BCMA-TARGETED TRISPECIFIC PROTEINS AND USES THEREOF

Abstract

Provided herein are B-cell maturation agent (BCMA)-targeted trispecific proteins comprising a domain that binds CD3, a half-life extending domain, and a domain that binds BCMA. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such BCMA-targeted trispecific proteins. Also disclosed are methods of using the disclosed BCMA-targeted trispecific proteins in the prevention and / or treatment of diseases, conditions, and disorders.

Description

[Technical field]

[0001] cross reference This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 196,595, filed June 3, 2021, and U.S. Provisional Patent Application No. 63 / 288,124, filed December 10, 2021, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] Cancer is the second leading cause of human death after coronary artery disease. Worldwide, millions of people die from cancer each year. In the United States alone, cancer claims the lives of well over 500,000 people each year, with approximately 1.4 million new cases diagnosed each year. Deaths from heart disease have declined significantly, but deaths due to cancer in general are increasing. At the beginning of the next century, cancer is predicted to become the leading cause of death.

[0003] Furthermore, cancer patients who survive early primary cancers share a common experience of having their lives dramatically altered. Many cancer patients experience intense anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients experience significant physical debilitation following treatment.

[0004] Generally speaking, a fundamental problem in the management of most deadly cancers is the lack of effective and non-toxic systemic therapies. Cancer is a complex disease characterized by genetic mutations that lead to uncontrolled cell proliferation. Cancer cells are present in all organisms and under normal circumstances, their excessive proliferation is tightly controlled by various physiological factors. Summary of the Invention

[0005] Described herein are methods of treating cancer, comprising administering to a subject an effective amount of a B-cell maturation agent (BCMA)-targeting trispecific protein, the protein comprising (a) a first domain (A) that specifically binds human CD3, (b) a second domain (B) that is a half-life extending domain, and (c) a third domain (C) that specifically binds BCMA, the domains being linked in the order H2N-(C)-(B)-(A)-COOH or by a linker L1 and a linker L2, and wherein the BCMA-targeting trispecific protein is administered at a dosage of about 1 μg to about 100 mg. In some embodiments, the BCMA-targeting trispecific protein is administered at a dosage of about 1 μg to about 5 mg. In some embodiments, the BCMA-targeting trispecific protein is administered at a dosage of about 1 μg to about 3 mg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of about 1 μg to about 2 mg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of about 1 μg to about 1 mg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of about 5 μg to about 2150 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of about 5 μg to about 2860 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of 2860 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of 2150 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of 1620 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of 810 μg. In some embodiments, the BCMA targeting trispecific protein is administered at a dosage of 270 μg. In some embodiments, the BCMA targeting trispecific protein is administered once a week. In some embodiments, the BCMA targeting trispecific protein is administered twice a week. In some embodiments, the BCMA targeting trispecific protein is administered every other week. In some embodiments, the BCMA targeting trispecific protein is administered every three weeks.In some embodiments, the BCMA-targeting trispecific is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0006] Described herein are methods of treating cancer, comprising administering to a subject an effective amount of a B-cell maturation agent (BCMA)-targeting trispecific protein, the protein comprising (a) a first domain (A) that specifically binds human CD3, (b) a second domain (B) that is a half-life extending domain, and (c) a third domain (C) that specifically binds BCMA, the domains being linked in the order H2N-(C)-(B)-(A)-COOH or by a linker L1 and a linker L2, and the BCMA-targeting trispecific protein is administered according to a schedule comprising the steps of (i) administering a first dose of the BCMA-targeting trispecific protein, (ii) administering a second dose of the BCMA-targeting trispecific protein, the second dose being higher than the first dose. In some embodiments, the first dose is about 1 μg to about 10 mg. In some embodiments, the first dose is about 1 μg to about 5 mg. In some embodiments, the first dose is about 1 μg to about 4 mg. In some embodiments, the first dose is about 1 μg to about 3 mg. In some embodiments, the first dose is about 1 μg to about 2 mg. In some embodiments, the first dose is about 1 mg. In some embodiments, the first dose is about 1.62 mg. In some embodiments, the first dose is about 1.5 mg. In some embodiments, the first dose is about 2.15 mg. In some embodiments, the first dose is about 2.86 mg. In some embodiments, the first dose is about 3.24 mg. In some embodiments, the first dose is administered for about 1 week to about 36 weeks. In some embodiments, the first dose is administered for about 1 week to about 27 weeks. In some embodiments, the first dose is administered for about 1 week to about 18 weeks. In some embodiments, the first dose is administered for about 1 week to about 9 weeks. In some embodiments, the first dose is administered once a day. In some embodiments, the first dose is administered twice a day. In some embodiments, the first dose is administered three times a day. In some embodiments, the first dose is administered five times a day. In some embodiments, the first dose is administered once a week. In some embodiments, the first dose is administered twice a week.In some embodiments, the first dose is administered every other week. In some embodiments, the first dose is administered every three weeks. In some embodiments, the first dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the second dose is about 1 mg to about 12 mg. In some embodiments, the second dose is about 1 mg to about 24 mg. In some embodiments, the second dose is about 1 mg to about 48 mg. In some embodiments, the second dose is about 5 mg to about 12 mg. In some embodiments, the second dose is about 10 mg to about 48 mg. In some embodiments, the second dose is about 10 mg. In some embodiments, the second dose is about 12 mg. In some embodiments, the second dose is about 24 mg. In some embodiments, the second dose is about 36 mg. In some embodiments, the second dose is about 48 mg. In some embodiments, the second dose is administered for about 1 week to about 36 weeks. In some embodiments, the second dose is administered for about 1 week to about 27 weeks. In some embodiments, the second dose is administered for about 1 week to about 18 weeks. In some embodiments, the second dose is administered for about 1 week to about 9 weeks. In some embodiments, the second dose is administered once a day. In some embodiments, the second dose is administered twice a day. In some embodiments, the second dose is administered three times a day. In some embodiments, the second dose is administered five times a day. In some embodiments, the second dose is administered once a week. In some embodiments, the second dose is administered twice a week. In some embodiments, the second dose is administered every other week. In some embodiments, the second dose is administered every three weeks. In some embodiments, the second dose is maintained until the end of the schedule after administration of the first dose. In some embodiments, the second dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0007] For any of the methods described herein, the BCMA-targeting trispecific protein has an elimination half-life of at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours. In some embodiments, the third domain comprises a VHH domain. In some embodiments, the VHH domain is human, humanized, affinity matured, or a combination thereof. In some embodiments, the third domain comprises one or more sequences selected from the group consisting of SEQ ID NOs: 346-460. In some embodiments, the first domain comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3. In some embodiments, the first domain is humanized or human. In some embodiments, the second domain binds human serum albumin. In some embodiments, the second domain comprises an scFv, a variable heavy domain (VH), a variable light domain (VL), a peptide, a ligand, or a small molecule. In some embodiments, the linker L1 and the linker L2 are each independently (GS) n (SEQ ID NO: 472), (GGS) n (SEQ ID NO: 473), (GGGS) n (SEQ ID NO:474), (GGSG) n (SEQ ID NO:475), (GGSGG) n (SEQ ID NO:476), (GGGGS) n(SEQ ID NO: 477), or GGGGSGGGS (SEQ ID NO: 602), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linker L1 and linker L2 are each independently (GGGGS)4 (SEQ ID NO: 480), (GGGGS)3 (SEQ ID NO: 481), or GGGGSGGGS (SEQ ID NO: 602). In some embodiments, the domains are linked in the order H2N-(C)-L1-(B)-L2-(A)-COOH. In some embodiments, the BCMA targeting trispecific protein is less than about 80 kDa. In some embodiments, the BCMA targeting trispecific protein is about 50 to about 75 kDa. In some embodiments, the BCMA targeting trispecific protein is less than about 60 kDa. In some embodiments, the BCMA targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NOs: 483-597. In some embodiments, the BCMA-targeting trispecific protein comprises a sequence set forth in SEQ ID NO: 520. In some embodiments, the cancer is a BCMA-associated neoplastic disease, autoimmune disease, or infectious disease. In some embodiments, the cancer is multiple myeloma, leukemia, lymphoma, or metastasis thereof. In some embodiments, the cancer is multiple myeloma.

