Methods for reducing alloantibody levels in a subject requiring a solid organ transplant

Abstract

B-cell maturation antigen (BCMA) is expressed on plasma cells. The present invention provides a method for desensitizing patients requiring solid organ transplantation to anti-HLA antibodies using a bispecific antibody that binds to both BCMA and CD3 and activates T cells via the CD3 complex in the presence of BCMA-expressing plasma cells. In certain embodiments, the bispecific antibody of the present invention reduces alloantibody levels and / or reduces calculated panel-reactive antibody levels to facilitate solid organ transplantation and / or improve long-term graft survival and / or function.

Description

[Technical Field]

[0001] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 11294WO01_Sequence, created on October 8, 2023, and containing 48,180 bytes.

[0002] The present invention relates to the pharmaceutical field and relates to bispecific antibodies (and antigen-binding fragments thereof) that bind to BCMA and CD3, and methods of using them to desensitize patients to anti-HLA alloantibodies, for example, prior to solid organ transplantation. [Background technology]

[0003] 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 distinct stages of development. BCMA is expressed exclusively in B-cell lineage cells, particularly in the interfollicular regions of germinal centers, as well as plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is required for optimal survival of long-lived plasma cells in the bone marrow.

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four distinct chains: epsilon, zeta, delta, and gamma. CD3 dimer configurations include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby triggering T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes, including T cell activation. Furthermore, bispecific antibodies capable of binding to CD3 and a target antigen have been proposed for therapeutic uses, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0005] Kidney transplantation is the preferred treatment for end-stage renal disease. Hemodialysis is lifesaving in the short term, but the 1- and 5-year mortality rates are high (25% and 65%), respectively, whereas the 5-year mortality rate for patients treated with kidney transplantation is low (3%). However, for patients who already have high levels of anti-human leukocyte antigen (HLA) antibodies, organ transplantation is rarely performed due to the increased risk of antibody-mediated rejection (AMR) and shorter graft survival. Approximately 30% of the approximately 95,000 patients on the kidney transplant waiting list in the United States are HLA-sensitized from previous transplants, blood transfusions, and pregnancies. Approximately half of these (approximately 13,000 patients) are considered highly sensitized (i.e., predicted to react to 80% or more of the donor HLA types). The calculated panel reactive antibody (cPRA) score represents the probability of encountering an incompatible donor for a potential organ transplant and is used as a measure of sensitization level. Although kidney allocation systems (KAS) have improved transplant rates for highly sensitized individuals overall, 30%–50% of patients with cPRA scores of 90% or higher remain on the kidney transplant waiting list for over 5 years, highlighting the unmet need for this subset of sensitized patients. Of the approximately 11,000 chronic kidney disease (CKD) patients with cPRA scores of 90% or higher on kidney transplant waiting lists in the United States, the majority (approximately 7,000) have cPRA scores of 99% or higher. These patients have a particularly high unmet need, as evidenced by more frequent removal from the waiting list and death due to medical comorbidities. Furthermore, for the approximately 2,000 patients on the kidney transplant waiting list with cPRA scores of 99.9% or higher, the average transplant rate is significantly lower than that of other HLA-sensitized patients, despite the implementation of KAS in 2014. Therefore, desensitization strategies to promote transplant success in these patients are a high priority.

[0006] Transplantation options for individuals sensitized to multiple or common HLAs are limited by the risk of AMR and subsequent graft loss when receiving an HLA-incompatible kidney. The pathogenesis of AMR involves antibodies against HLAs produced by plasma cells and, in some cases, B cells. Other effector molecules, including cytokines and complement, have also been shown to contribute to renal pathology. Antibody-mediated rejection manifests as hyperacute rejection, occurring within minutes of vascular anastomosis and often resulting in graft loss within hours. More commonly, AMR manifests as acute / active and chronic forms that develop over months or years. While hyperacute rejection is rare due to improved HLA screening and cross-matching, acute / active AMR rates have been reported to be as high as 40%–45% in HLA-sensitive patients, despite the use of plasma exchange and intravenous immunoglobulin (IVIG) desensitization techniques. While acute AMR can often be managed with short-term immunosuppression, it is a strong risk factor for the development of chronic AMR, the most common cause of graft loss in the United States. The Deterioration in Kidney Allograft Function (DeKAF) study showed that most patients with graft loss had evidence of chronic AMR, including deposition of the C4 complement component in biopsies (Matas et al., Am J Transplant, 18(5):1140-1150, 2018). Furthermore, the risk of chronic AMR is fourfold increased in previous transplant recipients who are highly HLA-sensitized, even after waiting for an HLA-matched kidney (Schinstock et al., Transplantation, 101(10):2429-2439, 2017). Sensitization and the presence of anti-HLA alloantibodies have also been implicated in 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.Thus, there remains a high unmet need for more effective therapies to promote engraftment and prevent post-transplant acute and chronic AMR in highly HLA-sensitized individuals. Summary of the Invention

[0007] The present disclosure generally relates to methods for safely depleting B-cell maturation antigen (BCMA)-expressing plasma cells and reducing anti-HLA alloantibodies to facilitate solid organ transplants (e.g., kidney transplants) using anti-BCMA x anti-CD3 bispecific antibodies (e.g., REGN5459 or REGN5458). By eliminating the source of anti-HLA alloantibodies, plasma cell-targeted therapy (described herein) has a longer-lasting HLA desensitization effect, facilitating transplantation and reducing the risk of post-transplant AMR. Anti-BCMA x CD3 antibodies (e.g., REGN5459 and REGN5458) target the surface protein BCMA, which is 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 impairing regulatory T cell responses that promote post-transplant tolerance. The restricted expression pattern of BCMA in tissues makes it an attractive therapeutic target for preventing AMR.

[0008] In one aspect, the invention provides a method of reducing alloantibody levels in a subject in need of a solid organ transplant, the method comprising administering to the subject a 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.

[0009] In some embodiments, reducing the alloantibody level in the subject comprises reducing the alloantibody level below a baseline alloantibody level measured before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the method further comprises measuring a baseline alloantibody level in the subject before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the alloantibody level and / or baseline alloantibody level are measured by a single-antigen bead assay, and the alloantibody level and / or baseline alloantibody level corresponds to a peak immunodominant anti-HLA antibody mean fluorescence intensity. In some cases, the decrease in alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity of 50% or more compared to the baseline alloantibody level.

[0010] In some embodiments, alloantibody levels are measured by a single antigen bead assay, and a decrease in alloantibody levels corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity to below 5000.

[0011] In one aspect, the invention provides a method for reducing calculated panel reactive antibody (cPRA) levels in a subject in need of a solid organ transplant, the method comprising administering to the subject a 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.

[0012] In some embodiments, reducing the subject's cPRA level comprises reducing the subject's cPRA level below a baseline cPRA level determined before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the subject's cPRA level is reduced by less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, or less than 80%.

[0013] In one aspect, the invention provides a method of reducing sensitization of a subject to anti-HLA antibodies prior to solid organ transplantation, comprising administering to the subject a 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 subject's risk of rejection upon organ transplantation is equal to or less than the risk of a control population having a pre-transplant calculated panel-reactive antibody (cPRA) level of 90%.

[0014] In some embodiments, the subject's risk of rejection at the time of organ transplant is equal to or less than the risk of a control population with a pre-transplant cPRA level of 80%. In some embodiments, the subject's risk of rejection at the time of organ transplant is equal to or less than the risk of a control population with a pre-transplant cPRA level of 50% to less than 80%.

[0015] In one aspect, the invention provides a method of reducing the risk of allograft rejection in a subject following solid organ transplantation, comprising administering to the subject a 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 subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population having a pre-transplant calculated panel-reactive antibody (cPRA) level of 90%.

[0016] In some embodiments, the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population with a pre-transplant cPRA level of 80%. In some embodiments, the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population with a pre-transplant cPRA level of 50% to less than 80%. In some cases, the post-transplant interval is a 3-month interval beginning one day after organ transplant. In some cases, the post-transplant interval is a 6-month interval beginning one day after organ transplant. In some cases, the post-transplant interval is a 12-month interval beginning one day after organ transplant.

[0017] In some embodiments, graft function is maintained in the subject at a functional level equal to or greater than that of a control population during the post-transplant interval.

[0018] In any of the various embodiments of the methods described above or discussed herein, the subject may be a human.

[0019] In any of the various embodiments of the method described above or discussed herein, the solid organ may be selected from the group consisting of kidney, lung, pancreas, or 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 more than 5 years.In some cases, the subject suffers from chronic kidney disease.In some cases, the subject is undergoing hemodialysis.In some cases, the subject is highly susceptible to end-stage renal failure that requires hemodialysis.

[0020] In any of the various embodiments of the methods described above or discussed herein, the bispecific antibody or antigen-binding fragment thereof may be as discussed herein below.

[0021] In some embodiments, the first antigen-binding domain comprises (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 5. In some cases, the first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4. In some cases, the first antigen-binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1, and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0022] 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) comprising 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) comprising the amino acid sequence of SEQ ID NO: 5. In some cases, the second antigen-binding domain comprises (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 15, and (c) an HCDR3 comprising 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 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some cases, the second antigen-binding domain comprises (a) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, or (b) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, the second antigen-binding domain comprises (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 9 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 13 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0023] 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.

[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: 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.

[0025] In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a human IgG heavy chain constant region. In some cases, the bispecific antibody comprises a heavy chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 33. In some cases, the bispecific antibody comprises a heavy chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 34. In some cases, the human IgG heavy chain constant region is of isotype IgG1. In some cases, the human IgG heavy chain constant region is of isotype IgG4. In some embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0026] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and a common light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0027] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 31, and a common light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0028] In any of the various embodiments of the methods described above or discussed herein, the bispecific antibody is administered in a dosing regimen comprising a divided initial dose, hi some embodiments, the bispecific antibody is administered to the subject once weekly at a dose of between 0.05 mg and 150 mg.

[0029] In one aspect, the invention provides a dosing regimen for use in any one of the embodiments of the method described above or discussed herein, wherein said dosing regimen comprises administering to said subject a bispecific antibody a first dose during week 1 of said dosing regimen, a second dose during week 2 of said dosing regimen, and a tertiary dose during week 3 of said dosing regimen, wherein said tertiary dose is equal to or greater than said second dose, and wherein the second dose is greater than the first dose.

