBCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3).

[0114] In certain embodiments, the CD3 binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3 binding arm weakly binds to human CD3 and induces human T cell activation. In other embodiments, the CD3 binding arm weakly binds to human CD3 and induces killing of cells expressing BCMA. In other embodiments, the CD3 binding arm binds or weakly binds to both human and cynomolgus macaque (monkey) CD3, but the binding interaction cannot be detected by in vitro assays known in the art.

[0115] As used herein, the term "BCMA" refers to the human BCMA protein, unless specified as from a non-human species (e.g., "mouse BCMA", "monkey BCMA", etc.). The human BCMA protein has the amino acid sequence shown in SEQ ID NO:22.

[0116] The bispecific antigen-binding molecules that specifically bind CD3 and BCMA as described above can include anti-CD3 antigen-binding molecules that bind to CD3 with a weak binding affinity, such as those exhibiting a K D greater than about 40 nM, as measured by an in vitro affinity binding assay.

[0117] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide, or molecular complex that comprises or consists of at least one complementarity-determining region (CDR) that specifically binds to a particular antigen, alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen-binding molecule is an antibody or an antibody fragment, as defined elsewhere herein.

[0118] As used herein, the expression "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., BCMA), and the second antigen-binding domain specifically binds to a second different antigen (e.g., CD3).

[0119] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy-chain variable domain (HCVR) and a light-chain variable domain (LCVR). In the case of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first antigen-binding domain and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "D1", and the CDRs of the second antigen-binding domain may be designated with the prefix "D2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

[0120] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:1; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:5. In some cases, the isolated bispecific antigen-binding molecule comprises an HCDR1 having the amino acid sequence of SEQ ID NO:2, an HCDR2 having the amino acid sequence of SEQ ID NO:3, and an HCDR3 having the amino acid sequence of SEQ ID NO:4. In some cases, the isolated bispecific antigen-binding molecule comprises an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:8. In some cases, the first antigen-binding domain comprises an HCVR having the amino acid sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:5.

[0121] In some embodiments, the isolated bispecific antigen-binding molecule comprises a second antigen-binding domain, the second antigen-binding domain comprising: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) that are contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 13; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) that are contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 5. In some cases, the second antigen-binding domain comprises: (a) an HCDR1 having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14; (b) an HCDR2 having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 15; and (c) an HCDR3 having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 16. In some cases, the second antigen-binding domain comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8. In some cases, the second antigen-binding domain comprises: (a) HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NO: 10, 11, and 12, respectively; and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NO: 6, 7, and 8, respectively; or (b) HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively; and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NO: 6, 7, and 8, respectively. In some cases, the second antigen-binding domain comprises: (a) an HCVR having the amino acid sequence of SEQ ID NO: 9 and an LCVR having the amino acid sequence of SEQ ID NO: 5; or (b) an HCVR having the amino acid sequence of SEQ ID NO: 13 and an LCVR having the amino acid sequence of SEQ ID NO: 5.

[0122] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 5; and (b) a second antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO: 9 and an LCVR having the amino acid sequence of SEQ ID NO: 5.

[0123] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 5; and (b) a second antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO: 13 and an LCVR having the amino acid sequence of SEQ ID NO: 5.

[0124] Exemplary bispecific antigen-binding molecules comprise: (a) a first antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:5; and (b) a second antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO:9 and an LCVR having the amino acid sequence of SEQ ID NO:5, being a bispecific antibody. This antibody is also referred to as REGN5458.

[0125] Exemplary bispecific antigen-binding molecules comprise: (a) a first antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO:13 and an LCVR having the amino acid sequence of SEQ ID NO:5; and (b) a second antigen-binding domain comprising an HCVR having the amino acid sequence of SEQ ID NO:13 and an LCVR having the amino acid sequence of SEQ ID NO:5, being a bispecific antibody. This antibody is also referred to as REGN5459.

[0126] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes to bind BCMA, or binds to the same epitope on BCMA as a reference antibody, wherein the reference antibody comprises: a first antigen-binding domain comprising an HCVR / LCVR pair having the amino acid sequences of SEQ ID NO:1 / 5, and a second antigen-binding domain comprising an HCVR / LCVR pair having the amino acid sequences of SEQ ID NO:9 / 5 or SEQ ID NO:13 / 5.

[0127] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes to bind human CD3, or binds to the same epitope on human CD3 as a reference antibody, wherein the reference antibody comprises: a first antigen-binding domain comprising an HCVR / LCVR pair having the amino acid sequences of SEQ ID NO:1 / 5, and a second antigen-binding domain comprising an HCVR / LCVR pair having the amino acid sequences of SEQ ID NO:9 / 5 or SEQ ID NO:13 / 5.

[0128] The bispecific antigen-binding molecules discussed above or herein can be bispecific antibodies. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region belongs to isotype IgG1, IgG2, IgG3 or IgG4. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to the wild-type hinge of the same isotype.

[0129] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be linked to separate polymeric domains. The binding of one polymeric domain to another polymeric domain promotes the binding between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "polymeric domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second polymeric domain of the same or similar structure or conformation. For example, a polymeric domain can be a polypeptide containing an immunoglobulin C H 3 domain. Non-limiting examples of polymeric components are the Fc portions of immunoglobulins (containing C H 2-C H 3 domains), such as Fc domains of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4 and any allotypes within each isotype group.

[0130] The bispecific antigen-binding molecule of the present invention generally comprises two polymeric domains, such as two Fc domains, which are parts of separate antibody heavy chains, respectively. The first polymeric domain and the second polymeric domain can have the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first polymeric domain and the second polymeric domain can have different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0131] In certain embodiments, the polymeric domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length that contains at least one cysteine residue. In other embodiments, the polymeric domain is a cysteine residue or a cysteine-containing short peptide. Other polymeric domains include peptides or polypeptides containing or consisting of leucine zippers, helix-loop motifs, or coiled-coil motifs.

[0132] Any bispecific antibody format or technology can be used to prepare the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent binding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetrabody, knob-in-hole, common light chain (e.g., common light chain with knob-in-hole, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (for a review of the foregoing formats, see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein).

[0133] In the context of the bispecific antigen-binding molecules of the present invention, a multimerization domain (e.g., an Fc domain) can contain one or more amino acid alterations (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring form of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is at C H 2 or C HRegion 3 contains modifications that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH ranging from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modification comprises 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0134] The invention also includes a bispecific antigen-binding molecule comprising a first C H H3 domain and a second Ig C H H3 domain, wherein at least one amino acid of the first and second Ig C H H3 domains differs from each other, and wherein at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H H3 domain binds protein A and the second Ig C H H3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second C H H3 may also contain the Y96F modification (according to IMGT; Y436F according to EU). See, for example, U.S. Patent No. 8,586,713. In some embodiments, one heavy chain or the other heavy chain (but not both) comprises a C H H3 domain having both the H435R and Y436F modifications. May be present in the second C HOther modifications within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU).