[0008] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0009] The novel features of the invention are set forth with particularity in the appended claims. To better understand the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings. [Figure 1]FIG. 1 is a schematic diagram of an exemplary BCMA-targeting trispecific antigen binding protein, which has a constant core element comprising an anti-CD3ε single chain variable fragment (scFv) and an anti-ALB variable heavy chain region, and an anti-BCMA binding domain that can be a VHH, VH, scFv, non-Ig binder, or ligand. [Diagram 2] FIG. 1 illustrates the effect of exemplary BCMA targeting molecules (01H08, 01F07, 02F02, and BH253) containing anti-BCMA binding proteins according to the present disclosure in killing purified human T cells expressing BCMA compared to a negative control. [Diagram 3] 1 is an image of an SDS-PAGE of a representative purified BCMA trispecific molecule. Lane 1: 01F07-M34Y TriTAC non-reduced; lane 2: 01F07-M34G-TriTAC non-reduced; lane 3: 02B05 TriTAC non-reduced; lane 4: 02G02-M34Y TriTAC non-reduced; lane 5: 02G02 M34G TriTAC non-reduced; lane 6: broad range SDS-PAGE standard (Bio-Rad #1610317); lane 7: 01F07-M34Y TriTAC non-reduced; lane 8: 01F07-M34G-TriTAC non-reduced; lane 9: 02B05 TriTAC non-reduced; lane 10: 02G02-M34Y TriTAC non-reduced; lane 11: 02G02 M34G TriTAC non-reduced; lane 12: broad range SDS-PAGE standard (Bio-Rad #1610317) [Figure 4A] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the present disclosure in killing BCMA-expressing Jeko1 cells compared to negative controls. [Figure 4B] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the present disclosure in killing BCMA-expressing Jeko1 cells compared to negative controls. [Figure 4C] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the present disclosure in killing BCMA-expressing Jeko1 cells compared to negative controls. [Figure 4D] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing MOLP-8 cells expressing BCMA compared to a negative control. [Figure 4E] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing MOLP-8 cells expressing BCMA compared to a negative control. [Figure 4F] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing MOLP-8 cells expressing BCMA compared to a negative control. [Figure 4G] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing OPM-2 cells expressing BCMA compared to a negative control. [Figure 4H] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing OPM-2 cells expressing BCMA compared to a negative control. [Figure 4I] Illustrates the efficacy of exemplary BCMA trispecific targeting molecules comprising anti-BCMA binding proteins of the disclosure in killing OPM-2 cells expressing BCMA compared to a negative control. [Figure 5A] Illustrates binding of an exemplary BCMA trispecific targeting protein (02B05) to purified T cells from four different human donors, Donor 02 (FIG. 5A), Donor 35 (FIG. 5B), Donor 81 (FIG. 5C), Donor 86 (FIG. 5D). [Figure 5B] Illustrates binding of an exemplary BCMA trispecific targeting protein (02B05) to purified T cells from four different human donors, Donor 02 (FIG. 5A), Donor 35 (FIG. 5B), Donor 81 (FIG. 5C), Donor 86 (FIG. 5D). [Figure 5C]Illustrates binding of an exemplary BCMA trispecific targeting protein (02B05) to purified T cells from four different human donors, Donor 02 (FIG. 5A), Donor 35 (FIG. 5B), Donor 81 (FIG. 5C), Donor 86 (FIG. 5D). [Figure 5D] Illustrates binding of an exemplary BCMA trispecific targeting protein (02B05) to purified T cells from four different human donors, Donor 02 (FIG. 5A), Donor 35 (FIG. 5B), Donor 81 (FIG. 5C), Donor 86 (FIG. 5D). [Figure 6A] Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 6B] Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 6C] Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 6D] Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 6E]Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 6F] Illustrated are binding of an exemplary BCMA trispecific targeting protein (02B05) to cells expressing BCMA, NCI-H929 (Figure 6A), EJM (Figure 6B), OPM2 (Figure 6D), RPMI8226 (Figure 6E), or cell lines that lack expression of BCMA, NCI-H510A (Figure 6C) and DMS-153 (Figure 6F). [Figure 7] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05) and BCMA-expressing EJM cells in the presence or absence of human serum albumin (HSA). [Figure 8] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05) and BCMA-expressing EJM cells using various effector to target cell ratios. [Figure 9] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05) and BCMA-expressing OPM2 cells using various effector to target cell ratios. [Figure 10] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05) and BCMA-expressing NCI-H929 cells using various time points and a 1:1 effector to target cell ratio. [Figure 11] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing EJM cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 12]Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing NCI-H929 cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 13] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing OPM2 cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 14] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing RPMI8226 cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 15] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA non-expressing OVCAR8 cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 16] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA non-expressing NCI-H510A cells, and T cells from four different donors in the presence of human serum albumin (HSA). [Figure 17] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing NCI-H929 cells, and peripheral blood mononuclear cells (PBMCs) from two different cynomolgus donors in the presence of human serum albumin (HSA). [Figure 18] Illustrates the results of a TDCC assay using an exemplary BCMA trispecific targeting protein (02B05), BCMA-expressing RPMI8226 cells, and peripheral blood mononuclear cells (PBMCs) from two different cynomolgus donors in the presence of human serum albumin (HSA). [Figure 19]Illustrates expression levels of the T cell activation biomarker CD69 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing EJM cells. [Figure 20] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD25 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing EJM cells. [Figure 21] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD69 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing OPM2 cells. [Figure 22] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD25 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing OPM2 cells. [Figure 23] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD69 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing RPMI8226 cells. [Figure 24] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD25 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA-expressing RPMI8226 cells. [Diagram 25] Illustrates expression levels of the T cell activation biomarker CD69 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA non-expressing OVCAR8 cells. [Figure 26] Illustrates expression levels of the T cell activation biomarker CD25 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA non-expressing OVCAR8 cells. [Figure 27] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD69 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA non-expressing NCI-H510A cells. [Figure 28] FIG. 1 illustrates the expression levels of the T cell activation biomarker CD25 following a TDCC assay using an exemplary BCMA-targeting trispecific protein (02B05) and BCMA non-expressing NCI-H510A cells. [Figure 29] Illustrates expression levels of the cytokine, TNF-α, in co-cultures of T cells and BCMA-expressing target cells (EJM cells) treated with increasing concentrations of an exemplary BCMA-targeting trispecific (02B05) protein, or a negative control GFP trispecific protein. [Diagram 30] Illustrates the reduction in tumor growth in an RPMI8226 xenograft model treated with various concentrations of an exemplary BCMA-targeting trispecific (02B05) protein or vehicle control. [Diagram 31] Illustrates the reduction in tumor growth in a Jeko1 xenograft model treated with various concentrations of an exemplary BCMA-targeting trispecific (02B05) protein or vehicle control. [Diagram 32] Illustrates the concentration of BCMA-targeting trispecific protein in serum samples from cynomolgus monkeys administered various concentrations of an exemplary BCMA-targeting trispecific (02B05) protein. [Diagram 33] TDCC assay results using BCMA trispecific targeting proteins from cynomolgus monkey serum samples taken 168 hours after administration of various concentrations of an exemplary BCMA targeting trispecific (02B05) protein, BCMA-expressing EJM cells, and purified human T cells in the presence of cynomolgus monkey serum that was not exposed to the BCMA trispecific targeting protein. [Diagram 34] Illustrates a BCMA trispecific antigen binding protein Phase 1 / 2 trial design. [Diagram 35] Treatment times are shown for all treated patients. [Diagram 36] The overall response rate is shown. [Figure 37A] 2 shows pharmacokinetic data of BCMA trispecific antigen binding proteins for different dosing cohorts. [Figure 37B] Evidence of BCMA trispecific accumulation shows a ∼1.5-2 fold increase in Cmax (Figure 37B) comparing C1D1 and C2D15. [Figure 37C] Evidence of BCMA trispecific accumulation is shown by a ∼1.5-2 fold increase in AUC (Figure 37C) comparing C1D1 and C2D15. [Figure 37D] Evidence for BCMA trispecific accumulation shows an approximately 2-3 fold increase in Clast (Figure 37D) compared to C1D1 and C2D15. [Figure 38A] Shown are serum cytokine concentrations of serum IL-6 (FIG. 38A) 5 hours after the first dose (C1D1) and the second dose (C1D8). [Figure 38B] Serum cytokine concentrations of serum TNFα (FIG. 38B) 5 hours after the first dose (C1D1) and the second dose (C1D8) are shown. [Figure 39]