[0030] In some embodiments, the initial dose is 0.05 mg to 5 mg. In some embodiments, the secondary dose is 0.15 mg to 25 mg. In some embodiments, the tertiary dose is 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 secondary dose is 0.15 mg, 0.5 mg, 1.5 mg, 3 mg, 5 mg, 15 mg, or 25 mg. In some cases, the tertiary dose is 0.5 mg, 1.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 40 mg, 50 mg, or 150 mg.

[0031] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another related value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0032] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 shows the reduction in serum IgG concentrations following treatment of highly sensitized chronic kidney disease patients with REGN5459, as discussed in Example 2. [Figure 2] Figures 2A and 2B show the experimental setup to assess in vivo targeting of plasma cells by BCMAxCD3 bispecific antibodies in BCMAhu / huCD3hu / hu mice (Figure 2A) and quantification of BMPCs (Figure 2B). [Figure 3] Figures 3A and 3B show the levels of IgA+ (Figure 3A) and IgM+ (Figure 3B) BMPCs upon administration of REGN5459 in BCMAhu / huCD3hu / hu mice. [Figure 4]FIG. 4 shows quantification of splenic antigen-experienced B cells 7 days after BCMA×CD3 bispecific antibody administration in BCMA hu / huCD3 hu / hu mice. [Figure 5] Figures 5A, 5B, and 5C show quantification of serum IgA (Figure 5A), IgM (Figure 5B), and IgG1 (Figure 5C) levels following BCMAxCD3 bispecific antibody administration in BCMAhu / huCD3hu / hu mice. [Figure 6-1] Figures 6A, 6B, and 6C show the study design showing 15 weeks of continuous HDM exposure with continuous anti-IL-4Ra administration starting at week 12 and transient BCMAxCD3 administration at week 15 (Figure 6A), quantification of IgE+ BMPC as a percent of viable cells (Figure 6B), and quantification of total BMPC (Figure 6C). [Figure 6-2] Same as above. [Figure 7] Figures 7A and 7B show the study design using cynomolgus monkeys (Figure 7A). Anti-IL-4Ra was administered weekly starting on day 1, and a single dose of BCMAxCD3 or isotype control antibody was administered on day 22. Bone marrow aspirates were collected on days 15 and 43. (Figure 7B) Flow cytometry quantification of BMPCs from bone marrow aspirates sampled at the indicated time points. [Figure 8] Figures 8A, 8B, and 8C show the mean concentrations of serum IgG (Figure 8B) and IgE (Figure 8C) levels over time in multiple myeloma patients receiving weekly BCMAxCD3 bispecific antibody at the indicated dose levels (Figure 8A). DETAILED DESCRIPTION OF THE INVENTION

[0034] Before describing the present invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific recited numerical value or range of values ​​means that the value can vary by 1% or less from the recited value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0036] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0037] definition As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence set forth in SEQ ID NO:23, human CD3-delta comprises the amino acid sequence set forth in SEQ ID NO:24, human CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:25, and CD3-gamma comprises the amino acid sequence set forth in SEQ ID NO:26. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless explicitly specified as being from a non-human species. Thus, the term "CD3" refers to human CD3 unless specified as being from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0038] As used herein, "antibodies that bind CD3" or "anti-CD3 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize dimeric complexes of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 protein as well as recombinant CD3 protein variants that lack the transmembrane domain or are otherwise not associated with the cell membrane, such as, for example, monomeric and dimeric CD3 constructs.

[0039] As used herein, the term "cell surface-expressed CD3" refers to one or more CD3 proteins expressed on the surface of a cell, either 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 accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes a CD3 protein contained in association with a functional T cell receptor on the cell membrane. The term "cell surface-expressed CD3" also includes a CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The term "cell surface-expressed CD3" also includes a CD3 chain expressed by itself, without other CD3 chain types, on the surface of a cell (e.g., CD3-epsilon, CD3-delta, or CD3-gamma). "Cell surface-expressed CD3" can include or consist of a CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0040] 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 set forth in SEQ ID NO: 22 and can also be found in GenBank accession number NP_001183.2.

[0041] As used herein, "antibodies that bind BCMA" or "anti-BCMA antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0042] "Plasma cells" are differentiated B lymphocytes capable of secreting antibodies, including alloantibodies.

[0043] "Human leukocyte antigens" (HLA) are cell surface proteins that present 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. Sensitization, the presence of anti-HLA antibodies, occurs in approximately 30% of patients requiring solid organ transplants. These alloantibodies develop after exposure to non-self HLA through pregnancy, blood transfusion, or previous organ transplantation.

[0044] "Alloantibodies" are antibodies produced in response to exposure to incompatible blood group antigens and are directed against non-self HLA proteins.

[0045] The "calculated panel reactive antibody" (cPRA) level is a value based on HLA antigens listed as non-sensitive for solid organ (e.g., kidney) transplant candidates. cPRA represents a method for determining a patient's risk of organ rejection prior to transplantation and is an estimate of the percentage of donors with whom a particular recipient would not be a match. A cPRA above 80% is considered highly sensitized.

[0046] As used herein, "subject" refers to an individual (e.g., a human) in need of a solid organ transplant (e.g., a kidney transplant) who is at risk of antibody-mediated rejection of the transplanted organ due to the presence of alloantibodies. The subject may also be an individual with chronic kidney disease or an individual with chronic kidney disease requiring hemodialysis.

[0047] As used herein, "chronic kidney disease" or CKD refers to kidney damage or a condition characterized by a rate of ≥ 60 mL / min / 1.73 m for at least 3 months. 2 CKD is a condition characterized by a decline in kidney function, either due to a decrease in glomerular filtration rate (GFR) below 1.73 m. The different stages of CKD define a continuum, classified as follows: Stage 1 (kidney damage with normal GFR), Stage 2 (GFR of 60-89 mL / min / 1.73 m). 2 stage 3a (mild decrease in GFR of 45-59 mL / min / 1.73 m 2 stage 3b (moderate decrease in GFR of 30-44 mL / min / 1.73 m 2 stage 4 (moderate decrease in GFR to 15-29 mL / min / 1.73 m 2 stage 1 (renal failure and GFR <15 mL / min / 1.73 m) 2 or dialysis).

[0048] As used herein, a "control population" refers to a group of individuals whose average calculated or measured parameters can be used as a comparison control. For example, a control population with a pre-transplant cPRA level at a specified value (e.g., 90%) refers to a group of individuals whose average cPRA level is at the specified value, and the cPRA level of the control population can be used as a control to evaluate the effect of antibody therapy (e.g., to reduce alloantibody levels or reduce the risk of antibody-mediated graft rejection), as discussed herein.

[0049] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0050] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex that contains 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" encompasses immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules that consist of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VVR herein). H The heavy chain constant region comprises three domains: C H 1. C H 2 and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are relatively conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the invention, the FRs of an anti-BCMA antibody or anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0051] As used herein, the term "antibody" also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "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 to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules using any suitable standard method, such as, for example, proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable regions and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0052] 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 mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0053] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may contain domains.

[0054] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the invention include: (i) a 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 H3, (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 may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (e.g., via disulfide bonds).

[0055] As with intact antibody molecules, antigen-binding fragments may be multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0056] The antibodies of the present invention can function through 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 present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC can be measured using assays well known and available in the art. (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 in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0057] In certain embodiments of the present invention, the anti-BCMA x anti-CD3 bispecific antibodies of the present invention are human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein 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 human framework sequences.

[0058] The antibodies of the present invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector (described in more detail below) transduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into 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 animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thereby altering the V and V regions of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Array and V L While derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0059] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, where the dimer is held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not linked via interchain disulfide bonds, and the approximately 75-80 kDa molecule is composed of covalently linked light and heavy chains (half antibodies). These forms have been very difficult to separate, even after affinity purification.

[0060] The frequency of occurrence of the second form in 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 a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for determining the frequency of occurrence of the second form in various intact IgG isotypes. H 2nd area or C H Antibodies with one or more mutations in three regions are included, which mutations may be desirable, for example, to improve the yield of the desired antibody form in production.

[0061] The antibody of the present invention may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0062] The anti-BCMA x anti-CD3 antibodies discussed herein may 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 compared to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found 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., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (in some cases), reduced immunogenicity, etc. Antibodies and antigen-binding fragments and those obtained in this general manner are encompassed by the present invention.

[0063] The present invention also includes anti-BCMAx anti-CD3 antibodies containing variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, with one or more conservative substitutions. For example, the present invention includes anti-BCMAx anti-CD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described herein, or the full-length heavy and light chain sequences described herein.

[0064] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on an antigen.

[0065] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, 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 antigen-binding fragment thereof is 90%, 95%, 96%, 97%, 98%, or 99% homologous to a sequence discussed herein (e.g., a CDR, HCVR, LCVR, heavy chain, or light chain sequence).

[0066] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" refer to two peptide sequences that share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence homology, when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weights. Preferably, non-homologous residue positions differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by 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 a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, 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: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0067] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, 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.

[0068] As used herein, the term "binding" in the context of binding of either an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0069] For example, binding affinities are typically about 10, as determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore 3000 instrument, with an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competitive 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).

[0070] Thus, the antibodies or antigen-binding proteins of the invention have a K that is at least 10-fold lower than the affinity for binding to a non-specific antigen (e.g., BSA, casein). D According to the present invention, the antibody binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is 10-fold or less than that of a non-specific antigen. D Although antibody affinities corresponding to values ​​may be considered undetectable binding, such antibodies may be paired with a second antigen-binding arm to generate bispecific antibodies of the invention.

[0071] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the 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, and therefore a smaller K DConversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and, therefore, a larger K D In some situations, the binding affinity (or K) of a particular molecule (e.g., an antibody) to an interaction partner molecule (e.g., antigen X) is used. D ) is higher compared to the binding affinity of the molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y), a larger K D The smaller the K value (lower, or weaker, affinity), the D This can be expressed as a binding ratio determined by dividing by a value (higher, or stronger, affinity), and in some cases can be expressed as, for example, 5-fold or 10-fold higher binding affinity.