[0135] In certain embodiments, the Fc domain can be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise portions or all of the C H 2 region C H 2 sequence from human IgG1, human IgG2, or human IgG4, and portions or all of the C H 3 sequence from human IgG1, human IgG2, or human IgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge can comprise an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that can be included in any antigen-binding molecule described herein include, from the N-terminus to the C-terminus: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any antigen-binding molecule described herein includes, from the N-terminus to the C-terminus: [IgG1 C H1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any antigen-binding molecule of the present invention are described in U.S. Publication 2014 / 0243504, published August 28, 2014, which is incorporated herein by reference in its entirety. Chimeric Fc domains having these general structural arrangements and their variants can have altered Fc receptor binding, which in turn affects Fc effector function. In various embodiments, a bispecific antigen-binding molecule can comprise a heavy chain constant region having a hinge domain, wherein positions 233-236 within the hinge domain can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where the positions are numbered according to EU numbering.

[0136] Sequence variants

[0137] Compared to the corresponding germline sequences from which the individual antigen-binding domains are derived, the antibodies and bispecific antigen-binding molecules of the present invention can comprise one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy chain variable domain and the light chain variable domain. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention can comprise antigen-binding domains derived from the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence variations are collectively referred to herein as "germline mutations"). One of ordinary skill in the art, starting from the heavy and light chain variable region sequences disclosed herein, can readily generate a variety of antibodies and antigen-binding fragments that comprise one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / 或VLAll framework and / or CDR residues within the domain are mutated back to the residues present in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., mutated residues that are only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues that are only within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain was originally derived). Additionally, the antigen-binding domain can contain any combination of two or more germline mutations within the framework region and / or CDR region, e.g., where certain individual residues are mutated to the corresponding residues of a specific germline sequence while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. After obtaining an antigen-binding domain containing one or more germline mutations, one or more desired properties of the antigen-binding domain, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity, etc., can be readily tested. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed by the present invention.

[0138] pH-dependent binding

[0139] The present invention includes anti-BCMA x anti-CD3 bispecific antigen-binding molecules having pH-dependent binding characteristics. For example, compared to neutral pH, the antibodies of the present invention can exhibit reduced binding to BCMA at acidic pH. Alternatively, compared to neutral pH, the antibodies of the present invention can exhibit enhanced binding to BCMA at acidic pH. The phrase "acidic pH" includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the phrase "neutral pH" refers to a pH of from about 7.0 to about 7.4. The phrase "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0140] In some cases, "reduced binding at acidic pH compared to neutral pH" is the K of the antibody binding to its antigen at acidic pH D value compared to the K of the antibody binding to its antigen at neutral pH Dexpressed as a ratio of values (and vice versa). For example, for the purposes of the present invention, if an antibody or an antigen-binding fragment thereof exhibits an acidic / neutral K D ratio of about 3.0 or greater, the antibody or antigen-binding fragment thereof can be considered to exhibit "reduced binding to BCMA at acidic pH compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K D ratio of the antibodies or antigen-binding fragments of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or higher.

[0141] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH compared to neutral pH. Additionally, modifying the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids (e.g., within a CDR) of the antigen-binding domain with histidine residues, an antibody can be obtained that has reduced antigen-binding at acidic pH relative to neutral pH.

[0142] Antibodies comprising Fc variants

[0143] In certain embodiments of the present invention, bispecific antigen-binding molecules that are anti-BCMA x anti-CD3 are provided, which comprise an Fc domain having one or more mutations, such as mutations that enhance or reduce the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention includes antibodies that have a mutation at C H 2 or C HRegion 3 contains mutations, one or more of which increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with a pH range from about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increased serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modification comprises 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). All positions are represented by EU numbering.

[0144] For example, the invention includes anti-BCMA x anti-CD3 bispecific antigen-binding molecules comprising an Fc domain having one or more pairs or one or more sets of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the invention.

[0145] Bioequivalent

[0146] The methods of the invention encompass antigen-binding molecules having an amino acid sequence different from those of the exemplary molecules disclosed herein but retaining the ability to bind CD3 and / or BCMA. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions when compared to the parental sequence, but exhibit substantially equivalent biological activity to the described bispecific antigen-binding molecules.

[0147] The present invention includes methods of administering an antigen-binding molecule that is bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins or antibodies are considered bioequivalents if they are pharmaceutical equivalents or pharmaceutical alternatives and do not show significant differences in the rate and extent of absorption when administered as a single dose and multiple doses at the same molar dose under similar experimental conditions. If some antigen-binding proteins are equivalent in extent of absorption but not in rate of absorption, they will be considered equivalent or pharmaceutical alternatives, but can be considered bioequivalents because this difference in rate of absorption is intentional and reflected in the labeling, and these antibodies are not necessary to achieve effective in vivo drug concentrations, for example, in long-term use, and are considered not to have clinical significance for the particular pharmaceutical product under study.

[0148] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences between them with respect to safety, purity, and potency.

[0149] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can switch between a reference product and a biological product one or more times without an increased risk of expected adverse reactions, including clinically significant changes in immunogenicity, or decreased efficacy, compared to a continuous therapy without switching between the reference product and the biological product.

[0150] In one embodiment, two antigen-binding proteins are bioequivalent if both act through one or more common mechanisms of action for one or more conditions of use to the extent that such mechanisms are known.

[0151] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence metrics include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolite in blood, plasma, serum, or other biological fluid is measured over time; (b) in vitro tests that are correlated with in vivo bioavailability data in humans and can reasonably predict in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of an antibody (or its target) is measured over time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0152] Bioequivalent variants of the exemplary bispecific antigen-binding molecules of the antigen-binding molecules described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bonds upon refolding. In other contexts, bioequivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the molecule (e.g., mutations that eliminate or remove glycosylation).

[0153] Therapeutic formulations and administrations

[0154] The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, diluents, and other agents that provide improved transfer, delivery, tolerance, and the like. Numerous suitable formulations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0155] The dosage of the antigen-binding molecule administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration, and the like. Preferred dosages are generally calculated based on body weight or body surface area. When the bispecific antigen-binding molecule of the invention is used for therapeutic purposes in an adult patient, it may be advantageous to administer the bispecific antigen-binding molecule of the invention intravenously in a single dose of from about 0.01 to about 20 mg / kg body weight, more preferably from about 0.02 to about 7 mg / kg body weight, from about 0.03 to about 5 mg / kg body weight, or from about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule of administration of the bispecific antigen-binding molecule can be determined empirically; for example, a patient's progress can be monitored by regular evaluation and the dosage adjusted accordingly. In addition, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0156] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, e.g., by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0157] The pharmaceutical compositions of the invention can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, for subcutaneous delivery, pen-type delivery devices are readily applicable to deliver the pharmaceutical compositions of the invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be used again. In a disposable pen-type delivery device, there is no replaceable cartridge. In fact, the disposable pen-type delivery device is prefilled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition within the reservoir is emptied, the entire device is discarded.

[0158] A variety of reusable pen-type delivery devices and autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the invention. Examples include but are not limited to AUTOPEN TM(Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (EliLilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA TM Pen (Abbott Labs, Abbott Park IL), etc.

[0159] In certain instances, a pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed near the target of the composition, so that only a fraction of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0160] The injectable preparation may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, etc. These injectable preparations can be prepared by well-known methods. For example, the injectable preparation can be prepared by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers (such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.). As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared is preferably filled in appropriate ampoules.