[0043] Figure 1 shows changes during treatment in serum BCMA. Changes during treatment were observed in serum BCMA (sBCMA) from baseline to pre-C1D15 (after 2 doses). [Figure 40A] Figure 40A shows the concentration-time profile of BCMA trispecific antigen binding protein after the sixth dose. [Figure 40B] Figure 40A shows the concentration-time profile of BCMA trispecific antigen binding protein. Figure 40B shows the concentration-time profile after the first dose. [Figure 41A] Serum cytokine and chemokine levels 5 hours after the first infusion are shown. Serum samples were taken before (baseline) and 5 hours after (5-hour EOI) the first infusion of C1D1. The median concentration increase from baseline for each cytokine (FIG. 41A) or chemokine (FIG. 41B) at 5-hour EOI is represented by the histogram bars and each patient's value is represented by the marker symbol. [Figure 41B]Serum cytokine and chemokine levels 5 hours after the first infusion are shown. Serum samples were taken before (baseline) and 5 hours after (5-hour EOI) the first infusion of C1D1. The median concentration increase from baseline for each cytokine (FIG. 41A) or chemokine (FIG. 41B) at 5-hour EOI is represented by the histogram bars and each patient's value is represented by the marker symbol. [Figure 42A] Kinetics of changes in serum cytokines and chemokines in fixed-dose and graded-dose cohorts are shown. Cytokines (FIG. 42A) and chemokines (FIG. 42B) in serum taken prior to the first dose (Pre) and at 5 hours EOI on the indicated dosing days are shown along with the administered dose. Median cytokine or chemokine concentrations are represented by histogram bars and individual patient values ​​are represented by marker symbols. [Figure 42B] Kinetics of changes in serum cytokines and chemokines in fixed-dose and graded-dose cohorts are shown. Cytokines (FIG. 42A) and chemokines (FIG. 42B) in serum taken prior to the first dose (Pre) and at 5 hours EOI on the indicated dosing days are shown along with the administered dose. Median cytokine or chemokine concentrations are represented by histogram bars and individual patient values ​​are represented by marker symbols. [Diagram 43] Cytokine induction and clinical response in the 2150 and 2860 μg dose cohorts are shown. Serum samples were taken at baseline and 5 hours after the first infusion. The increase in cytokine or chemokine concentration from baseline for each subject in the 2150 and 2860 μg dose cohorts is indicated by a marker (triangle or circle). Subjects are divided into two groups by their most recent clinical response - patients with progressive disease (PD) or stable disease (SD) and patients with a partial response (PR), very good partial response (VGPR), or complete response (CR) to treatment. The significance of differences in cytokine or chemokine concentrations between these two groups is determined using an unpaired two-tailed Student's t-test. [Figure 44A]Figure 44A shows the change in circulating T cell counts following BCMA trispecific antigen binding protein infusion for fixed dose cohorts. Figure 44A shows CD4+ T cells. Flow cytometry analysis was performed on whole blood to determine cell counts of each T cell subset pre-infusion and 5, 24 or 48 hours post-infusion as indicated. Cell counts at post-dose time points are expressed as % of pre-dose. The % of median pre-dose cell counts for each cohort are represented by the histogram bars, with each patient's value indicated by the marker symbol. [Figure 44B] Figure 44B shows the change in circulating T cell counts following BCMA trispecific antigen binding protein infusion for fixed dose cohorts. Figure 44B shows CD8+ T cells. Flow cytometry analysis was performed on whole blood to determine cell counts of each T cell subset pre-infusion and 5, 24 or 48 hours post-infusion as indicated. Cell counts at post-dose time points are expressed as % of pre-dose. The median % of pre-dose cell counts for each cohort are represented by the histogram bars, with each patient's value indicated by the marker symbol. [Diagram 45] Figure 1 shows the change in circulating T cell counts following BCMA trispecific antigen binding protein infusion for step-dose cohorts. Flow cytometry analysis was performed on whole blood to determine cell counts of each T cell subset pre-infusion and 5, 24 or 48 hours post-infusion as indicated. Cell counts at post-dose time points are expressed as % of pre-dose. The % of median pre-dose cell counts for each cohort are represented by the histogram bars, with values ​​for each patient indicated by the marker symbols. [Figure 46A] Figure 46A shows upregulation of CD69 expression on T cells following BCMA trispecific antigen binding protein infusion for fixed dose cohorts. Figure 46B shows CD4+ T cells. Flow cytometry analysis was performed on whole blood to determine the percentage of CD4+ and CD8+ T cells expressing CD69 before and after the indicated BCMA trispecific antigen binding protein doses. The median % of CD69+ for each cohort is represented by the histogram bars and each patient's value is indicated by the marker symbol. [Figure 46B]Figure 46A shows upregulation of CD69 expression on T cells following BCMA trispecific antigen binding protein infusion for fixed dose cohorts. Figure 46B shows CD8+ T cells. Flow cytometry analysis was performed on whole blood to determine the percentage of CD4+ and CD8+ T cells expressing CD69 before and after the indicated BCMA trispecific antigen binding protein doses. The median % of CD69+ for each cohort is represented by the histogram bars, and each patient's value is indicated by the marker symbol. [Figure 47] Figure 1 shows T cell activation and clinical responses in the 2150 and 2860 μg dose cohorts. Flow cytometry analysis was performed on whole blood to measure expression of CD69 on T cells at baseline and 24 hours after the first BCMA trispecific antigen binding protein dose. The fold change in CD69+ T cell percentage for each subject in the 2150 and 2860 μg dose cohorts is indicated by a marker (triangle or circle). Subjects are divided into two groups according to their most recent clinical response - patients with progressive disease (PD) or stable disease (SD) and patients with a partial response (PR), very good partial response (VGPR), or complete response (CR) to treatment. The significance of differences in cytokine or chemokine concentrations between these two groups is determined using unpaired two-tailed Student's t-tests. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in implementing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of the claims, and equivalents thereof, be covered thereby.