[0072] "k d The term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment or molecular interaction. This value is expressed as k off Also called value.

[0073] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0074] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d It is obtained by dividing by

[0075] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of antibody that elicits a response halfway between baseline and maximum after a specified exposure time. 50 The EC essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC50 The EC value is equal to the concentration of an antibody of the invention that confers half-maximal binding to cells expressing CD3 or BCMA, as determined, for example, by a FACS binding assay. 50 Alternatively, the greater the half-maximal effective concentration value, the lower or weaker binding is observed.

[0076] In one embodiment, the reduction in binding is determined by measuring the EC2 concentration that allows binding to half the maximum amount of target cells. 50 It can be defined as an increase in antibody concentration.

[0077] In another embodiment, EC 50 The values ​​represent the concentration of the antibody of the present invention that induces half-maximal depletion of target cells by T cell cytotoxicity. Thus, an increase in cytotoxicity (e.g., T cell-mediated killing of plasma cells) is associated with an increase in EC 50 , or a decrease in half-maximal effective concentration values ​​is observed. A method for desensitizing patients to anti-HLA alloantibodies to enhance the success of solid organ transplantation.

[0078] The present disclosure provides methods for safely depleting B-cell maturation antigen (BCMA)-expressing cells (e.g., plasma cells) and reducing anti-HLA alloantibodies to facilitate solid organ transplants (e.g., kidney transplants) using anti-BCMA x anti-CD3 bispecific antibodies (e.g., REGN5459 or REGN5458). By removing the source of anti-HLA alloantibodies, plasma cell-targeted therapy (described herein) has a longer-lasting HLA desensitization effect, facilitating transplantation and reducing the risk of antibody-mediated rejection (AMR) after transplantation. Anti-BCMA x CD3 antibodies (e.g., REGN5459 and REGN5458) target the surface protein BCMA, which is 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 impairing regulatory T cell responses that promote tolerance after transplantation. Desensitization of a patient to anti-HLA alloantibodies can be measured, for example, by reducing alloantibody levels in the subject, by reducing calculated panel reactive antibody (cPRA) levels in the subject, or by comparing the subject's risk of rejection at the time of transplant or within a specified period after transplantation with a control population.

[0079] Desensitization can be achieved, for example, via a method of reducing alloantibody levels in a subject requiring a solid organ transplant, the method comprising administering to the subject a 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 connection with such methods are discussed in more detail herein.

[0080] In some embodiments, reducing the alloantibody level in the subject comprises reducing the alloantibody level below a baseline alloantibody level measured before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the method further comprises measuring a baseline alloantibody level in the subject before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the alloantibody level and / or baseline alloantibody level are measured by a single-antigen bead assay, and the alloantibody level and / or baseline alloantibody level corresponds to a peak immunodominant anti-HLA antibody mean fluorescence intensity. In some cases, the decrease in alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity (MFI) of 50% or more compared to the baseline alloantibody level. In some cases, the decrease in alloantibody levels indicates that the peak immunodominant anti-HLA antibody MFI is 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%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, %, or 99%, or at least a 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%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% decrease.

[0081] In some embodiments, the alloantibody level is measured by a single antigen bead assay, and the decrease in alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity to less than 5000. In some cases, the alloantibody level is measured by a single antigen bead assay, and the decrease in alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity to less than 10000 mcg, less than 9500, less than 9000, less than 8500, less than 8000, less than 7500, less than 7000, less than 6500, less than 6000, less than 5500, less than 5000, less than 4900, less than 4800, less than 4700, less than 4600, less than 4500, less than 4400, less than 4300, less than 4200, less than 4100, less than 4000, less than 3900, less than 3800, less than 3700, less than 3600, less than 3500. This corresponds to a decrease to less than 3400, less than 3300, less than 3200, less than 3100, less than 3000, less than 2900, less than 2800, less than 2700, less than 2600, less than 2500, less than 2400, less than 2300, less than 2200, less than 2100, less than 2000, less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1400, less than 1300, less than 1200, less than 1100, less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 490, less than 480, less than 470, less than 460, or less than 450.

[0082] Desensitization can be achieved, for example, via a method of reducing computed panel-reactive antibody (cPRA) levels in a subject requiring a solid organ transplant, comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) on cells (e.g., plasma cells) and a second antigen-binding domain that specifically binds to human CD3 on T cells. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in connection with such methods are discussed in more detail herein.

[0083] In some embodiments, reducing the subject's cPRA level comprises reducing the subject's cPRA level below a baseline cPRA level determined before administration of the bispecific antibody or antigen-binding fragment thereof. In some cases, the subject's cPRA level is reduced by less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, or less than 80%. In some cases, the subject's cPRA level may be 99.5% or less, 99% or less, 98.5% or less, 98% or less, 97.5% or less, 97% or less, 96.5% or less, 96% or less, 95.5% or less, 95% or less, 94.5% or less, 94% or less, 93.5% or less, 93% or less, 92.5% or less, 92% or less, 91.5% or less, 91% or less, 90.5% or less, 90% or less, 89.5% or less, 89% or less, 88.5% or less, 88% or less , 87.5% or less, 87% or less, 86.5% or less, 86% or less, 85.5% or less, 85% or less, 84.5% or less, 84% or less, 83.5% or less, 83% or less, 82.5% or less, 82% or less, 81.5% or less, 81% or less, 80.5% or less, 80% or less, 79.5% or less, 79% or less, 78.5% or less, 78% or less, 77.5% or less, 77% or less, 76.5% or less, 76% or less, 75.5% or less, 75% or less, 74.5 % or less, 74% or less, 73.5% or less, 73% or less, 72.5% or less, 72% or less, 71.5% or less, 71% or less, 70.5% or less, 70% or less, 69.5% or less, 69% or less, 68.5% or less, 68% or less , 67.5% or less, 67% or less, 66.5% or less, 66% or less, 65.5% or less, 65% or less, 64.5% or less, 64% or less, 63.5% or less, 63% or less, 62.5% or less, 62% or less, 61.5% or less, 6 1% or less, 60.5% or less, 60% or less, 59.5% or less, 59% or less, 58.5% or less, 58% or less, 57.5% or less, 57% or less, 56.5% or less, 56% or less, 55.5% or less, 55% or less, 54.5 % or less, 54% or less, 53.5% or less, 53% or less, 52.5% or less, 52% or less, 51.5% or less, 51% or less, 50.5% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25%.Decrease by 20% or less, 15% or less, or 10% or less.

[0084] The present disclosure also provides a method for reducing a subject's sensitization to anti-HLA antibodies prior to solid organ transplantation, the method comprising administering to the subject a 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 subject's risk of rejection upon organ transplantation is equal to or less than that of a control population having a pre-transplant calculated panel reactive antibody (cPRA) level of 90%. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in connection with such methods are discussed in more detail herein.

[0085] In some embodiments, the subject's risk of rejection at the time of organ transplant is equal to or less than the risk of a control population with a pre-transplant cPRA level of 80%. In some embodiments, the subject's risk of rejection at the time of organ transplant is equal to or less than the risk of a control population with a pre-transplant cPRA level of 50% to less than 80%. In some embodiments, the subject's risk of rejection at the time of organ transplantation is reduced by assessing whether pre-transplant cPRA levels are 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%, 75.5%, 75.5%, 76 ... 3.5%, 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% risk below that of the control population.

[0086] The present disclosure also provides a method for reducing the risk of allograft rejection in a subject after solid organ transplantation, comprising administering to the subject a 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 subject's risk of rejection during the post-transplant interval is equal to or less than that of a control population having a pre-transplant calculated panel-reactive antibody (cPRA) level of 90%. Exemplary bispecific antibodies and antigen-binding fragments thereof that can be used in connection with such methods are discussed in more detail herein.

[0087] In some embodiments, the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population with a pre-transplant cPRA level of 80%. In some embodiments, the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population with a pre-transplant cPRA level of 50% to less than 80%. In some embodiments, the subject's risk of rejection during the post-transplant interval is reduced by 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%, 75.5%, 75.5%, 76 ... 3.5%, 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%, The rejection risk is less than or equal to 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% of the control population. In some cases, the post-transplant interval is a 3-month interval beginning 1 day after organ transplantation. In some cases, the post-transplant interval is a 6-month interval beginning 1 day after organ transplantation. In some cases, the post-transplant interval is a 12-month interval beginning 1 day after organ transplantation.In various embodiments, the post-transplant interval is from 1 day after organ transplant to 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, 60 months, 61 months, 62 months, 63 months, 64 months, 65 months, 66 months, 67 months, 68 months, 69 months, 70 months, 71 months, 72 months, 73 months, 74 months, 75 months, 76 months, 77 months, 78 months, 79 months, 80 months, 81 months, 82 months, 83 months, 84 months, 85 months, 86 months, 87 months, 88 months, 89 months, 90 months, 91 months, 92 months, 93 months, 94 months, 95 months, 96 months, 97 months, 98 months, 99 months, 100 months, The periods ending in 0 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, and 60 months.

[0088] In some embodiments, graft function is maintained in the subject at a functional level equal to or greater than that of a control population during the post-transplant interval.

[0089] In any of the various embodiments of the methods described above or discussed herein, the subject may be a human.

[0090] In any of the various embodiments of the methods described above or discussed herein, the solid organ may be selected from the group consisting of a kidney, a lung, a pancreas, a heart, a colon or a portion thereof, a liver or a portion thereof, or skin. In some cases, the solid organ is a kidney.