[0161] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a dosage form suitable for unit doses corresponding to the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injection solutions (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is usually about 0.05 mg to about 500 mg per dosage form in a unit dose; especially in the injectable form, it is preferred to contain the aforementioned antibody in an amount of about 0.05 mg to about 150 mg.

[0162] Administration regimens

[0163] In certain embodiments of the present invention, multiple doses of an antigen-binding molecule (e.g., a bispecific anti-BCMA x anti-CD3 antibody) can be administered to a subject over a defined time course. Methods according to this aspect of the present invention include sequentially administering multiple doses of the antigen-binding molecule of the present invention to a subject. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to the subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient a single initial dose of the antigen-binding molecule, then one or more second doses of the antigen-binding molecule, and optionally subsequently one or more third doses of the antigen-binding molecule.

[0164] The terms "initial dose", "second dose", and "third dose" refer to the chronological order of administration of the antigen-binding molecules of the present invention. Thus, the "initial dose" is the dose administered at the start of a treatment regimen (also referred to as the "baseline amount"); the "second dose" is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial dose, second dose, and third dose can all contain the same amount of antigen-binding molecules, but typically can differ from each other in terms of administration frequency. However, in certain embodiments, the amounts of antigen-binding molecules contained in the initial dose, second dose, and / or third dose differ from each other during the course of treatment (e.g., are appropriately upregulated or downregulated). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of a treatment regimen as a "loading dose", followed by subsequent doses administered at a lower frequency (e.g., a "maintenance dose"). In any of the embodiments, the initial dose (e.g., the first weekly dose) can be divided into two doses administered on separate days (e.g., consecutive days) separated by no more than three days. In any of the embodiments, the first nominal dose (i.e., the second dose) can be divided into two doses administered on separate days (e.g., consecutive days) separated by no more than three days. For example, if the initial dose or second dose is 1 mg, the dose can be divided into two 0.5 mg doses administered, for example, on consecutive days or on separate days separated by no more than three days. In various embodiments, the dose (e.g., the dose administered weekly, as a single dose or as two divided portions of a dose) is or is at least 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg,53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg970 mg, 980 mg, 990 mg, 1000 mg, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, 10 g or more. Any of these amounts can be used to define the range of the initial dose, second dose, or third dose discussed herein and are encompassed within the scope of the present disclosure. In some embodiments, all doses are administered as a single dose (e.g., a single infusion), including the doses administered during the first and second weeks of the dosing regimen. For example, an initial dose of 0.05 mg to 5 mg can be administered as a single dose during the first week, a second dose of 0.15 mg to 25 mg can be administered as a single dose during the second week, and a third dose of 0.5 mg to 150 mg can be administered as a single dose during the third week. Additional doses (e.g., the same dose as the third dose) can be administered weekly for some period of time thereafter. In another example, an initial dose of 0.05 mg to 5 mg can be administered as a single dose during the first week, a second dose of 0.15 mg to 25 mg can be administered as a single dose during the second week, and a third dose of 0.5 mg to 150 mg can be administered as a single dose during the third week. In another example, an initial dose of 0.05 mg to 1 mg can be administered as a single dose during the first week, a second dose of 0.15 mg to 5 mg can be administered as a single dose during the second week, and a third dose of 0.5 mg to 40 mg can be administered as a single dose during the third week. In some cases, the dosing schedule thereafter (e.g., after weeks 1 - 3) can include administration weekly, bi - weekly, tri - weekly, monthly, etc.

[0165] Example

[0166] The following examples are provided to give a complete disclosure and description of how to make and use the methods and compositions of the present invention to one of ordinary skill in the art and are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric or near atmospheric.

[0167] Example 1: Generation of a Bispecific Antibody that Binds BCMA and CD3

[0168] The present invention utilizes bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind CD3 and BCMA; such bispecific antigen-binding molecules are also referred to herein as "anti-BCMA x anti-CD3 or anti-CD3xBCMA or anti-BCMA x anti-CD3 bispecific molecules". The anti-BCMA portion of the anti-BCMA x anti-CD3 bispecific molecule can be used to target plasma cells expressing BCMA (also known as CD269), and the anti-CD3 portion of the bispecific molecule can be used to activate T cells. Binding of BCMA on plasma cells and CD3 on T cells promotes the directed killing (cytolysis) of targeted plasma cells by activated T cells.

[0169] Bispecific antibodies comprising an anti-BCMA specific binding domain and an anti-CD3 specific binding domain are constructed using standard methods, wherein the anti-BCMA antigen-binding domain and the anti-CD3 antigen-binding domain each comprise different, distinct HCVR pairs and a common LCVR. In an exemplary bispecific antibody, the molecule is constructed using the heavy chain from an anti-CD3 antibody, the heavy chain from an anti-BCMA antibody, and the common light chain from an anti-CD3 antibody (e.g., SEQ ID NO:5). In other cases, the bispecific antibody can be constructed using the heavy chain from an anti-CD3 antibody, the heavy chain from an anti-BCMA antibody, and an antibody light chain known to be promiscuous or that pairs efficiently with many heavy chain arms. Additional details regarding the anti-CD3 portion of the bispecific antibody can be found in WO 2017 / 053856, which is incorporated herein by reference.

[0170] Table 1: Summary of components of anti-BCMA x anti-CD3 bispecific antibodies

[0171]

[0172]

[0173] Tables 2A and 2B show the amino acid sequence identifiers of the bispecific anti-BCMA x anti-CD3 antibodies exemplified herein.

[0174] Table 2A: Amino acid sequences (HCVR) of anti-BCMA x anti-CD3 bispecific antibodies

[0175]

[0176] Table 2B: Amino acid sequences (LCVR) of anti-BCMA x anti-CD3 bispecific antibodies

[0177]

[0178] The REGN5458 bispecific antibody identified in Tables 2A and 2B comprises a first heavy chain (containing a first antigen-binding domain) having the amino acid sequence of SEQ ID NO:29, a second heavy chain (containing a second antigen-binding domain) having the amino acid sequence of SEQ ID NO:30, and a common light chain having the amino acid sequence of SEQ ID NO:32. The first heavy chain of the REGN5458 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:33. The second heavy chain of the REGN5458 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:34. The common light chain of the REGN5458 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:35.

[0179] The REGN5459 bispecific antibody identified in Tables 2A and 2B comprises a first heavy chain (containing a first antigen-binding domain) having the amino acid sequence of SEQ ID NO:29, a second heavy chain (containing a second antigen-binding domain) having the amino acid sequence of SEQ ID NO:31, and a common light chain having the amino acid sequence of SEQ ID NO:32. The first heavy chain of the REGN5459 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:33. The second heavy chain of the REGN5459 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:34. The common light chain of the REGN5459 bispecific antibody comprises a constant region having the amino acid sequence of SEQ ID NO:35.

[0180] Example 2: Desensitization of chronic kidney disease patients in need of kidney transplantation who are highly sensitive to human leukocyte antigen with an anti-BCMA x anti-CD3 bispecific antibody

[0181] A phase 1 / 2 study of a bispecific anti-BCMA x anti-CD3 antibody for desensitization of chronic kidney disease patients in need of kidney transplantation who are highly sensitive to human leukocyte antigen (HLA).

[0182] Objective: To explore primary and secondary endpoints.