[0011] Described herein are trispecific proteins targeting B-cell maturation antigen (BCMA), pharmaceutical compositions thereof (referred to herein as BCMA-binding trispecific proteins, BCMA-targeting trispecific proteins, or BCMA trispecific antigen-binding proteins), as well as nucleic acids, recombinant expression vectors, and host cells for making such proteins. Additionally, methods of using the disclosed BCMA-targeting trispecific proteins in the prevention and / or treatment of diseases, illnesses, and disorders are also provided. The BCMA-targeting trispecific proteins can specifically bind to BCMA as well as CD3, and have a half-life extending domain, such as a domain binding to human albumin (ALB). Figure 1 depicts a non-limiting example of a trispecific BCMA-binding protein.

[0012] An "antibody" typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), which can be readily classified as kappa or lambda based on amino acid sequence and gene locus. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the classification of IgG, IgA, and IgD, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length (usually about 10 to about 60 amino acids in IgG). The variable domains of both the light and heavy chains are joined to the constant domains by a "J" region of about 12 or more amino acids, with the heavy chains having a "D" region of about 10 additional amino acids. Each class of antibody contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues. There are two types of natural disulfide bridges or bonds in immunoglobulin molecules: interchain disulfide bonds and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary with the immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to solvent, and are usually relatively easy to reduce. In the human IgG1 isotype, there are four interchain disulfide bonds, one from each heavy chain to the light chain and two between heavy chains. Interchain disulfide bonds are not required for chain attachment. As is known, the cysteine-rich IgG1 hinge region of the heavy chain is generally held to consist of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will understand that the IgG1 hinge region contains cysteines in the heavy chain that contain interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide structural flexibility that facilitates Fab movement. The interchain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain.The interchain disulfide bonds between the heavy chains are at positions C226 and C229 (all numbered according to the EU index according to Kabat, et al., below).