[0091] The methods discussed herein address the high unmet need for facilitating solid organ transplants (e.g., kidney transplants) for highly HLA-sensitized patients. Using kidney transplantation as an example, due to the difficulty of finding a compatible organ donor, fewer than 10% of highly sensitized patients requiring a kidney receive a kidney transplant. Highly HLA-sensitized patients are frequently defined as having a cPRA of ≥80%. The cPRA score represents the probability of encountering an incompatible donor for a potential organ transplant and is used as a measure of sensitization level. While kidney allocation systems have improved transplant rates for highly sensitized individuals overall, 30% to 50% of patients with a cPRA score of ≥90% remain on the kidney transplant waiting list for over five years, highlighting the high unmet need for this subset of sensitized patients. Of the approximately 11,000 chronic kidney disease patients with a cPRA of ≥90% on the kidney transplant waiting list in the United States, the majority (approximately 7,000) have a cPRA score above 98%. These patients have a particularly high unmet need, as evidenced by more frequent removal from the waiting list and death due to medical comorbidities. Furthermore, for the approximately 2,000 patients on the kidney transplant waiting list with a cPRA of 99.9% or higher, the average transplant rate is significantly lower than for other HLA-sensitized patients, despite the implementation of a kidney allocation system in 2014. Highly HLA-sensitized patients spend years on the kidney transplant waiting list dependent on hemodialysis, which is associated with reduced quality of life, high rates of illness, and a mortality rate of up to 20%-25%. Therefore, treatments that increase the likelihood of kidney transplantation would be beneficial. In some cases, the methods discussed herein may be applied to subjects seeking solid organ transplants who have been on the waiting list (e.g., kidney transplant waiting list) for at least one year, at least two years, at least three years, at least four years, or at least five years or longer. While desensitization via the methods discussed herein does not necessarily result in organ transplantation due to limited availability, reducing the number of unacceptable HLA matches will improve each patient's chances of receiving a transplant.Additionally, organs are donated during the trial, and patients with an acceptable crossmatch can receive organ (e.g., kidney) transplants.

[0092] Plasma cells are promising targets for the treatment of antibody-mediated autoimmune and alloimmune diseases, including desensitization of highly HLA-sensitized individuals, which prevents safe transplantation. By eliminating the source of anti-HLA alloantibodies, plasma cell-targeted therapy may have a longer-lasting HLA desensitization effect than plasma exchange and other antibody reduction therapies. 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. While both REGN5459 and REGN5458 contain the same BCMA-binding arm, the difference between these two molecules is their respective affinity for CD3; REGN5459 binds to CD3 on T cells with lower affinity than REGN5458. In vitro, each of these bispecific antibodies induces activation of Jurkat T lymphocytes only in the presence of human BCMA-expressing cells. REGN5459 and REGN5458 induce T cell activation and cytotoxicity directed against tumor cells with a wide range of BCMA protein expression levels, and the BCMA cell surface expression level on target cells does not affect the efficacy of either REGN5459 or REGN5458. REGN5459 and REGN5458 can mediate killing of multiple cell lines and primary cells with varying BCMA cell surface expression, including normal plasma cells in bone marrow and secondary lymphoid organs, as demonstrated in healthy cynomolgus macaques. Due to its weaker affinity for CD3, REGN5459 is hypothesized to be less likely to induce CRS and have similar efficacy in plasma cell depletion.

[0093] Clinically relevant indicators used in sensitized patients requiring solid organ (e.g., kidney) transplants include donor-specific antibodies (DSA), particularly the range of anti-HLA alloantibody reactivity measured with the Luminex-based single antigen bead (SAB) assay. The SAB assay measures the reactivity of a patient's serum or plasma against a panel of HLA-coated beads, which is expressed as mean fluorescence intensity (MFI). By using a center-specific cutoff value and algorithm, HLA proteins are identified for each patient at which the individual has an unacceptable threshold level of preformed alloantibodies. The patient's own HLA genotype is thought to distinguish genuine alloreactivity from background fluorescence signals. Based on the number of unacceptable HLA matches and estimated HLA allele frequencies within the relevant population, a cPRA level (range: 0%–100%) representing the probability of encountering an incompatible donor for organ transplantation can be calculated and used as a measure of sensitization level.

[0094] Anti-HLA alloantibodies are well-characterized prognostic biomarkers that can predict clinical outcomes in solid organ (e.g., kidney) transplants, and anti-HLA monitoring is part of the consensus guidelines established by the Transplantation Society's Antibody Consensus Group. The risk of AMR and graft loss is significantly increased by the presence of anti-HLA alloantibodies against donor-specific HLAs above a certain threshold, expressed as MFI. A peak anti-HLA MFI above 3,000 is associated with a significant increase in AMR and decreased graft survival at 1, 3, and 5 years, whereas patients with anti-HLA MFIs between 465 and approximately 3,000 exhibited rates of AMR and graft survival similar to those of controls with no anti-HLA (highest MFI ≤ 465). Other studies have also found a significantly increased risk of post-transplant AMR in patients with pre-transplant peak anti-HLA MFIs above 3,000 or 8,000. Thus, there is a clear correlation between high peak anti-HLA MFI, AMR, and decreased graft survival. Anti-HLA MFIs above 3,000–5,000 are generally considered high-risk, and MFIs above 10,000 are almost universally considered unacceptable for transplants from patients with HLA types (without desensitization). Additionally, there are other less standardized DSA detection techniques, including measuring anti-HLA alloantibody titers by serially diluting serum samples for SAB assays, specific measurement of C1q-binding anti-HLA alloantibodies using a similar Luminex platform, and cell-based physical crossmatch assays for T cells (expressing class I HLA), B cells (expressing class II HLA), and endothelial progenitor cells (used to test reactivity to non-HLA antigens). Cell-based crossmatch assays can be flow cytometry-based and cytotoxicity-based.

[0095] Although plasma cells are the target cells for anti-BCMA x anti-CD3 antibodies (e.g., REGN5459 and REGN5458), the rarity of these cells in the circulation can limit reliable quantification. Therefore, Ig subclass concentrations (IgA, IgM, IgE, IgG, IgG1, IgG2, and IgG3) and pathogen-specific antibody titers can be monitored as potential surrogates for antibody-producing plasma cell levels. Hyposensitized patients treated according to the methods discussed herein and undergoing solid organ (e.g., kidney) transplants can be followed for a period of time (e.g., 12 months) after transplantation without additional bispecific anti-BCMA x CD3 antibodies to assess post-transplant outcomes. Graft function after transplantation (e.g., kidney transplantation) is a primary measure of transplant success, and the occurrence of graft loss 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 renal allograft pathology may be determined from kidney biopsies obtained as standard of care or cause of suspected rejection (e.g., in cases of rising serum creatinine, worsening hypertension, or increasing proteinuria). Anti-HLA MFI and cPRA may also be determined, as 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 is also assessed, as these viruses are common after transplantation and the impact of plasma cell-depleting therapy on their incidence is unknown.

[0096] In the context of kidney transplantation, the incidence of delayed graft function, defined as the need for dialysis within 7 days after transplantation, may be assessed because it is associated with higher acute rejection rates and is one of the strongest risk factors for chronic allograft nephropathy, leading to a 40% reduction in long-term graft survival. The incidence and classification of biopsy-proven TCMR and AMR may be monitored because both types of rejection alter graft histology and adversely affect graft survival. Furthermore, because 25%–45% of highly sensitized patients experience post-transplant AMR, and 80% of these AMR episodes occur in the first month after transplantation, preliminary assessment of the rate of AMR in sensitized transplant recipients previously treated with REGN5459 or REGN5458 may be performed. Finally, estimated glomerular filtration rate (eGFR) over time may be monitored as an important functional indicator of post-transplant graft function.

[0097] Combination therapy The bispecific anti-BCMA x anti-CD3 antibodies and antigen-binding fragments discussed herein may be combined with other therapeutic agents to desensitize patients to anti-HLA alloantibodies. Such combinations may include the use of IVIG and / or plasma exchange in addition to the bispecific antibody. Protocols combining plasma exchange with low-dose IVIG (100 mg / kg) have shown a transient reduction in anti-HLA levels and, in some cases, facilitated engraftment after repeated administration, depending on the anti-HLA alloantibody response. Other combinations include bispecific antibodies plus rituximab, interleukin (IL)-6 / IL-6 receptor antibody blockers (e.g., clazakizumab / tocilizumab), proteasome inhibitors (e.g., carfilzomib, bortezomib), other plasma cell-targeted therapies (e.g., daratumumab), and irifidase / IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS).

[0098] The present disclosure provides methods comprising administering a pharmaceutical composition comprising any of the exemplary bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. The additional therapeutically active ingredient may be administered immediately before, simultaneously with, or immediately after administration of the bispecific antigen-binding molecule of the present invention; (for purposes of this disclosure, such administration regimens are considered administration of the bispecific antigen-binding molecule "in combination with" the additional therapeutically active ingredient).

[0099] The present invention includes pharmaceutical compositions in which the bispecific antigen-binding molecules of the invention are co-formulated with one or more of the additional therapeutically active ingredients discussed elsewhere herein.

[0100] Bispecific antibodies and antigen-binding fragments thereof The present invention includes bispecific antigen binding molecules that specifically bind to CD3 and BCMA. Such molecules may be referred to herein as, for example, "anti-BCMA x anti-CD3," or "anti-CD3 / anti-BCMA," or "anti-CD3 x BCMA," or "CD3 x BCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3).

[0101] 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 BCMA-expressing cell killing. In other embodiments, the CD3 binding arm weakly binds or associates with human and cynomolgus monkey (monkey) CD3, but the binding interaction is not detectable by in vitro assays known in the art.

[0102] As used herein, the term "BCMA" refers to the human BCMA protein, unless specified to be from a non-human species (e.g., "mouse BCMA," "monkey BCMA," etc.). The human BCMA protein has the amino acid sequence set forth in SEQ ID NO:22.

[0103] The bispecific antigen-binding molecules that specifically bind to CD3 and BCMA may include anti-CD3 antigen-binding molecules that bind to CD3 with weak binding affinity, such as exhibiting a KD of greater than about 40 nM as measured by an in vitro affinity binding assay.

[0104] As used herein, the term "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity-determining region (CDR), alone or in combination with one or more additional CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. In certain embodiments, the antigen-binding molecule is an antibody or antibody fragment, as those terms are defined elsewhere herein.

[0105] As used herein, the term "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either 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, distinct antigen (e.g., CD3).

[0106] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context 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.

[0107] 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) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1, and (b) three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 5. In some cases, the isolated bispecific antigen-binding molecule comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4. In some cases, the isolated bispecific antigen-binding molecule comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1, and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0108] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a second antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising 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) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 5. In some cases, the second antigen-binding domain comprises (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 15, and (c) an HCDR3 comprising 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 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some cases, the second antigen-binding domain comprises (a) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, or (b) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively. In some cases, the second antigen-binding domain comprises (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 9 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 13 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0109] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising 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 comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising 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 comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 9 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0110] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising 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 comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising 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 comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 13 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5.