[0183] This Investigational The primary objective is to evaluate the safety and tolerability of REGN5459 (Part A) or REGN5458 (Part B) as monotherapy in chronic kidney disease (CKD) patients in need of kidney transplantation who are highly sensitive to human leukocyte antigen (HLA).

[0184] This study's Secondary objectivesTo determine / evaluate the following for REGN5459 (Part A) or REGN5458 (Part B): (i) dosing regimens that result in a clinically significant decrease in anti-HLA alloantibody levels; (ii) the effect on calculated panel reactive antibody (cPRA) levels; (iii) the time at which anti-HLA alloantibody levels reach maximal and clinically significant decreases; (iv) the duration of the effect of the study drug on the decrease in anti-HLA alloantibodies; (v) the effect on circulating immunoglobulin (Ig) classes (isotypes); (vi) pharmacokinetic (PK) properties; and (vii) immunogenicity.

[0185] Study Design: This is a Phase 1 / 2 study of REGN5459 (Part A) or REGN5458 (Part B) in chronic kidney disease (CKD) patients on hemodialysis who are highly sensitized to HLA. For both Part A and Part B, a 3 + 3 dose-escalation design will be used to determine dose levels with acceptable safety, PK, and pharmacologic effects. Part A will be started first, and Part B of the study may be started based on safety data emerging from Part A of this study.

[0186] Each patient will receive REGN5459 (Part A) or REGN5458 (Part B), but not both, at a designated dose level as 3 infusions in 1 cycle over 15 days, as shown in Part A of Table 3 or Part B of Table 4. At least 1 hour before the start of each study drug infusion, patients will be premedicated with 40 mg of dexamethasone by intravenous (IV) infusion to limit the likelihood of cytokine release syndrome (CRS). If any infusion is not well tolerated, the subsequent infusion doses for this patient and patients enrolled in additional cohorts may be modified.

[0187] The planned duration of the study for each patient is up to approximately 30 weeks, including a screening period (up to 14 days), a dosing period (15 days), a safety observation period (28 days, starting on Day 1), and a follow-up period (approximately 26 weeks, or until the patient cross-matches with a potential transplant donor). Patients who successfully cross-match with a potential transplant donor will discontinue this study and be enrolled in a separate protocol (Example 3) for additional evaluation after kidney transplantation.

[0188] Safety will be evaluated by the incidence and severity of treatment-emergent adverse events (TEAEs), which will be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, Version 5.0 (NCI–CTCAEv5.0), except for CRS, which will be graded according to Table 5. Drug activity will be evaluated by the mean fluorescence intensity (MFI) of anti-HLA alloantibodies obtained by single-antigen bead (SAB) assay. Pharmacokinetic, anti-drug antibody (ADA), and pharmacodynamic (PD) parameters will be analyzed from serum / plasma and peripheral blood mononuclear cells at several time points before and after REGN5459 or REGN5458 infusion.

[0189] Each cohort will enroll a minimum of 3 patients. Additional patients (up to a total of 6 evaluable patients) may be enrolled in any given cohort to further explore safety, PK, and PD characteristics.

[0190] Modifications to the dosing regimens for Parts A and B may be explored based on data observed in previous cohorts (e.g., smaller dose increments between lower and higher doses within and / or between cohorts).

[0191] Table 3: Dose Escalation Scheme for Part A (REGN5459)

[0192]

[0193] *If Cohort 1 is determined to be intolerable, Cohort 1 may be evaluated.

[0194] Table 4: Dose Escalation Scheme for Part B (REGN5458)

[0195]

[0196] *If Cohort 1 is determined to be intolerable, Cohort 1 may be evaluated.

[0197] Dose escalation for Parts A and B will continue until an acceptable dose is determined based on safety assessments and any other data available at that time or the maximum dose level is reached. The maximum full dose evaluated in this study is 150 mg of REGN5459 or 40 mg of REGN5458. However, during the 14-day safety observation period after the full dose, if 1 patient in a dose cohort experiences an adverse event of interest (AEI), or 2 or more patients experience adverse events (AEs) of grade ≥2 (except for AEs of grade ≥2 that are clearly unrelated to the study drug), but the dosing regimen is still determined to be tolerable, the full dose escalation will not exceed 2-fold (i.e., a 100% increase) of the full dose of the previously evaluated dose cohort.

[0198] Acute IRR is defined as starting after the infusionLess than 6 hours any AE that occurs within 2 hours after the infusion (whichever is later), and is accompanied by typical signs and symptoms, including but not limited to: flushing, tachycardia, hypotension, dyspnea, bronchospasm, back pain, fever, urticaria, edema, nausea, and rash. Cytokine release syndrome is a condition characterized by fever, tachypnea, headache, tachycardia, hypotension, rash, and / or hypoxia; in this study, it is defined as occurring 6 hours or longer or an event that occurs more than 2 hours after the infusion (whichever is later). Guidelines for grading and management of cytokine release syndrome, including clinical assessment and monitoring, are shown in Table 5.

[0199] Table 5: Toxicity Grading of Cytokine Release Syndrome*

[0200]

[0201] BiPAP = bilevel positive airway pressure ventilation, CPAP = continuous positive airway pressure ventilation, CRS = cytokine release syndrome

[0202] *Adapted from the American Society for Transplantation and Cellular Therapy (ASTCT) Consensus Grading of Cytokine Release Syndrome.

[0203] Organ toxicities associated with CRS can be graded according to CTCAE v5.0, but they do not affect CRS grading.

[0204] 1 Fever is defined as a body temperature ≥38°C not caused by any other reason. In patients with CRS who then receive antipyretics or anti-cytokine therapies (such as tocilizumab or steroids), fever is no longer a necessary condition for subsequent CRS severity grading. In such cases, CRS grading is driven by hypotension and / or hypoxia.

[0205] 2 The CRS level is determined by the more severe event: hypotension or hypoxia not caused by any other reason. For example, a patient with a body temperature of 39.5°C, hypotension requiring 1 vasopressor, and hypoxia requiring low-flow nasal cannula is classified as grade 3 CRS.

[0206] 3 Low-flow nasal cannula is defined as an oxygen delivery rate ≤6 liters per minute. Low flow also includes blow-by oxygen delivery, sometimes used in pediatrics. High-flow nasal cannula is defined as an oxygen delivery rate >6 liters per minute.

[0207] Study Duration: The study is planned to last up to approximately 30 weeks per patient, including a screening period (up to 14 days), a dosing period (15 days), a safety observation period (28 days starting from Day 1), and a follow-up period (approximately 26 weeks or until the patient cross-matches with a potential transplant donor). Patients who successfully cross-match with a potential transplant donor will discontinue this study and be enrolled in a separate protocol (Example 3) for additional evaluation after kidney transplantation.

[0208] Study Population: There will be approximately 6 to 60 evaluable patients in this study; 6 to 30 for each of Part A and Part B.

[0209] The study population will consist of adult CKD patients aged 18 to 70 years who require hemodialysis and are waiting for a kidney transplant on the United Network for Organ Sharing (UNOS) list, with cPRA ≥ 99.9%, or for those patients with cPRA > 98% (98.1% to 99.8%), they have been on the waiting list for 5 years or longer.