[0013] As used herein, the term "antibody" includes polyclonal, multiclonal, monoclonal, chimeric, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific, bispecific, monovalent, polyvalent, anti-idiotypic, synthetic antibodies, immunospecific antibody fragments, e.g., Fd, Fab, F(ab')2, F(ab') fragments, single chain fragments (e.g., ScFv and ScFvFc), disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (VH, VL, or VHH domains), such as sdAbs, including muteins and variants thereof, and any other immunoreactive molecule so long as it contains a domain that has a binding site for preferential association or binding to a BCMA protein. Moreover, unless contextual constraints dictate otherwise, the term further includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy-chain constant domains that correspond to the various classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domains.

[0014] In some embodiments, the BCMA binding domain of the BCMA-targeting trispecific protein of this disclosure comprises a heavy chain-only antibody, such as a VH or VHH domain. In some instances, the BCMA-binding trispecific protein comprises a heavy chain-only antibody that is an engineered human VH domain. In some instances, the engineered human VH domain is generated by panning a phage display library. In some embodiments, the BCMA binding domain of the BCMA-targeting trispecific protein of this disclosure comprises a VHH. The term "VHH" as used herein refers to a single chain antibody binding domain that lacks light chains. In some instances, the VHH is derived from a type of antibody found in camelids or cartilaginous fish that naturally lack light chains, or a synthetic non-immune VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by V, D, and J exons. The VHH is optionally a naturally occurring VHH, e.g., a VHH from a camelid, or a recombinant protein that comprises a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camel, llama, vicuña, guanaco, and cartilaginous fish (such as, but not limited to, shark). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, Huacaya Alpaca and Suri alpaca).

[0015] As used herein, "variable region" or "variable domain" refers to the fact that certain portions of the variable domain vary widely in sequence in antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called the complementarity determining regions (CDRs) or hypervariable regions in both the light chain (VL) and heavy chain (VH) variable domains. The more highly conserved portions of the variable domain are called the framework (FR). Native heavy and light chain variable domains each contain four FR regions that largely adopt a β-sheet configuration and are connected by three CDRs, forming loop junctions and, in some cases, forming part of the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. In some cases, the ScFv fragments (or variable single chain fragments) obtained by genetic engineering associate in a single polypeptide chain the VH and VL domains of an antibody separated by a peptide linker.

[0016] In some embodiments of the disclosure, the BCMA-binding domain of the BCMA-targeting trispecific protein comprises a heavy chain-only antibody, such as a VH or VHH domain, and comprises three CDRs. Such heavy chain-only antibodies, in some embodiments, bind BCMA as monomers that are not dependent on dimerization with the VL (variable light) region for optimal binding affinity. In some embodiments of the disclosure, the CD3-binding domain of the BCMA-targeting trispecific protein comprises an scFv. In some embodiments of the disclosure, the albumin-binding domain of the BCMA-targeting trispecific protein comprises a heavy chain-only antibody, such as a single domain antibody comprising a VH or VHH domain.

[0017] The assignment of amino acids to each domain, framework region and CDR, in some embodiments, follows one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, Chothia et al., 1987, PMID: 3681981, Chothia et al., 1989, PMID: 2687698, MacCallum et al., 1996, PMID: 8876650, or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopia), unless otherwise indicated. The CDRs of this disclosure are not intended to necessarily correspond to the Kabat numbering convention.

[0018] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0019] As used herein, the term "percent (%) amino acid sequence identity" for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0020] As used herein, "elimination half-life" is used in its ordinary sense as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because the drug concentration does not usually reach the concentration required for saturation of the elimination process. The rate of an exponential process can be expressed by its rate constant k, which represents the fractional rate of change per unit of time, or by its half-life t1 / 2, which is the time required for 50% of the process to be completed. The units of these two constants are time-1 and time, respectively. The first-order rate constant and the half-life of a reaction are simply related (k×t1 / 2=0.693) and can be interchanged accordingly. First-order elimination kinetics dictates that a constant fraction of the drug is lost per unit of time, so that a plot of the logarithm of drug concentration versus time is linear for all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). Drug elimination half-life can be accurately determined from such a graph.

[0021] As used herein, the term "binding affinity" refers to the affinity of a protein described in this disclosure for a binding target, and is expressed numerically using a "Kd" value. When two or more proteins are shown to have comparable binding affinities for their binding targets, the Kd values ​​for the binding of each protein to the binding targets are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinities for a single binding target, the Kd values ​​for the binding of each protein to the single binding target are within ±2-fold of each other. When a protein is shown to bind to two or more targets with comparable binding affinities, the Kd values ​​for the binding of the protein to the two or more targets are within ±2-fold of each other. Generally, a higher Kd value corresponds to weaker binding. In some embodiments, "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, the "on-rate" or "rate of association or association rate" or "k" and the "off-rate" or "rate of dissociation or dissociation rate" or "k" are also determined with surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In further embodiments, "Kd", "k" and "k" are measured using OCTET® Systems (Pall Life Sciences).In an exemplary method of measuring binding affinity using OCTET® Systems, a ligand, e.g., biotinylated human or cynomolgus BCMA, is immobilized on the OCTET® streptavidin capillary sensor tip surface, and the streptavidin tip is then activated with about 20-50 μg / ml of human or cynomolgus BCMA protein according to the manufacturer's instructions. A solution of PBS / casein is also introduced as a blocker. For association response rate measurements, BCMA binding protein variants are introduced at concentrations ranging from about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, BCMA binding single domain proteins are used at concentrations ranging from about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the case of negative control assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, for example ForteBio software.

[0022] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., on the constraints of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to convention, for any value. When a particular value is described in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0023] The terms "individual," "patient," or "subject" are used interchangeably. None of the terms require or are limited to a situation characterized by the supervision (e.g., continuous or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, floor worker, or hospice worker).

[0024] The terms used herein are intended to describe particular instances only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising."

[0025] In one embodiment, the BCMA-targeting trispecific protein comprises a domain that specifically binds to CD3 (A), a domain that specifically binds to human albumin (ALB) (B), and a domain that specifically binds to BCMA (C). The three domains in the BCMA-targeting trispecific protein are arranged in any order. Thus, it is contemplated that the domain order of the BCMA-targeting trispecific protein is as follows: H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, HN-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH.