[0111] An exemplary bispecific antigen-binding molecule is a bispecific antibody comprising (a) a first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 9 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5. This antibody is also referred to herein as REGN5458.

[0112] An exemplary bispecific antigen-binding molecule is a bispecific antibody comprising (a) a first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 13 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5. This antibody is also referred to herein as REGN5459.

[0113] In certain exemplary embodiments, the isolated bispecific antigen binding molecule competes for binding to 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 comprising the amino acid sequence of SEQ ID NO: 1 / 5, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 9 / 5 or SEQ ID NO: 13 / 5.

[0114] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes for binding to 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 comprising the amino acid sequence of SEQ ID NO:1 / 5, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO:9 / 5 or SEQ ID NO:13 / 5.

[0115] The bispecific antigen-binding molecules described above or herein may be bispecific antibodies. In some cases, the bispecific antibodies comprise a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is of the isotype IgG1, IgG2, IgG3, or IgG4. In some cases, the human IgG heavy chain constant region is of the isotype IgG1. In some cases, the human IgG heavy chain constant region is of the isotype IgG4. In various embodiments, the bispecific antibodies comprise a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0116] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected 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 connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain can be connected to the immunoglobulin C. H The multimer-forming component may be a polypeptide comprising three domains. H 2~C H The Fc portion of an immunoglobulin (comprising three domains), for example, the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0117] The bispecific antigen-binding molecules of the present invention typically comprise two multimerization domains, e.g., two Fc domains, each of which is part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0118] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0119] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the present invention. For example, an antigen-binding molecule or fragment thereof having a first antigen-binding specificity can be operatively linked (e.g., by chemical conjugation, genetic fusion, or noncovalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to generate the bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in connection with 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, quadroma, knob-into-hole, common light chain (such as a common light chain with knobs-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a discussion of the foregoing formats).

[0120] In the context of the bispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid alterations (e.g., insertions, deletions, or substitutions) compared to a naturally occurring version of the wild-type Fc domain. For example, the present invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2nd area or C HThe FcRn-binding domain contains modifications in three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., 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 positions 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 positions 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 positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 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 and / or 308P).

[0121] The present invention also provides a first C H 3 domain and second Ig C H a bispecific antigen-binding molecule comprising three domains, a first and a second Ig C H In one embodiment, the three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or eliminates protein A binding, for example, an H95R modification (according to IMGT exon numbering; H435R according to EU numbering).H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Patent No. 8,586,713. In some embodiments, one heavy chain or the other heavy chain has both an H435R modification and a Y436F modification, but not both. H Contains 3 domains. The second C H Further modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0122] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4. HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be comprised in any of the antigen-binding molecules described herein comprises, from N- to C-terminus, [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be comprised in any of the antigen-binding molecules described herein comprises, from N- to C-terminus, [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in U.S. Patent Application Publication No. 2014 / 0243504, published August 28, 2014, and incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, thereby affecting Fc effector function. In various embodiments, the bispecific antigen-binding molecule can include a heavy chain constant region including a hinge domain, where positions 233-236 within the hinge domain can be G, G, unoccupied, and unoccupied, G, unoccupied, and unoccupied, or all unoccupied, as numbered by EU numbering.

[0123] Sequence variants The antibodies and bispecific antigen-binding molecules of the present invention may 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 compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may contain antigen-binding domains derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can easily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the residues found 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., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline variant sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (in some cases), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained by this general method are encompassed by the present invention.

[0124] pH dependent binding The present invention includes anti-BCMA x anti-CD3 bispecific antigen-binding molecules with pH-dependent binding properties. For example, antibodies of the present invention may exhibit decreased binding to BCMA at acidic pH compared to neutral pH. Alternatively, antibodies of the present invention may exhibit enhanced binding to BCMA at acidic pH compared to neutral pH. The term "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, and 5.0 or less. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "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.

[0125] In certain instances, "reduced binding at acidic pH compared to neutral pH" is expressed as the ratio of the K value of the antibody that binds to its antigen at acidic pH to the K value of the antibody that binds to that antigen at neutral pH (or vice versa). For example, an antibody or antigen-binding fragment thereof can be considered to exhibit "reduced binding to BCMA at acidic pH compared to neutral pH for the purposes of this disclosure if the antibody or antigen-binding fragment thereof exhibits an acidic / neutral K ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic / neutral K ratio for an antibody or antigen-binding fragment of the invention is D The ratio 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 more.

[0126] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modifying the antigen-binding domain in amino acid concentration can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody can be obtained that has reduced antigen binding at acidic pH compared to neutral pH. Antibodies containing Fc variants

[0127] According to certain embodiments of the present invention, there are provided anti-BCMA x anti-CD3 bispecific antigen binding molecules comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present invention provides an anti-BCMA x anti-CD3 bispecific antigen binding molecule comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HAntibodies containing mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0) can result in an extended serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 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 positions 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 positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 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 and / or 308P). All positions are listed in EU numbering.

[0128] For example, the present invention includes anti-BCMA x anti-CD3 bispecific antigen binding molecules comprising an Fc domain comprising one or more 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 in antibody variable domains disclosed herein, are contemplated as being within the scope of the present invention.

[0129] biological equivalent The methods of the present invention encompass antigen-binding molecules having amino acid sequences that differ from the exemplary molecules disclosed herein but that retain the ability to bind to BCMA and / or CD3. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.

[0130] The present invention encompasses administering an antigen-binding molecule that is bioequivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that exhibit no significant difference in the rate and extent of absorption when administered in either a single dose or multiple doses at the same molar dose under similar experimental conditions. Some antigen-binding proteins are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their absorption rate is not, and furthermore, such differences in absorption rate are considered bioequivalent because they are intentional, reflected in labeling, and are not considered medically significant for the particular pharmaceutical product being studied, for example, because they are not essential for achieving effective body drug concentrations in chronic use.

[0131] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0132] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy, compared to continuous therapy without such switching.

[0133] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms and to the known extent of such mechanisms for one or more conditions of use.

[0134] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0135] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by 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 substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent 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, for example, mutations that eliminate or remove glycosylation.

[0136] Therapeutic Formulations and Administration 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 improve transportation, delivery, tolerability, etc. Numerous suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0137] The dose of an antigen-binding molecule administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously in a single dose of typically about 0.01 to about 20 mg per kg of body weight, more preferably about 0.02 to about 7 mg, about 0.03 to about 5 mg, or about 0.05 to about 3 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering bispecific antigen-binding molecules can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0138] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods 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, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0139] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in 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 delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold in a pre-filled state, with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0140] A number of reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN, to name just a few. STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices that have application in subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR™ Pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name a few.

[0141] In certain circumstances, pharmaceutical compositions can be delivered in a controlled-release system. In one embodiment, a pump can be used (see Langer and Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201, supra). 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, a controlled-release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, 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.

[0142] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable solution prepared in this manner is preferably filled into appropriate ampoules.

[0143] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody per unit dose is generally about 0.05 mg to about 500 mg per dosage form, and particularly in the case of injections, it is preferably about 0.05 mg to about 150 mg.

[0144] Dosing regimen According to certain embodiments of the 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. The method according to this aspect of the invention comprises sequentially administering multiple doses of an antigen-binding molecule of the invention to a subject. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to a subject at a different time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient a single initial dose of the antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally, followed by one or more tertiary doses of the antigen-binding molecule.

[0145] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of an antigen-binding molecule of the present invention. Thus, an "initial dose" is a dose administered at the start of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses can all contain the same amount of antigen-binding molecule, but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecule contained in the initial, secondary, and / or tertiary doses differs from one another (e.g., adjusted accordingly) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the start of a treatment regimen, with subsequent doses administered less frequently (e.g., "maintenance doses"). In any of the embodiments, the initial dose (e.g., the first weekly dose) may be divided into two doses administered on separate days (e.g., consecutive days) spaced no more than three days apart. In any of the embodiments, the first nominal dose (i.e., the secondary dose) may be divided into two doses administered on separate days (e.g., consecutive days) spaced no more than three days apart. For example, if the initial or secondary dose is 1 mg, the dose may be divided into, for example, two 0.5 mg doses administered on consecutive days, or on separate days spaced no more than three days apart. In various embodiments, the dose (e.g., a weekly dose, as a single dose, or as two divided fractions of that dose) 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 , 0.8mg, 0.85mg, 0.9mg, 0.95mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 3.5mg, 4mg, 4.5mg, 5mg, 5.5mg, 6mg, 6.5mg, 7mg, 7. 5mg, 8mg, 8.5mg, 9mg, 9.5mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg,24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg , 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg ,74mg,75mg,76mg,77mg,78mg,79mg,80mg,81mg,82mg,83mg,84mg,85mg,86mg,87mg,88mg,89mg,90mg,91mg,92mg,93mg,94mg,95mg,96mg,97mg,98mg,99mg,100mg,,or at least,0.05mg,0.06mg,0.07mg,0.08mg,0.09mg,0.1mg,0.15mg,0.2mg,0.25mg,0.3mg,0.35mg,0.4mg,0.45mg,0.5mg,0.55mg,0.6 mg, 0.65mg, 0.7mg, 0.75mg, 0.8mg, 0.85mg, 0.9mg, 0.95mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 3.5mg, 4mg, 4.5mg, 5mg, 5.5mg, 6mg, 6.5mg, 7mg, 7.5mg, 8mg, 8 .5mg, 9mg, 9.5mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg,82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 510mg, 520mg, 530mg, 540mg, 550mg, 560mg, 570mg, 580mg, 590mg, 600mg, 610mg, 620mg, 630mg, 640mg, 650mg, 660mg, 670mg, 680mg, 690mg, 700mg, 710mg, 720mg, 730mg, 740mg, 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 mg, 970 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 administer the initial dose, secondary dose,Alternatively, a tertiary dose range may be defined and is encompassed within the scope of the present disclosure. In some embodiments, all doses are administered as a single administration (e.g., a single infusion), including the doses administered in weeks 1 and 2 of the dosing regimen. For example, an initial dose of 0.05 mg to 5 mg may be administered as a single dose in week 1, a secondary dose of 0.15 mg to 25 mg may be administered as a single dose in week 2, and a tertiary dose of 0.5 mg to 150 mg may be administered as a single dose in week 3. Additional doses may be administered weekly thereafter (e.g., at the same dose as the tertiary dose) for a period of time. In another example, an initial dose of 0.05 mg to 5 mg may be administered as a single dose in week 1, a secondary dose of 0.15 mg to 25 mg may be administered as a single dose in week 2, and a tertiary dose of 0.5 mg to 150 mg may be administered as a single dose in week 3. In another example, an initial dose of 0.05 mg to 1 mg may be administered as a single dose in week 1, a secondary dose of 0.15 mg to 5 mg may be administered as a single dose in week 2, and a tertiary dose of 0.5 mg to 40 mg may be administered as a single dose in week 3. In some cases, the dosing schedule may include administration thereafter (e.g., after weeks 1-3) weekly, every two weeks, every three weeks, monthly, or the like. [Example]