[0210] Inclusion criteria - Patients must meet the following criteria at screening to be eligible for inclusion in this study:

[0211] 1. Aged 18 to 70 years

[0212] 2. Have CKD requiring hemodialysis and be waiting for a kidney transplant on the UNOS list, with cPRA ≥ 99.9%, or cPRA > 98% (98.1% to 99.8%) and have been on the waiting list for 5 years or longer

[0213] 3. Adequate blood function, as measured by: platelet count > 50 × 10 9 / L. The patient may not have received a platelet transfusion within 7 days to meet this platelet eligibility requirement; absolute neutrophil count (ANC) > 1.0 × 10 9 / L; hemoglobin > 8.0 g / dL

[0214] 4. Adequate liver function, defined as: total bilirubin ≤ 1.5x ULN*; transaminases (ALT, AST) ≤ 2.5x ULN; alkaline phosphatase ≤ 2.5x ULN (upper limit of normal value) - *Patients with a documented history of Gilbert syndrome do not need to meet this total bilirubin requirement if the total bilirubin has not changed compared to the baseline value

[0215] 5. Willing and able to comply with clinical visits and study-related procedures

[0216] 6. Provide an informed consent form signed by the study patient or the legally authorized representative

[0217] Exclusion criteriaPatients who meet any of the following criteria, or who have any other medical condition that may preclude the safe administration of the study treatment, will be excluded from this study:

[0218] 1. Current or active malignancy with no remission for at least 1 year

[0219] 2. History of CNS lesions or CNS neurodegenerative or movement disorders

[0220] 3. Patients who have had their spleen removed, including those with functional asplenia

[0221] 4. Patients who have received a stem cell transplant within 5 years

[0222] 5. Body mass index ≥ 35 kg / m² at screening 2

[0223] 6. Hypogammaglobulinemia, defined as total plasma IgG < 300 mg / dL at screening

[0224] 7. Continuous systemic corticosteroid treatment with prednisone (or anti-inflammatory equivalent) at a dose of more than 10 mg per day within 72 hours after the start of study drug administration

[0225] 8. Received a calcineurin inhibitor (e.g., tacrolimus, cyclosporine) within 30 days of study drug administration

[0226] 9. Received cyclophosphamide, rituximab, obinutuzumab, other anti-CD20 or B-cell depleting agents, or proteasome inhibitors or anti-CD38 therapies (e.g., isatuximab, daratumumab) within 6 months of study drug administration

[0227] 10. Previous treatment with any anti-BCMA antibody (including antibody-drug conjugates or bsAbs) or BCMA-directed CAR-T cell therapy

[0228] 11. Use of a study agent within 8 weeks or 5 half-lives (whichever is longer) of study drug administration

[0229] 12. Evidence of active infection on screening chest X-ray and appropriate clinical evidence of infection (patients may be rescreened after the complete treatment course and resolution of the active infection)

[0230] 13. Any infection that requires hospitalization or intravenous anti-infective treatment within 4 weeks after study drug administration

[0231] 14. Patients who are positive for human immunodeficiency virus (HIV) at screening

[0232] 15. Latent or active tuberculosis infection that was not fully treated and diagnosed by skin test, interferon-γ release assay, and / or chest imaging within the past 5 years according to local practice guidelines or Centers for Disease Control and Prevention guidelines. Unclear cases should be discussed with the medical / research principal investigator before enrollment.

[0233] 16. Patients with a history of chronic hepatitis B, and those who test positive for hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (HBcAb) at screening

[0234] 17. Patients with a history of hepatitis C or positive for hepatitis C virus (HCV) antibody are excluded. However, patients who are positive for HCV antibody and have successfully completed a previous anti-HCV therapy course, and those in whom HCV ribonucleic acid (RNA) is undetectable by polymerase chain reaction (PCR) are permitted.

[0235] 18. Vaccination with a vector with replication potential within 28 days before the first study drug administration

[0236] 19. Received a COVID-19 vaccination within 1 week of the planned start of study drug, or did not complete the planned COVID-19 vaccination 1 week before starting study drug

[0237] 20. Any recent recipient of a licensed or investigational live / attenuated vaccine within 2 months of the screening visit, including but not limited to: adenovirus (oral live adenovirus vaccine type 7); varicella-zoster virus (VZV; ); rotavirus vaccine yellow fever vaccine measles and mumps vaccine (live measles and mumps virus vaccine); measles, mumps, and rubella vaccine ( II); BCG vaccine; Sabin oral polio vaccine; rabies vaccine vaccine

[0238] 21. History of a severe allergic reaction or acute hypersensitivity reaction attributed to previous IVIG, anti-cytokine therapy (e.g., tocilizumab), study-required vaccines, or premedication

[0239] 22. Known hypersensitivity to any component of the formulated product

[0240] 23. Patients who are institutionalized at the time of screening due to an order issued by a judicial or administrative authority

[0241] 24. Members of the clinical research center study team and / or their immediate family members, unless approved in advance by the sponsor

[0242] 25. Women who are pregnant or breastfeeding at the time of screening

[0243] 26. Women of childbearing potential and men who are unwilling to use highly effective contraceptive measures before the first dose / start of the first treatment, during the study period, and for at least 4 months after the last dose. Highly effective contraceptive measures for women include: starting stable use of combined (estrogen and progestin) hormonal contraception (oral, intravaginal, transdermal) or progestin-only hormonal contraception (oral, injectable, implantable) and suppressing ovulation starting 2 or more menstrual cycles before screening; intrauterine device (IUD); intrauterine hormone-releasing system (IUS); bilateral tubal ligation; a partner with vasectomy (provided that the male partner with vasectomy is the sole partner of the study participant and this partner has received a medical evaluation of successful surgery); and / or abstinence.

[0244] 27. Any medical condition, comorbidity, physical examination finding, or metabolic dysfunction or clinical laboratory abnormality that the investigator deems makes the patient ineligible to participate in the clinical study due to high safety risks and / or potential impact on the interpretation of study results

[0245] 28. Cardiac ejection fraction < 40% measured by echocardiogram or multiple-gated acquisition scan (MUGA)

[0246] 29. Major cardiovascular diseases (e.g., New York Heart Association class III or IV heart disease, myocardial infarction, stroke or transient ischemic attack within the previous 6 months, unstable arrhythmia or unstable angina) and / or major pulmonary diseases (e.g., obstructive lung disease and history of symptomatic bronchospasm)

[0247] 30. History of autonomic dysfunction (e.g., type 1 diabetes) that has been documented and may interfere with the patient's ability to tolerate the protocol therapy

[0248] Treatment: Placebo will not be used in this study. The administered dose will be a fixed dose and will not depend on the patient's weight or body surface area. The study drug will be administered by intravenous infusion.

[0249] REGN5459 or REGN5458 (B cell maturation antigen [BCMA] × cluster of differentiation 3 [CD3] bispecific antibody [bsAb]) for intravenous infusion (3 infusions in 1 cycle over a 15-day period) will be provided as a liquid in a sterile single-use vial. Each vial will contain REGN5459 or REGN5458 at a concentration of 10 mg / mL. The diluent for REGN5459 or REGN5458 is provided as a sterile liquid solution in a glass vial for intravenous injection administration.

[0250] Dexamethasone 40 mg intravenous injection; antihistamine (diphenhydramine [H-1 blocker]) 25 mg intravenous injection or orally; acetaminophen 650 mg orally. According to Table 6, the patient should receive premedication with dexamethasone before the administration of REGN5459 or REGN5458.