[0026] In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(A)-(B)-(C)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(A)-(C)-(B)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(B)-(A)-(C)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(B)-(C)-(A)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the BCMA targeting trispecific protein has a domain order of H2N-(C)-(A)-(B)-COOH. In some embodiments, the anti-BCMA domain (anti-targeting domain, T), the anti-CD3 domain (C), and the anti-ALB domain (A) are in an orientation of anti-CD3:anti-ALB:anti-BCMA (CAT). In some embodiments, the anti-BCMA domain (anti-targeting domain, T), the anti-CD3 domain (C), and the anti-ALB domain (A) are in an orientation of anti-BCMA:anti-ALB:anti-CD3 (TAC).

[0027] In some embodiments, the BCMA-targeting trispecific protein has the HSA binding domain as an intermediate domain, such that the domain order is H2N-(A)-(B)-(C)-COOH, or H2N-(C)-(B)-(A)-COOH. In such embodiments in which the ALB binding domain is the intermediate domain, it is contemplated that the CD3 and BCMA binding domains are afforded additional flexibility to bind to their respective targets.

[0028] In some embodiments, the BCMA-targeting trispecific proteins described herein comprise polypeptides having the sequences set forth in the Sequence Listing (SEQ ID NOs: 438-597) and subsequences thereof. In some embodiments, the trispecific antigen binding proteins comprise polypeptides having at least 70%-95% or more homology to the sequences set forth in the Sequence Listing (SEQ ID NOs: 483-597). In some embodiments, the trispecific antigen binding proteins comprise polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequences set forth in Sequence Table 1 (SEQ ID NOs: 483-597).

[0029] The BCMA-targeted trispecific proteins described herein are designed to be able to specifically target cells expressing BCMA by recruiting cytotoxic T cells. This improves efficacy compared to ADCC (antibody-dependent cellular cytotoxicity), which uses full-length antibodies directed against a single antigen and cannot directly recruit cytotoxic T cells. In contrast, by binding CD3 molecules specifically expressed on these cells, the BCMA-targeted trispecific proteins can crosslink cytotoxic T cells with cells expressing BCMA in a highly specific manner, thereby directing the cytotoxic potential of T cells to the target cells. The BCMA-targeted trispecific proteins described herein bind to cytotoxic T cells by binding to the surface-expressed CD3 protein, which forms part of the TCR. The simultaneous binding of multiple BCMA trispecific antigen-binding proteins to CD3 and BCMA expressed on the surface of a particular cell causes T cell activation and mediates the subsequent lysis of the particular BCMA-expressing cell. Thus, the BCMA-targeted trispecific proteins are contemplated to display potent, specific, and efficient target cell killing. In some embodiments, the BCMA-targeting trispecific proteins described herein stimulate target cell killing by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing BCMA). In some such embodiments, the cells are selectively eliminated, thereby reducing the potential for toxic side effects.

[0030] The BCMA-targeting trispecific proteins described herein further confer advantages over conventional monoclonal antibodies and smaller bispecific molecules. In general, the efficacy of recombinant protein pharmaceuticals is highly dependent on the intrinsic pharmacokinetics of the protein itself. One such advantage is that the BCMA-targeting trispecific proteins described herein have a half-life extension domain, such as a domain specific for HSA, thereby extending the pharmacokinetic elimination half-life. In this regard, the BCMA-targeting trispecific proteins described herein have an extended serum elimination half-life of about 2, 3, about 5, about 7, about 10, or about 14 days, depending on the embodiment. This is in contrast to other binding proteins, such as BiTE or DART molecules, which have a relatively very short elimination half-life. For example, the CD19xCD3 bispecific scFv-scFv fusion molecule of BiTE requires drug delivery by continuous infusion (intravenous) due to its short elimination half-life. The longer intrinsic half-time of BCMA-targeting trispecific proteins solves this problem, thereby increasing therapeutic possibilities such as lower dose pharmaceutical formulations, reduced regular dosing, and / or novel pharmaceutical compositions.

[0031] The BCMA targeting trispecific proteins described herein further have an optimal size for enhanced tissue penetration and tissue distribution. Larger sizes limit or prevent the penetration or distribution of the protein in the target tissue. The BCMA targeting trispecific proteins described herein avoid this by having a small size that enhances tissue penetration and distribution. Accordingly, the BCMA targeting trispecific proteins described herein have a size of about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kD to about 70 kD, or about 50 kD to about 65 kD in some embodiments. Thus, the size of the BCMA targeting trispecific proteins is more advantageous than IgG antibodies, which are about 150 kD, and than BiTE and DART bispecific antibody molecules, which are about 55 kD but do not have an extended half-life and are therefore quickly cleared by the kidney.

[0032] In further embodiments, the BCMA targeting trispecific proteins described herein have an optimal size for enhanced tissue penetration and distribution. In these embodiments, the BCMA targeting trispecific proteins are constructed to be as small as possible while retaining specificity for their targets. Accordingly, in these embodiments, the BCMA targeting trispecific proteins described herein have a size of about 20 kD to about 40 kD, or about 25 kD to about 35 kD, to about 40 kD, to about 45 kD, to about 50 kD, to about 55 kD, to about 60 kD, to about 65 kD. In some embodiments, the BCMA targeting trispecific proteins described herein have a size of about 50kD, 49kD, 48kD, 47kD, 46kD, 45kD, 44kD, 43kD, 42kD, 41kD, 40kD, about 39kD, about 38kD, about 37kD, about 36kD, about 35kD, about 34kD, about 33kD, about 32kD, about 31kD, about 30kD, about 29kD, about 28kD, about 27kD, about 26kD, about 25kD, about 24kD, about 23kD, about 22kD, about 21kD, or about 20kD. A typical approach to small size is by using single domain antibody (sdAb) fragments for each of the domains. For example, a particular BCMA trispecific antigen binding protein has an anti-CD3 sdAb, an anti-ALB sdAb, and an sdAb for BCMA. This reduces the size of a typical BCMA trispecific antigen binding protein to less than 40 kD. Thus, in some embodiments, the domains of the BCMA-targeting trispecific protein are all single domain antibody (sdAb) fragments. In other embodiments, the BCMA-targeting trispecific proteins described herein include small molecule entity (SME) binders for ALB and / or BCMA. The SME binders are small molecules, on average about 500-2000 Da in size, that are attached to the BCMA-targeting trispecific protein by known methods such as sortase ligation or conjugation. In these examples, one of the domains of the BCMA-targeting trispecific antigen binding protein is a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 482).To bind an SME binder to a BCMA-targeted trispecific antigen binding protein having a sortase recognition sequence, the protein is incubated with a sortase and an SME binder, whereby the sortase binds the SME binder to the recognition sequence. Known SME binders include MIP-1072 and MIP-1095, which bind to BCMA.