[0146] The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0147] Generation of bispecific antibodies that bind BCMA and CD3 The present invention uses bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind to CD3 and BCMA; such bispecific antigen-binding molecules are also referred to herein as "anti-BCMA x anti-CD3 or anti-CD3 x BCMA or anti-BCMA x anti-CD3 bispecific molecules." The anti-BCMA portion of the anti-BCMA x anti-CD3 bispecific molecule is useful for targeting plasma cells that express BCMA (also known as CD269), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of BCMA on plasma cells and CD3 on T cells promotes directed killing (cytolysis) of the targeted plasma cells by the activated T cells.

[0148] Bispecific antibodies comprising an anti-BCMA-specific binding domain and an anti-CD3-specific binding domain are constructed using standard methodologies, with the anti-BCMA antigen-binding domain and the anti-CD3 antigen-binding domain each consisting of a different HCVR paired with a common LCVR. In the illustrated bispecific antibody, the molecule was constructed using an anti-CD3 antibody heavy chain, an anti-BCMA antibody heavy chain, and a common anti-CD3 antibody light chain (e.g., SEQ ID NO: 5). In other examples, bispecific antibodies can be constructed using a heavy chain derived from an anti-CD3 antibody, a heavy chain derived from an anti-BCMA antibody, and a promiscuous antibody light chain or an antibody light chain known to effectively pair with various heavy chain arms. Further details regarding the anti-CD3 portion of bispecific antibodies can be found in WO 2017 / 053856, incorporated herein by reference.

[0149] [Table 1]

[0150] Tables 2A and 2B show the amino acid sequence identifiers of the bispecific anti-BCMA x anti-CD3 antibodies exemplified herein.

[0151] [Table 2A]

[0152] [Table 2B]

[0153] The REGN5458 bispecific antibodies identified in Tables 2A and 2B comprise a first heavy chain (containing a first antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain (containing a second antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 30, and a common light chain comprising the amino acid sequence of SEQ ID NO: 32. The first heavy chain of the REGN5458 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 33. The second heavy chain of the REGN5458 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 34. The common light chain of the REGN5458 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 35.

[0154] The REGN5459 bispecific antibody identified in Tables 2A and 2B comprises a first heavy chain (containing a first antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain (containing a second antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 31, and a common light chain comprising the amino acid sequence of SEQ ID NO: 32. The first heavy chain of the REGN5459 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 33. The second heavy chain of the REGN5459 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 34. The common light chain of the REGN5459 bispecific antibody comprises a constant region comprising the amino acid sequence of SEQ ID NO: 35. [Example]

[0155] Desensitization of patients with chronic kidney disease requiring kidney transplantation who are highly sensitive to human leukocyte antigen with anti-BCMA × anti-CD3 bispecific antibodies A phase 1 / 2 trial of a bispecific anti-BCMA × anti-CD3 antibody for desensitization of patients with chronic kidney disease requiring kidney transplantation who are highly sensitized to human leukocyte antigens (HLA).

[0156] Objectives: To explore both primary and secondary endpoints.

[0157] The primary objective of this study is to evaluate the safety and tolerability of REGN5459 (Part A) or REGN5458 (Part B) as monotherapy in patients with chronic kidney disease (CKD) who require a kidney transplant and are highly human leukocyte antigen (HLA) sensitized.

[0158] Secondary objectives of this study are to determine / evaluate the following for REGN5459 (Part A) or REGN5458 (Part B): (i) the dosing regimen(s) that result in a clinically meaningful reduction in anti-HLA alloantibody levels, (ii) the effect on calculated panel reactive antibody (cPRA) levels, (iii) the time to maximal and clinically meaningful reduction in anti-HLA alloantibody levels, (iv) the duration of effect of the study drug on reducing anti-HLA alloantibodies, (v) the effect on circulating immunoglobulin (Ig) classes (isotypes), (vi) pharmacokinetic (PK) properties, and (vii) immunogenicity.

[0159] Study Design: This is a Phase 1 / 2 study of REGN5459 (Part A) or REGN5458 (Part B) in patients with chronic kidney disease (CKD) who are on hemodialysis and are highly HLA-sensitized. A 3+3 dose-escalation design will be used for both Parts A and B to determine the dose level with acceptable safety, PK, and pharmacological effects. Part A will be initiated first, and Part B of the study may be initiated based on emerging safety data from Part A of the study.

[0160] Each patient will receive either REGN5459 (Part A) or REGN5458 (Part B) at the designated dose level in a cycle of three infusions over a 15-day period, as shown in Table 3, Part A or Table 4, Part B. Patients will be premedicated with a 40 mg intravenous (IV) dexamethasone infusion at least 1 hour before the start of each study drug infusion to limit the potential for cytokine release syndrome (CRS). If any infusion is not well tolerated, the dose for subsequent infusions may be modified for that patient and for patients enrolled in additional cohorts.

[0161] The planned study duration for each patient is 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 is cross-matched with a potential transplant donor). Patients who are successfully cross-matched with a potential transplant donor will be discontinued from the study and offered enrollment in a separate protocol (Example 3) for additional evaluation after kidney transplantation.

[0162] Safety will be assessed by the incidence and severity of treatment-emergent adverse events (TEAEs) graded by the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0 (NCI-C TCAE v5.0), except for CRS, which will be graded according to Table 5. Drug activity will be assessed by anti-HLA alloantibody mean fluorescence intensity (MFI) by single antigen bead (SAB) assay. Pharmacokinetics, anti-drug antibody (ADA), and pharmacodynamic (PD) measurements will be analyzed from serum / plasma and peripheral blood mononuclear cells before REGN5459 or REGN5458 infusion and at several time points thereafter.

[0163] At least three patients will be enrolled in each cohort. Additional patients (up to a total of six evaluable patients) may be enrolled in any given cohort to further explore safety, PK, and PD characteristics.

[0164] Modifications to the Part A and Part B dosing schemes (e.g., smaller dose increments between cohorts and / or dose increments between lower and higher doses within a cohort) may be investigated based on data observed in earlier cohorts.

[0165] [Table 3]

[0166] [Table 4]

[0167] Dose escalation will proceed for Parts A and B based on an evaluation of safety and any other data available at that time until an acceptable dose is determined or a maximum dose level is reached. The maximum total dose evaluated in this study is 150 mg of REGN5459 or 40 mg of REGN5458. However, if there is an occurrence of an adverse event of interest (AEI) in one patient or a Grade ≥ 2 adverse event (AE) (excluding Grade ≥ 2 AEs clearly unrelated to study drug) in two or more patients in a dose cohort during the 14-day safety observation period following the full dose, the dosing regimen will still be determined to be tolerable. Then, all dose escalations will be no more than two-fold (i.e., a 100% increase across all doses from the previously evaluated dose cohort).

[0168] Acute IRR is defined as any AE occurring within 6 hours of initiating the infusion or within 2 hours after completion of the infusion (whichever occurs later) and associated with typical signs and symptoms, including, but not limited to, tidal flow, tachycardia, hypotension, dyspnea, bronchospasm, back pain, fever, urticaria, edema, nausea, and rash. Cytokine release syndrome is a disorder characterized by fever, tachypnea, headache, tachycardia, hypertension, rash, and / or hypoxia, and in this study, is defined as such an event occurring 6 hours or more after initiating the infusion or 2 hours or more after completion of the infusion, whichever occurs later. Cytokine release syndrome grading and management guidelines, including clinical evaluation and monitoring, are detailed in Table 5.

[0169] [Table 5]

[0170] Study Duration: The planned study duration for each patient is 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 is cross-matched with a potential transplant donor). Patients who are successfully cross-matched with a potential transplant donor will be discontinued from this study and offered enrollment in a separate protocol (Example 3) for additional evaluation after kidney transplantation.

[0171] Study Population: There will be approximately 6-60 evaluable patients in the study, with 6-30 in each of Part A and Part B.