[0251] Table 6: Premedication with antihistamine and acetaminophen

[0252]

[0253] Study endpoints:

[0254] Investigational The primary endpoints are:

[0255] · Incidence of AEI from the first study drug dose to the end of the safety observation period

[0256] · Incidence and severity of TEAE (including AESI and serious adverse events (SAE)) from the first study drug dose to the end of the study

[0257] Investigational The secondary endpoints are:

[0258] · Proportion of patients with a clinically significant decrease in anti-HLA alloantibodies during the study, defined as: decrease in cPRA relative to baseline; or peak (immunodominant) anti-HLA mean fluorescence intensity (MFI) decreased to <5,000, or decreased by ≥50% (measured by single antigen bead [SAB])

[0259] · Maximum decrease in peak (immunodominant) MFI relative to baseline

[0260] · Percentage change in peak (immunodominant) MFI relative to baseline

[0261] · Percentage change in the sum of MFI of anti-HLA alloantibodies relative to baseline when measured by SAB

[0262] · Time to first clinically significant decrease in anti-HLA alloantibody level (defined as peak anti-HLA alloantibody MFI <5,000 or decreased by ≥50%) measured by SAB

[0263] · Time to maximum decrease in anti-HLA alloantibody level (defined as peak anti-HLA alloantibody MFI <5,000 or decreased by ≥50%) measured by SAB

[0264] · Maximum decrease in cPRA relative to baseline

[0265] · Time to first clinically significant decrease in cPRA

[0266] ·Time to maximal reduction of cPRA relative to baseline

[0267] ·Duration during which peak anti-HLA alloantibodies are reduced to MFI < 5,000 or by ≥ 50% as measured by SAB, and duration of maximal reduction of anti-HLA alloantibody MFI and % cPRA

[0268] ·Change in serum concentration of Ig classes (IgG, IgA, IgE, IgM, IgG1, IgG2, and IgG3) over time

[0269] ·Percentage change in serum concentration of Ig classes (IgG, IgA, IgE, IgM, IgG1, IgG2, and IgG3) over time relative to baseline

[0270] ·Concentration of REGN5459 or REGN5458 in serum

[0271] ·Change over time in the incidence of antidrug antibodies against REGN5459 or REGN5458 that develop during treatment

[0272] Preliminary results: A total of 7 highly sensitized participants with chronic kidney disease requiring hemodialysis received REGN5459 administration at full doses of 1.5 mg to 5 mg. All participants enrolled in the 5 mg cohort ( Figure 1 squares, triangles, circles) showed a decrease in total serum IgG concentration relative to baseline ( Figure 1 ).

[0273] Example 3: Evaluation of renal transplant recipients previously desensitized with anti-BCMA x anti-CD3 bispecific antibody

[0274] Non-interventional extension study of patients who received renal transplantation and were treated with REGN5459 or REGN5458 (BCMA x CD3 bispecific antibody) in the study discussed in Example 2

[0275] Objective: To explore primary and secondary endpoints

[0276] This study Primary objectives is to evaluate adverse events (AEs) and serious adverse events (SAEs) in renal transplant recipients previously treated with REGN5459 or REGN5458 in the study discussed in Example 2

[0277] This study Secondary objectives is to evaluate the following in renal transplant recipients previously treated with REGN5459 or REGN5458

[0278] · Incidence and classification of antibody-mediated and T cell-mediated renal allograft rejection

[0279] · Graft survival

[0280] · Allograft function

[0281] · Delayed allograft function

[0282] · Levels of anti-human leukocyte antigen (HLA) alloantibodies and calculation of panel reactive antibody (cPRA)

[0283] · Appearance of de novo donor-specific antibodies

[0284] · Circulating immunoglobulin (Ig) classes (isotypes)

[0285] · Pharmacokinetics (PK) of REGN5459 or REGN5458

[0286] Study design: All patients who were treated with REGN5459 or REGN5458 and received a kidney transplant in the study discussed in Example 2 can be enrolled in this study and will be followed up for AE, clinical outcomes, and biomarkers as two cohorts / treatment groups (patients who previously received REGN5459 and patients who received REGN5458). The study drug will no longer be administered.

[0287] Study duration: This study will continue until the last patient completes the 12-month follow-up assessment after transplantation, or until all patients discontinue the study.

[0288] Study population: Six to twelve patients are expected to be enrolled in this study to collect safety and outcome data of patients aged 18 to 70 years who received a kidney transplant and were administered REGN5459 or REGN5458 in the study discussed in Example 2.

[0289] Inclusion criteria - Patients must meet the following criteria to be eligible for inclusion in the study:

[0290] 1. Received at least 1 dose of treatment with REGN5459 or REGN5458 in the study discussed in Example 2

[0291] 2. Received or planned to receive a kidney transplant after an acceptable crossmatch at the time of enrollment in the study discussed in Example 2

[0292] 3. Willing and able to comply with clinical visits and study-related procedures

[0293] 4. Provide an informed consent form signed by the study patient or the legally authorized representative

[0294] Exclusion criteria - There are no exclusion criteria for this study.

[0295] Study endpoints:

[0296] The primary endpoint of this study The incidence of AEs and SAEs within 12 months from the start of kidney transplantation.

[0297] Of this study The secondary endpoints were to evaluate the following post-transplantation:

[0298] · The incidence, time to diagnosis, and response to therapy at 12 months for each of the following biopsy-proven kidney allograft rejection types according to the Banff classification:

[0299] ○ Active antibody-mediated rejection (AMR) (Banff classification, class 2)

[0300] ○ Chronic active AMR (Banff classification, class 2)

[0301] ○ Acute T cell-mediated rejection (TCMR) (Banff classification, class 4)

[0302] ○ Chronic active TCMR (Banff classification, class 4)

[0303] · The incidence and timing of graft failure (defined as dialysis dependence) at 12 months

[0304] · Changes in estimated glomerular filtration rate (eGFR) at 1, 2, 3, 6, and 12 months

[0305] · The incidence of delayed graft function (defined as dialysis use within 7 days after transplantation)

[0306] · Changes in anti-HLA alloantibodies (measured using single antigen beads [SAB]) compared to pre-transplant levels (%, and mean fluorescence intensity [MFI]) at 2, 3, 6, and 12 months and at the clinical onset of suspected allograft rejection

[0307] · Changes in cPRA at 1, 2, 3, 6, and 12 months

[0308] · Changes in donor-specific anti-HLA alloantibodies (measured using SAB and donor HLA type) compared to the recipient's pre-transplant anti-HLA alloantibody levels (%, and mean fluorescence intensity [MFI]) at 1, 2, 3, 6, and 12 months and at the clinical onset of suspected allograft rejection

[0309] · The cumulative incidence of de novo anti-HLA alloantibody appearance measured by SAB at 12 months

[0310] ·Changes in the circulating serum concentration of Ig classes (IgG, IgA, and IgM) over time

[0311] ·Percentage change in the circulating serum concentration of Ig classes (IgG, IgA, and IgM) over time relative to baseline

[0312] ·Concentration of REGN5459 or REGN5458 in serum for up to 12 months

[0313] Results: The incidence of AEs and SAEs after kidney transplantation was comparable to that in the population of kidney transplant patients who did not receive REGN5459 or REGN5458, and graft failure and graft function were comparable to those in the population of kidney transplant patients who did not receive REGN5459 or REGN5458.