[0033] In yet other embodiments, the BCMA-binding domain of the BCMA-targeting trispecific proteins described herein comprises a knottin peptide for binding to BCMA. Knottins are disulfide-stable peptides with a cysteine ​​knot scaffold and have an average size of about 3.5 kD. Knottins have been contemplated for binding to certain tumor molecules, such as BCMA. In further embodiments, the BCMA-binding domain of the BCMA-targeting trispecific proteins described herein comprises a natural BCMA ligand.

[0034] Another feature of the BCMA-targeting trispecific proteins described herein is that they are single polypeptide designs with flexible domain connections. This allows for easy production and manufacturing of the BCMA-targeting trispecific proteins, since they can be encoded by a single cDNA molecule that can be easily incorporated into a vector. Furthermore, because the BCMA-targeting trispecific proteins described herein are monomeric single polypeptide chains, there is no problem with chain pairing and no requirement for dimerization. The BCMA-targeting trispecific proteins described herein have a reduced tendency to aggregate, unlike other reported molecules, such as bispecific proteins with Fcγ immunoglobulin domains.

[0035] In the BCMA targeting trispecific proteins described herein, the domains are linked by internal linkers L1 and L2, where L1 binds to the first and second domains of the BCMA targeting trispecific protein, and L2 binds to the second and third domains of the BCMA targeting trispecific protein. The linkers L1 and L2 have an optimized length and / or amino acid composition. In some embodiments, the linkers L1 and L2 have the same length and amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, the internal linkers L1 and / or L2 are "short", i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain instances, the internal linker consists of about 12 amino acid residues or less. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linkers L1 and / or L2 are "long", i.e., "consist of" 15, 20, or 25 amino acid residues. In some embodiments, these internal linkers consist of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. With regard to the amino acid composition of the internal linkers L1 and L2, peptides are selected with properties that confer flexibility to the BCMA targeting trispecific protein, do not interfere with the binding domains, as well as resist cleavage from proteases. For example, glycine and serine residues generally confer protease resistance. Examples of suitable internal linkers for linking domains in a BCMA targeting trispecific protein include, but are not limited to, (GS) n (SEQ ID NO: 472), (GGS) n (SEQ ID NO: 473), (GGGS) n (SEQ ID NO:474), (GGSG) n (SEQ ID NO:475), (GGSGG) n (SEQ ID NO:476), (GGGGS) n (SEQ ID NO: 477), (GGGGG) n (SEQ ID NO: 478), or (GGG) n(SEQ ID NO:479), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the internal linker L1 and / or linker L2 is (GGGGS)4 (SEQ ID NO:480) or, alternatively, (GGGGS)3 (SEQ ID NO:481).

[0036] CD3-binding domain The specificity of T cell responses is mediated by the recognition of antigens (displayed in the context of the major histocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 is a protein complex present on the cell surface that includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. To comprise the complete TCR, CD3 binds together with the TCR α (alpha) and β (beta) chains, as well as CD3ζ (zeta). Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, triggers T cell activation that resembles T cell receptor engagement but is independent of the specificity typical of that clone.

[0037] In one aspect, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds to human CD3. In some embodiments, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3γ. In some embodiments, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3δ. In some embodiments, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3ε.

[0038] In further embodiments, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds the TCR. In one example, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds the alpha chain of the TCR. In one example, the BCMA-targeting trispecific proteins described herein comprise a domain that specifically binds the beta chain of the TCR.

[0039] In some embodiments, the CD3 binding domain of the BCMA trispecific antigen binding protein can be any domain that binds to CD3, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some instances, it is beneficial for the CD3 binding domain to be derived from the same species in which the BCMA trispecific antigen binding protein will ultimately be used. For example, for human use, it may be beneficial for the CD3 binding domain of the BCMA trispecific antigen binding protein to include human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0040] Thus, in one aspect, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises the light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of one or more (e.g., all three) of the humanized or human-CD3 binding domains described herein, and / or the heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of one or more (e.g., all three) of the humanized or human anti-CD3 binding domains described herein, such as one or more, e.g., all three LC CDRs and one or more, e.g., all three HC CDRs.

[0041] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, the light chain variable region specific for CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In some examples, the light chain framework region is a λ (lambda) light chain framework. In other examples, the light chain framework region is a κ (kappa) light chain framework.

[0042] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, where the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0043] In certain examples, the complementarity determining regions of the heavy and / or light chains are selected from, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66, or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12. 5, derived from known anti-CD3 antibodies such as F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0044] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of the amino acid sequences provided herein. As used herein, a "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable regions of the light and heavy chains are contiguously linked by a short, flexible polypeptide linker, expressible as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In embodiments, the anti-CD3 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, but not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity to an amino acid sequence provided herein; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, but not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD3 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein is linked to a heavy chain variable region comprising an amino acid sequence described herein by an scFv linker. The light and heavy chain variable regions of the scFv can be, for example, in either of the following orientations: light chain variable region-scFv linker-heavy chain variable region, or heavy chain variable region-scFv linker-light chain variable region.

[0045] In some examples, scFvs that bind to CD3 are prepared according to known methods. For example, scFv molecules can be made by linking together VH and VL regions using a flexible polypeptide linker. The scFv molecules include an scFv linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can bind intermolecularly with another variable domain to form a CD3 binding site. In certain embodiments, such scFv linkers are "short", i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the scFv linker consists of about 12 or less amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the scFv linker, a peptide is selected that provides flexibility and does not interfere with the variable domains, as well as allows interchain folding to join the two variable domains to form a functional CD3 binding site. For example, scFv linkers that contain glycine and serine residues generally provide protease resistance. In some embodiments, the linker in the scFv contains glycine and serine residues. The amino acid sequence of the scFv linker can be optimized, for example, by phage display methods to improve CD3 binding and production yield of the scFv. Examples of peptide scFv linkers that are suitable for linking the variable light and variable heavy domains in scFvs include, but are not limited to, (GS) n (SEQ ID NO: 472), (GGS) n (SEQ ID NO: 473), (GGGS) n (SEQ ID NO:474), (GGSG) n (SEQ ID NO:475), (GGSGG) n (SEQ ID NO:476), (GGGGS) n (SEQ ID NO: 477), (GGGGG) n (SEQ ID NO: 478), or (GGG) n(SEQ ID NO: 479), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the internal linker L1 and / or linker L2 is (GGGGS)4 (SEQ ID NO: 480), or (GGGGS)3 (SEQ ID NO: 481). Variation in linker length may retain or enhance activity, resulting in superior efficacy in activity studies.