[0172] The study population consisted of adult patients aged 18–70 years with CKD requiring hemodialysis and awaiting kidney transplantation on the United Network for Organ Sharing (UNOS) list with a cPRA of 99.9% or greater, or adult patients with a cPRA of >98% (98.1%–99.8%) who had spent 5 years or more on the waiting list. Participation Criteria - Patients must meet the following criteria to be eligible for enrollment in this study: 1. 18 to 70 years old 2. Patients with CKD who require hemodialysis and are awaiting kidney transplantation on the UNOS list with a cPRA ≥ 99.9% or a cPRA > 98% (98.1%-99.8%) who have spent 5 years or more on the waiting list 3. Adequate hematological function as measured by: platelet count >50 x 10 9 / L. Patients 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 4. Adequate liver function, defined as: total bilirubin ≤ 1.5 x ULN*, transaminases (ALT, AST) ≤ 2.5 x ULN, alkaline phosphatase ≤ 2.5 x ULN (upper limit of normal) - *Patients with a documented history of Gilbert syndrome do not need to meet this total bilirubin requirement if their total bilirubin remains unchanged from baseline. 5. Willing and able to comply with clinic visit and study-related procedures 6. Provide informed consent signed by the study patient or legally acceptable representative

[0173] Exclusion Criteria - Patients who meet any of the following criteria or have any other medical condition that may prevent the safe administration of the study drug will be excluded from the study. 1. Current or active malignancy that has not been in remission for at least one year 2. History of CNS pathology or CNS neurodegenerative or movement disorder 3. Patients who have had their spleen removed, including those with functional apnea. 4. Patients who have undergone stem cell transplantation within the past 5 years 5. Body mass index ≥ 35 kg / m at screening 2 6. Hypogammaglobulinemia defined as total plasma IgG <300 mg / dL at screening 7. Continuous systemic corticosteroid treatment with prednisone (or anti-inflammatory equivalent) >10 mg per day within 72 hours of initiating study drug administration 8. Received calcineurin inhibitors (e.g., tacrolimus, cyclosporine) within 30 days of study drug administration 9. Received cyclophosphamide, rituximab, obinutuzumab, other anti-CD20 or B-cell depleting agents, or proteasome inhibitor or anti-CD38 therapy (e.g., isatuximab, daratumumab) within 6 months of study drug administration 10. Previous treatment with any anti-BCMA antibody (including antibody-drug conjugates or bsAbs) or BCMA-specific CAR-T cell therapy 11. Use of any investigational drug within 8 weeks or within 5 half-lives of study drug administration (whichever is longer) 12. Chest radiography evidence of active infection and adequate clinical evidence of infection (patients may be rescreened after a complete course of treatment and resolution of active infection) 13. Any infection requiring hospitalization or treatment with IV anti-infectives within 4 weeks of study drug administration 14. Human immunodeficiency virus (HIV) positive patients at screening 15. Incompletely treated latent or active tuberculosis infection 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 guidelines. Equivocal cases should be discussed with the Medical / Study Director prior to enrollment. 16. Patients with a history of hepatitis B and patients who test positive for hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (HBcAb) at screening 17. Patients with a history of hepatitis C or who are hepatitis C virus (HCV) antibody positive will be excluded. However, patients who are HCV antibody positive, have successfully completed a previous course of anti-HCV therapy, and have undetectable HCV ribonucleic acid (RNA) by polymerase chain reaction (PCR) will be accepted. 18. Vaccination with a replication-competent vector within 28 days prior to the first dose of study drug 19. Received COVID-19 vaccination within 1 week of starting the study drug schedule or did not complete the COVID-19 vaccination schedule 1 week before the study drug schedule 20. Recent recipient of licensed or investigational live / attenuated vaccines within 2 months of the screening visit. This includes, but is not limited to: adenovirus (Adenovirus Vaccine Live Oral Type 7); Calycella-Zoster Virus (VZV; Varivax®, Zostavax®); rotavirus vaccine (RotaShield®); yellow fever vaccine (YF-Vax®); measles and mumps vaccine (Measles and Mumps Virus Vaccine Live); measles, mumps, rubella vaccine (MMR® II); SIP vaccine; Sabin Oral Polio Vaccine; rabies vaccine (Imovax®, RabAvert®); FluMist® vaccine 21. History of severe allergic or acute hypersensitivity reactions due to previous IVIG, anti-cytokine therapy (e.g., tocilizumab), investigational vaccines, or prior drug treatment 22. Known hypersensitivity to any component of the formulated product 23. Patients who are institutionalized pursuant to an order issued by a judicial or administrative authority at the time of screening. 24. Members of the clinical trial site study team and / or their immediate family members, only prior to approval by the sponsor 25. Women who are pregnant or breastfeeding at the time of screening 26. Women of childbearing potential and men who are not willing to use highly effective contraception before the first dose / initiation of first treatment, during the study, and for at least 4 months after the last dose. For women, highly effective contraceptive methods include combined (estrogen- and progestogen-containing) hormonal contraception (oral, intravaginal, transdermal) or progestogen-only hormonal contraception (oral, injectable, implantable associated with the inhibition of ovulation initiated ≥2 menstrual cycles prior to screening); intrauterine device (IUD), intrauterine hormone-releasing system (IUS) with bilateral tubal ligation, vasectomized partner (provided that the vasectomized male partner is the study participant's only sexual partner and that partner has undergone a medical evaluation for the surgical success of the procedure); and / or sexual abstinence. 27. Has any medical condition, comorbidity, physical examination findings, or metabolic dysfunction or laboratory abnormality that, in the opinion of the investigator, makes the patient unsuitable for participation in the clinical trial because it poses a high safety risk and / or may affect the interpretation of the trial results. 28. Cardiac ejection fraction <40% by echocardiogram or multi-gated acquisition scan (MUGA) 29. Significant cardiovascular disease (e.g., New York Heart Association class III or IV heart disease, myocardial infarction, stroke, or transient ischemic attack, unstable arrhythmia, or unstable angina within the past 6 months) and / or significant pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm) 30. A documented history of autonomic dysfunction (e.g., type 1 diabetes) that may prevent the patient from tolerating protocol therapy.

[0174] Treatment: No placebo will be used in this trial. The dose administered will be a fixed dose and will not be determined by the patient's weight or body surface area. The study drug will be administered as an IV infusion.

[0175] REGN5459 or REGN5458 (B-cell maturation antigen [BCMA] x cluster of differentiation 3 [CD3] bsAb) for IV infusion (one cycle of three infusions over a 15-day period) is supplied as a liquid in a sterile, single-use vial. Each vial contains REGN5459 or REGN5458 at a concentration of 10 mg / mL. The diluent for REGN5459 or REGN5458 is supplied as a sterile liquid solution in a glass vial for IV administration.

[0176] Dexamethasone 40 mg IV; antihistamine (diphenhydramine [H-1 blocker]) 25 mg IV or PO; acetaminophen (paracetamol) 650 mg PO. Patients should be premedicated with dexamethasone according to Table 6 before receiving REGN5459 or REGN5458.

[0177] [Table 6]

[0178] Test evaluation items

[0179] The primary endpoints of this study are as follows: Occurrence of AEIs from the first dose of study drug to the end of the safety observation period Incidence and severity of TEAEs, including AESIs and serious adverse events (SAEs), from the first study dose to the end of the study

[0180] The secondary endpoints of this study were: The proportion of patients with a clinically meaningful decrease in anti-HLA alloantibodies during the study, as measured by a reduction in cPRA from baseline or a reduction in peak (immunodominant) anti-HLA mean fluorescence intensity (MFI) to less than 5,000 or a reduction of 50% or more (by single antigen bead [SAB] assay) Maximum decrease in peak (immunodominant) MFI from baseline Percent change from baseline in peak (immunodominant) MFI Percent change from baseline in the sum of MFIs of anti-HLA alloantibodies using the SAB assay Time to first clinically meaningful decrease in anti-HLA alloantibody levels by SAB assay (defined as peak anti-HLA alloantibody MFI <5,000 or a ≥50% decrease) Time to maximum decrease in anti-HLA alloantibody levels by SAB assay (defined as peak anti-HLA alloantibody MFI <5,000 or a decrease of ≥50%) Maximum decrease in cPRA from baseline Time to first clinically meaningful decrease in cPRA Time to maximum decrease in cPRA from baseline Duration and duration of reduction of peak anti-HLA alloantibody MFI < 5,000 by SAB assay, or maximum reduction of anti-HLA alloantibody MFI and cPRA % ≥ 50% Serum concentrations of Ig classes (IgG, IgA, IgE, IgM, IgG1, IgG2, and IgG3) over time Percent change from baseline over time in serum concentrations of Ig classes (IgG, IgA, IgE, IgM, IgG1, IgG2, and IgG3) Serum concentrations of REGN5459 or REGN5458 · The incidence of treatment-emergent anti-drug antibodies to REGN5459 or REGN5458 over time.

[0181] Preliminary Results: A total of seven highly sensitized participants with chronic kidney disease requiring hemodialysis were treated with REGN5459 at doses ranging from 1.5 mg to 5 mg. All participants enrolled in the 5 mg cohort (Figure 1, squares, triangles, and circles) demonstrated a decrease in total serum IgG concentrations from baseline (Figure 1). [Example]

[0182] Evaluation of kidney transplant recipients previously desensitized with anti-BCMA × anti-CD3 bispecific antibodies A non-interventional extension study for patients treated in the study discussed in Example 2 with REGN5459 or REGN5458 (BCMAxCD3 bispecific antibodies) who underwent kidney transplantation.

[0183] Objectives: To explore both primary and secondary endpoints.

[0184] The primary objective of this study is to evaluate adverse events (AEs) and serious adverse events (SAEs) in kidney transplant recipients previously treated with REGN5459 or REGN5458 in the study discussed in Example 2.

[0185] Secondary objectives of this study are to evaluate each of the following in kidney transplant recipients previously treated with REGN5459 or REGN5458: Rates and classification of antibody-mediated and T-cell-mediated renal allograft rejection · Graft survival · Allograft function Delayed allograft function Anti-human leukocyte antigen (HLA) alloantibody levels and calculated panel reactive antibodies (cPRA) Emergence of new donor-specific antibodies Circulating immunoglobulin (Ig) class (isotype) Pharmacokinetics (PK) of REGN5459 or REGN5458

[0186] Study Design: All patients who have undergone kidney transplantation and were treated with REGN5459 or REGN5458 in the study discussed in Example 2 can be enrolled in this study and will be followed for AEs, clinical outcomes, and biomarkers in two cohorts / treatment arms (patients who previously received REGN5459 and patients who received REGN5458). No additional study medication will be administered.

[0187] Study Duration: The study will continue until the last patient completes the 12-month post-transplant follow-up assessment or until all patients have discontinued the study.

[0188] Study Population: In the study discussed in Example 2, it is expected that 6-12 patients will be enrolled in the study to collect safety and outcome data in patients aged 18-70 years who have undergone kidney transplantation and received REGN5459 or REGN5458.

[0189] Participation Criteria - Patients must meet the following criteria to be eligible for enrollment in this study: 1. Received at least one treatment with REGN5459 or REGN5458 in the study discussed in Example 2 2. Have undergone or plan to undergo a kidney transplant after permissive cross-matching while enrolled in the study discussed in Example 2 3. Willing and able to comply with clinic visit and study-related procedures 4. Provide informed consent signed by the study patient or legally acceptable representative

[0190] Exclusion Criteria - There are no exclusion criteria for this trial.