[0314] Example 4: REGN5459 causes plasma cell depletion and reduces serum Ig levels in humanized mice

[0315] This example demonstrates T cell-mediated killing of plasma cells (PCs) and reduction of serum Ig levels by the human BCMAxCD3 bispecific antibody (REGN5459), which binds to BCMA on PCs via the targeting arm and to CD3 on T cells via the effector arm. The activity of REGN5458 was evaluated in genetically humanized BCMAhu / hu / CD3hu / hu mice (US11,384,153) ( Figure 2A ).

[0316] Administration of the BCMAxCD3 bispecific antibody resulted in a robust and statistically significant reduction in bone marrow plasma cells (BMPCs), which was comparable at all doses between 5 mg / kg and 25 mg / kg and was equivalent among different isotype-specific PC subsets ( Figure 2B , Figure 3A and 3B ). Importantly, splenic B cells (including antigen-experienced CD38+IgD− follicular B cells) were not significantly affected by the BCMAxCD3 bispecific antibody ( Figure 4 ). Plasma cell depletion was reflected by a significant reduction in serum IgA, IgM, and IgG1 levels, and the magnitude of the reduction matched their expected half-lives. ( Figure 5A , 5B and 5C). This finding is consistent with IgA having the shortest half-life in circulation relative to IgM and IgG1, which results in a ~10- to 20-fold reduction in pre-existing IgA within one week after elimination of its source.

[0317] In a second experiment, the effect of the BCMAxCD3 bispecific antibody on antigen-specific Ig was evaluated in a 15-week model of intranasal house dust mite (HDM) exposure (Asrat et al. 2020, Sci Immunol 5)( Figure 6A ). Within two weeks of administration of the bispecific antibody, all BMPCs (including IgE+BMPCs) were almost completely depleted( Figure 6B and 6C ). Total circulating IgA and IgM, as well as IgG1 levels, were evaluated and found to be transiently reduced after treatment with the BCMAxCD3 bispecific antibody.

[0318] Thus, the BCMA x CD3 bispecific antibody effectively depletes (transiently) plasma cells (including IgE plasma cells) but not antigen-experienced B cells in BCMAhu / huCD3hu / hu mice.

[0319] Example 5: Effect of BCMAxCD3 bispecific antibody on simian plasma cells and circulating antibody levels

[0320] The effect of the BCMAxCD3 bispecific antibody (known to also bind simian BCMA (Haber et al. 2021, Sci Rep 11, 14397)) alone or in combination with a simian IL-4Ra antibody (REGN646) was evaluated in cynomolgus monkeys( Figure 7A ). A single dose of the BCMAxCD3 bispecific antibody led to a rapid and robust reduction in BMPCs( Figure 7B ). Consistent with the observations in mice (in Example 4), this reduction in BMPCs was closely associated with a rapid reduction in IgE and IgA levels, which reached a nadir 5 weeks after administration of the BCMAxCD3 bispecific antibody. Notably, after the nadir, IgA levels gradually recovered in animals treated with the BCMAxCD3 bispecific antibody alone and in combination with anti-IL-4Ra, while IgE levels recovered in animals treated with the BCMAxCD3 bispecific antibody alone but not in animals treated with the combination of BCMAxCD3 bispecific antibody and anti-IL-4Ra.

[0321] Example 6: Effect of BCMAxCD3 bispecific antibody on human serum immunoglobulins

[0322] The effect of weekly administration of the BCMAxCD3 bispecific antibody (REGN5458) was evaluated in patients with multiple myeloma( Figure 8A ). REGN5458 led to a robust reduction in immunoglobulin levels over time, with IgE levels being completely reduced as early as 4 weeks after treatment

[0323] ( Figure 8C)。Between 4 and 8 weeks, the IgG level was also depleted by more than 50%,

[0324] consistent with the half-life in the human body ( Figure 8B ).

[0325] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description, other than those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0326] *****

[0327] Table 7: Sequences excluded from the ST.26 format sequence listing

[0328] SEQ ID NO: Sequence 7 AAS

Claims

1. A method for reducing the level of allospecific antibodies in a subject in need of a solid organ transplant, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells, and a second antigen-binding domain that specifically binds to human CD3 on T cells.

2. The method according to claim 1, wherein reducing the level of allospecific antibodies in the subject comprises reducing the level of allospecific antibodies to below the baseline allospecific antibody level measured prior to administering the bispecific antibody or antigen-binding fragment thereof.

3. The method according to claim 1 or 2, further comprising measuring the baseline allospecific antibody level in the subject prior to administering the bispecific antibody or antigen-binding fragment thereof.

4. The method according to claim 2 or 3, wherein the allospecific antibody level and / or the baseline allospecific antibody level is measured by a single antigen bead assay, and the allospecific antibody level and / or the baseline allospecific antibody level corresponds to the mean fluorescence intensity of the peak immunodominant anti-HLA antibodies.

5. The method according to claim 4, wherein the reduction in the allospecific antibody level corresponds to a reduction in the mean fluorescence intensity of the peak immunodominant anti-HLA antibodies of ≥ 50% relative to the baseline allospecific antibody level.

6. The method according to claim 1, wherein the allospecific antibody level is measured by a single antigen bead assay, and the reduction in the allospecific antibody level corresponds to a reduction in the mean fluorescence intensity of the peak immunodominant anti-HLA antibodies to < 5000.

7. A method for reducing the level of calculated panel reactive antibodies (cPRA) in a subject in need of a solid organ transplant, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells, and a second antigen-binding domain that specifically binds to human CD3 on T cells.

8. The method according to claim 7, wherein reducing the cPRA level in the subject comprises reducing the cPRA level in the subject to below the baseline cPRA level measured prior to administering the bispecific antibody or antigen-binding fragment thereof.

9. The method according to claim 7 or 8, wherein the cPRA level in the subject: (a) is reduced to < 99%; (b) is reduced to < 98%; (c) is reduced to < 97%; (d) is reduced to < 96%; (e) is reduced to < 95%; (f) is reduced to < 94%; (g) is reduced to < 93%; (h) is reduced to < 92%; (i) is reduced to < 91%; (j) is reduced to < 90%; (k) is reduced to < 89%; (l) is reduced to < 88%; (m) is reduced to < 87%; (n) is reduced to < 86%; (o) is reduced to < 85%; (p) is reduced to < 84%; (q) is reduced to < 83%; (r) is reduced to < 82%; (s) is reduced to < 81%; or (t) is reduced to <80%.

10. A method for reducing the sensitivity of a subject to anti-HLA antibodies prior to solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells and a second antigen-binding domain that specifically binds to human CD3 on T cells, wherein the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population with a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation.

11. The method of claim 10, wherein the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population with a cPRA level of 80% prior to transplantation.

12. The method of claim 11, wherein the risk of rejection in the subject at the time of organ transplantation is not greater than the risk of rejection in a control population with a cPRA level of 50% to <80% prior to transplantation.

13. A method for reducing the risk of allograft rejection in a subject after solid organ transplantation, the method comprising administering to the subject a bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on plasma cells and a second antigen-binding domain that specifically binds to human CD3 on T cells, wherein the risk of rejection in the subject during the post-transplantation interval is not greater than the risk of rejection in a control population with a calculated panel reactive antibody (cPRA) level of 90% prior to transplantation.

14. The method of claim 13, wherein the risk of rejection in the subject during the post-transplantation interval is not greater than the risk of rejection in a control population with a cPRA level of 80% prior to transplantation.

15. The method of claim 14, wherein the risk of rejection in the subject during the post-transplantation interval is not greater than the risk of rejection in a control population with a cPRA level of 50% to <80% prior to transplantation.

16. The method of any one of claims 13-15, wherein the post-transplantation interval is a 3-month interval starting one day after organ transplantation.

17. The method of any one of claims 13-15, wherein the post-transplantation interval is a 6-month interval starting one day after organ transplantation.

18. The method of any one of claims 13-15, wherein the post-transplantation interval is a 12-month interval starting one day after organ transplantation.

19. The method of any one of claims 13-18, wherein the graft function in the subject remains at a functional level equal to or higher than the functional level of the control population during the post-transplantation interval.

20. The method of any one of claims 1-19, wherein the subject is a human.

21. The method of any one of claims 1-20, wherein the solid organ is selected from the group consisting of kidney, lung, pancreas, or heart.

22. The method of claim 21, wherein the solid organ is a kidney.

23. The method according to any one of claims 1-22, wherein the subject has chronic kidney disease and requires hemodialysis.

24. The method according to any one of claims 1-23, wherein the first antigen-binding domain comprises: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), which are contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:1; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), which are contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

5.

25. The method according to claim 24, wherein the first antigen-binding domain comprises HCDR1 having the amino acid sequence of SEQ ID NO:2, HCDR2 having the amino acid sequence of SEQ ID NO:3, and HCDR3 having the amino acid sequence of SEQ ID NO:

4.

26. The method according to claim 24 or 25, wherein the first antigen-binding domain comprises LCDR1 having the amino acid sequence of SEQ ID NO:6, LCDR2 having the amino acid sequence of SEQ ID NO:7, and LCDR3 having the amino acid sequence of SEQ ID NO:

8.

27. The method according to claim 24, wherein the first antigen-binding domain comprises an HCVR having the amino acid sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence of SEQ ID NO:

5.

28. The method according to any one of claims 1-27, wherein the second antigen-binding domain comprises: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), which are contained within a heavy-chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:13; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), which are contained within a light-chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

5.

29. The method according to claim 28, wherein the second antigen-binding domain comprises: (a) HCDR1 having the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:14; (b) HCDR2 having the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:15; and (c) HCDR3 having the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:

16.

30. The method according to claim 28 or 29, wherein the second antigen-binding domain comprises an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO:

8.

31. The method according to claim 30, wherein the second antigen-binding domain comprises: (a) an HCDR1, HCDR2, and HCDR3 domain having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; and an LCDR1, LCDR2, and LCDR3 domain having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; or (b) an HCDR1, HCDR2, and HCDR3 domain having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; and an LCDR1, LCDR2, and LCDR3 domain having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

32. The method according to claim 31, wherein the second antigen-binding domain comprises: (a) an HCVR having the amino acid sequence of SEQ ID NO: 9 and an LCVR having the amino acid sequence of SEQ ID NO: 5; or (b) an HCVR having the amino acid sequence of SEQ ID NO: 13 and an LCVR having the amino acid sequence of SEQ ID NO:

5.

33. The method according to any one of claims 1-23, wherein: (a) the first antigen-binding domain comprises an HCDR1, HCDR2, and HCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and an LCDR1, LCDR2, and LCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding domain comprises an HCDR1, HCDR2, and HCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and an LCDR1, LCDR2, and LCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

34. The method according to any one of claims 1-23, wherein: (a) the first antigen-binding domain comprises an HCDR1, HCDR2, and HCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and an LCDR1, LCDR2, and LCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; and (b) the second antigen-binding domain comprises an HCDR1, HCDR2, and HCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and an LCDR1, LCDR2, and LCDR3 domain consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

35. The method according to claim 33, wherein: (a) the first antigen-binding domain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO:1 and a LCVR consisting of the amino acid sequence of SEQ ID NO:5; and (b) the second antigen-binding domain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO:9 and a LCVR consisting of the amino acid sequence of SEQ ID NO:

5.

36. The method according to claim 34, wherein: (a) the first antigen-binding domain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO:1 and a LCVR consisting of the amino acid sequence of SEQ ID NO:5; and (b) the second antigen-binding domain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO:13 and a LCVR consisting of the amino acid sequence of SEQ ID NO:

5.

37. The method according to any one of claims 1-36, wherein the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a human IgG heavy chain constant region.

38. The method according to claim 37, wherein the bispecific antibody comprises a heavy chain having a constant region comprising the amino acid sequence of SEQ ID NO:

33.

39. The method according to claim 37 or 38, wherein the bispecific antibody comprises a heavy chain having a constant region comprising the amino acid sequence of SEQ ID NO:

34.

40. The method according to claim 37, wherein the human IgG heavy chain constant region is isotype IgG1.

41. The method according to claim 37, wherein the human IgG heavy chain constant region is isotype IgG4.

42. The method according to claim 40 or claim 41, wherein the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

43. The method according to any one of claims 1-36, wherein the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain having the amino acid sequence of SEQ ID NO:29, a second heavy chain having the amino acid sequence of SEQ ID NO:30, and a common light chain having the amino acid sequence of SEQ ID NO:

32.

44. The method according to any one of claims 1-36, wherein the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain having the amino acid sequence of SEQ ID NO:29, a second heavy chain having the amino acid sequence of SEQ ID NO:31, and a common light chain having the amino acid sequence of SEQ ID NO:

32.

45. The method according to any one of claims 1-44, wherein the bispecific antibody is administered according to a dosing regimen comprising a fractional initial dose.

46. The method according to any one of claims 1-45, wherein the bispecific antibody is administered to the subject at a dose of 0.05 mg to 150 mg per week.

47. A dosing regimen for the method according to any one of claims 1-46, wherein the dosing regimen comprises administering a bispecific antibody to the subject at an initial dose in the first week of the dosing regimen, at a second dose in the second week of the dosing regimen, and at a third dose in the third week of the dosing regimen, wherein the third dose is equal to or greater than the second dose, and the second dose is greater than the initial dose.

48. The dosing regimen according to claim 47, wherein the initial dose is 0.05 mg to 5 mg.

49. The dosing regimen according to claim 47 or 48, wherein the second dose is 0.15 mg to 25 mg.

50. The dosing regimen according to any one of claims 47-49, wherein the third dose is 0.5 mg to 150 mg.

51. The dosing regimen according to any one of claims 47-50, wherein the initial dose is 0.05 mg, 0.15 mg, 0.5 mg, 1 mg, 1.5 mg or 5 mg.

52. The dosing regimen according to any one of claims 47-51, wherein the second dose is 0.15 mg, 0.5 mg, 1.5 mg, 3 mg, 5 mg, 15 mg or 25 mg.

53. The dosing regimen according to any one of claims 47-52, wherein the third dose is 0.5 mg, 1.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 40 mg, 50 mg or 150 mg.

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