[0191] Study endpoints:

[0192] The primary endpoint of the study was the incidence of AEs and SAEs over a 12-month period, beginning at the time of kidney transplant.

[0193] Secondary endpoints of this study include post-transplant assessment of: • Incidence up to 12 months, time to diagnosis, and response to therapy for each of the following types of biopsy-proven renal allograft rejection according to the Banff classification: Active antibody-mediated rejection (AMR) (Banff classification, category 2) Chronic active AMR (Banff classification, category 2) ○ Acute T cell-mediated rejection (TCMR) (Banff classification, category 4) ○ Chronically active TCMR (Banff classification, category 4) • Incidence and timing of graft loss (defined as dialysis-dependent) up to 12 months Change in estimated glomerular filtration rate (eGFR) at 1, 2, 3, 6, and 12 months • The occurrence of delayed graft function (defined as the use of dialysis within 7 days of transplantation). • Changes (in % and mean fluorescence intensity [MFI]) in anti-HLA alloantibodies (using single antigen bead [SAB] assay) compared with pretransplant levels at 2, 3, 6, and 12 months, and at the time of a clinical episode of suspected allograft rejection. Change in cPRA at 1, 2, 3, 6, and 12 months • Changes (% and MFI) in donor-specific anti-HLA alloantibodies (using SAB assay and donor HLA type) compared with recipient pre-transplant anti-HLA alloantibody levels at 1 month, 2 months, 3 months, 6 months, and 12 months, and at the time of the clinical episode of suspected allograft rejection Cumulative incidence of new anti-HLA alloantibody development by SAB assay over 12 months • Circulating serum concentrations of Ig classes (IgG, IgA, and IgM) over time. Percent change from baseline over time in circulating serum concentrations of Ig classes (IgG, IgA, and IgM) Serum concentrations of REGN5459 or REGN5458 for up to 12 months

[0194] Results: The incidence of AEs and SAEs after kidney transplantation is comparable to that in kidney transplant patient populations not receiving REGN5459 or REGN5458, and graft loss and graft function are comparable to that in kidney transplant patient populations not receiving REGN5459 or REGN5458. [Example]

[0195] REGN5459 causes plasma cell depletion and reduces serum Ig levels in humanized mice This example demonstrates T cell-mediated killing of plasma cells (PCs) and reduction of serum Ig levels by a human BCMAxCD3CD3 bispecific antibody (REGN5459) that binds BCMA on PCs via its targeting arm and CD3 on T cells via its effector arm. The activity of REGN5459 was evaluated in genetically humanized BCMA hu / hu / CD3 hu / hu mice (US 11,384,153) (Figure 2A).

[0196] Administration of BCMAxCD3 bispecific antibody resulted in a robust and statistically significant reduction in bone marrow plasma cells (BMPCs) that was comparable across all doses from 5 to 25 mg / kg and across different isotype-specific PC subsets (Figures 2B, 3A, and 3B). Importantly, splenic B cells, including antigen-experienced CD38+IgD- follicular B cells, were not significantly affected by BCMAxCD3 bispecific antibody (Figure 4). Plasma cell depletion was reflected by a significant decrease in serum IgA, IgM, and IgG1 levels, the magnitude of which is consistent with their expected half-lives (Figures 5A, 5B, and 5C). This finding is consistent with IgA having the shortest circulating half-life compared to IgM and IgG1, allowing existing IgA to be reduced approximately 10-20-fold within one week of removal of its source.

[0197] In a second experiment, the effect of the BCMAxCD3 bispecific antibody on antigen-specific Ig production was evaluated in a 15-week model of nasal house dust mite (HDM) exposure (Asrat et al. 2020, Sci Immunol 5) (Figure 6A). Overall BMPC, including IgE+ BMPC, was nearly eliminated within 2 weeks of bispecific antibody administration (Figures 6B and 6C). Total circulating IgA, IgM, and IgG1 levels were assessed and found to transiently decrease after BCMAxCD3 bispecific antibody treatment.

[0198] Thus, BCMAxCD3 bispecific antibodies (transiently) deplete plasma cells in BCMAhu / huCD3hu / hu mice, including IgE plasma cells, but do not efficiently deplete antigen-experienced B cells. [Example]

[0199] Effects of BCMAxCD3 bispecific antibody on plasma cell and circulating antibody levels in monkeys The effects of a BCMAxCD3 bispecific antibody (also known to bind monkey BCMA (Haber et al., 2021, Sci Rep 11, 14397) alone or in combination with a monkey IL-4Ra antibody (REGN646) were evaluated in cynomolgus monkeys (Figure 7A). A single administration of the BCMAxCD3 bispecific antibody resulted in a rapid and robust reduction in BMPCs (Figure 7B). Consistent with observations in mice (Example 4), this reduction in BMPCs was closely mirrored by a rapid decrease in IgE and IgA levels, which reached a nadir 5 weeks after BCMAxCD3 bispecific antibody administration. Notably, after the nadir, IgA levels gradually recovered in animals treated with the BCMAxCD3 bispecific antibody, both alone and in combination with anti-IL-4Ra, whereas IgE levels recovered in animals treated with the BCMAxCD3 bispecific antibody alone but not in animals treated in combination with anti-IL-4Ra. [Example]

[0200] Effect of BCMAxCD3 bispecific antibody on human serum immunoglobulins The effect of weekly administration of the BCMAxCD3 bispecific antibody (REGN5458) was evaluated in patients with multiple myeloma (Figure 8A). REGN5458 resulted in a robust reduction in immunoglobulin levels over time, with IgE levels completely reduced as early as 4 weeks post-treatment (Figure 8C). IgG levels were also depleted by more than 50% between 4 and 8 weeks, consistent with their half-life in humans (Figure 8B).

[0201] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0202]

Table 7

Claims

1. 1. A method of reducing alloantibody levels in a subject in need of a solid organ transplant, comprising administering to the subject a 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. 2. The method of claim 1, wherein reducing the alloantibody level in the subject comprises reducing the alloantibody level below a baseline alloantibody level measured before administration of the bispecific antibody or antigen-binding fragment thereof.

3. 3. The method of claim 1 or 2, further comprising measuring baseline alloantibody levels in the subject prior to administration of the bispecific antibody or antigen-binding fragment thereof.

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

5. 5. The method of claim 4, wherein the decrease in the alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity of 50% or more compared to the baseline alloantibody level.

6. 2. The method of claim 1, wherein the alloantibody level is measured by a single antigen bead assay and a decrease in the alloantibody level corresponds to a decrease in peak immunodominant anti-HLA antibody mean fluorescence intensity to less than 5000.

7. 1. A method of reducing calculated panel reactive antibody (cPRA) levels in a subject requiring a solid organ transplant, comprising administering to the subject a 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. 8. The method of claim 7, wherein reducing the cPRA level in the subject comprises reducing the cPRA level in the subject below a baseline cPRA level determined prior to administration of the bispecific antibody or antigen-binding fragment thereof.

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

10. 1. A method for reducing a subject's sensitization to anti-HLA antibodies prior to solid organ transplantation, the method comprising administering to the subject a bispecific antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment 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 subject's risk of rejection upon organ transplantation is equal to or less than the risk of a control population having a pre-transplant calculated panel-reactive antibody (cPRA) level of 90%.

11. 11. The method of claim 10, wherein the subject's risk of rejection upon organ transplantation is equal to or less than the risk of a control population having a pre-transplant cPRA level of 80%.

12. 12. The method of claim 11, wherein the subject's risk of rejection upon organ transplant is equal to or less than the risk of a control population having a pre-transplant cPRA level of 50% to less than 80%.

13. 1. 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 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 subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population having a pre-transplant calculated panel-reactive antibody (cPRA) level of 90%.

14. 14. The method of claim 13, wherein the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population having a pre-transplant cPRA level of 80%.

15. 15. The method of claim 14, wherein the subject's risk of rejection during the post-transplant interval is equal to or less than the risk of a control population having a pre-transplant cPRA level of 50% to less than 80%.

16. 16. The method of any one of claims 13 to 15, wherein the post-transplant interval is a three-month interval beginning one day after organ transplantation.

17. 16. The method of any one of claims 13 to 15, wherein the post-transplant interval is a 6-month interval beginning 1 day after organ transplantation.

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

19. 19. The method of any one of claims 13 to 18, wherein graft function is maintained in said subject at a functional level equal to or greater than that of said control population during said post-transplant interval.

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

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

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

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

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

5.

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

4.

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

8.

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

5.

28. the second antigen-binding domain (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising 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) comprising the amino acid sequence of SEQ ID NO:

5.

29. the second antigen-binding domain (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 14; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 15; and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:

16.

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

8.

31. the second antigen-binding domain (a) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; or (b) HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

32. the second antigen-binding domain (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 9 and an LCVR comprising the amino acid sequence of SEQ ID NO: 5; or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 13, and an LCVR comprising the amino acid sequence of SEQ ID NO:

5.

33. (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; (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. The method of any one of claims 1 to 23.

34. (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; (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.

35. (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; (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.

36. (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; (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.

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

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

33.

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

34.

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

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

42. 42. The method of claim 40 or 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 of any one of claims 1 to 36, wherein the bispecific antibody or antigen-binding fragment thereof is a bispecific antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 29, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 30, and a common light chain comprising the amino acid sequence of SEQ ID NO:

32.

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

32.

45. 45. The method of any one of claims 1 to 44, wherein the bispecific antibody is administered in a dosing regimen comprising divided initial doses.

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

47. 47. A dosing regimen for use in the method of any one of claims 1 to 46, comprising administering to the subject a first dose during week 1 of the dosing regimen, a second dose during week 2 of the dosing regimen, and a tertiary dose during week 3 of the dosing regimen, wherein the tertiary dose is equal to or greater than the second dose, and the second dose is greater than the first dose.

48. 48. The dosing regimen of claim 47, wherein the initial dose is between 0.05 mg and 5 mg.

49. 49. The dosing regimen of claim 47 or 48, wherein the secondary dose is between 0.15 mg and 25 mg.

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

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

53. 53. The dosing regimen of any one of claims 47-52, wherein the tertiary 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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