[0046] In some embodiments, the CD3 binding domain of the BCMA trispecific antigen binding protein has a K of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. D In some embodiments, the CD3 binding domain of the BCMA trispecific antigen binding protein has a K of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. D for CD3 epsilon, gamma, or delta. In a further embodiment, the CD3 binding domain of the BCMA trispecific antigen binding protein has low affinity for CD3 (i.e., about 100 nM or greater).

[0047] Affinity for binding to CD3 can be determined, for example, by the ability of the BCMA trispecific antigen binding protein itself or its CD3 binding domain to bind to CD3 coated on an assay plate, CD3 displayed on a microbial cell surface, CD3 in solution, etc. The binding activity of the BCMA trispecific antigen binding protein of the present disclosure itself or its CD3 binding domain to CD3 can be analyzed by immobilizing a ligand (e.g., CD3) or the BCMA trispecific antigen binding protein itself or its CD3 binding domain to beads, substrates, cells, etc. The agent can be added to the binding partner in an appropriate buffer and incubated for a period of time at a predetermined temperature. After washing to remove unbound material, the binding protein can be released, for example, with SDS, high pH buffer, etc., and analyzed, for example, by surface plasmon resonance (SPR).

[0048] Half-life extension domain Contemplated herein are domains that extend the half-life of an antigen-binding domain, including, but not limited to, albumin-binding domains, Fc domains, small molecules, and other half-life extending domains known in the art.

[0049] Human albumin (ALB) (molecular mass of about 67 kDa) is the most abundant protein in plasma, present at about 50 mg / ml (600 μM), with a half-life of about 20 days in humans. ALB helps maintain plasma pH, contributes to colloidal blood pressure, functions as a carrier for many metabolites and fatty acids, and serves as the major drug transport protein of plasma.

[0050] Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment resulted in a 25-fold and 58-fold increase in in vivo clearance and a 26-fold and 37-fold increase in half-life when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin was acylated with fatty acids to promote association with albumin, a delayed effect was observed when injected subcutaneously in rabbits or pigs. Collectively, such studies demonstrate the link between albumin binding and delayed action.

[0051] In one aspect, the BCMA-targeting trispecific protein described herein comprises a half-life extending domain, e.g., a domain that specifically binds to ALB. In some embodiments, the ALB-binding domain of the BCMA trispecific antigen-binding protein can be any domain that binds to ALB, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the ALB-binding domain is a single-chain variable fragment (scFv) specific for HSA, a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a single-domain antibody derived from camelid (VHH), a peptide, a ligand, or a small molecule entity. In certain embodiments, the ALB-binding domain is a single-domain antibody. In other embodiments, the HSA-binding domain is a peptide. In some embodiments, the HSA-binding domain is a small molecule. It is contemplated that the HSA binding domain of the BCMA trispecific antigen binding protein is fairly small, in some embodiments being 25 kD or less, 20 kD or less, 15 kD or less, or 10 kD or less. In certain instances, the ALB binding domain, when a peptide or small molecule entity, is 5 kD or less.

[0052] The half-life prolonging domain of the BCMA trispecific antigen binding protein results in changes in the pharmacokinetics and pharmacodynamics of the BCMA trispecific antigen binding protein itself. As described above, the half-life prolonging domain increases the elimination half-life. The half-life prolonging domain also changes the pharmacokinetic properties of the trispecific antigen binding protein, including changes in tissue distribution, penetration, and diffusion. In some embodiments, the half-life prolonging domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissues, and enhanced efficacy compared to proteins without the half-life prolonging domain. In one embodiment, the method of treatment effectively and efficiently utilizes reduced amounts of the trispecific antigen binding protein, resulting in reduced side effects, such as reduced cytotoxicity to non-tumor cells.

[0053] Furthermore, the binding affinity of the half-life prolonging domain can be selected to target the specific elimination half-life of a particular trispecific antigen binding protein. Thus, in some embodiments, the half-life prolonging domain has a high binding affinity. In other embodiments, the half-life prolonging domain has a moderate binding affinity. In yet other embodiments, the half-life prolonging domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (medium), and above 100 nM (low). As described above, the binding affinity to ALB is determined by known methods, such as surface plasmon resonance (SPR).

[0054] In some embodiments, the ALB binding domain described herein comprises a single domain antibody.

[0055] B-cell maturation antigen (BCMA) binding domain B-cell maturation antigen (BCMA, TNFRSF17, CD269) is a transmembrane protein belonging to the tumor necrosis family receptor (TNFR) superfamily that is expressed primarily on terminally differentiated B cells. BCMA expression is restricted to the B-cell lineage, present primarily on plasma cells and plasmablasts, and to some extent on memory B cells, but is virtually absent from peripheral naive B cells. BCMA is expressed on multiple myeloma (MM) cells, leukemia cells, and lymphoma cells.

[0056] BCMA was identified by molecular analysis of the t(4;16)(q26;p13) translocation found in human intestinal T-cell lymphomas, and the in-frame sequence was mapped to chromosomal band 16p13.1.

[0057] The human BCMA cDNA has an open reading frame of 552 bp encoding a 184 amino acid polypeptide. The BCMA gene is organized into three exons separated by two introns, each flanked by a GT donor and an AG receptor consensus splice site, and encodes a 1.2 kb transcript. The structure of the BCMA protein comprises an integral transmembrane protein based on a central 24 amino acid hydrophobic region in an α-helical structure.

[0058] The mouse BCMA gene is located on chromosome 16 syntenic to the human 16p13 region and further contains three exons separated by two introns. The gene encodes a 185 amino acid protein. Mouse BCMA mRNA is expressed as a 404 bp transcript with maximal levels in plasmacytoma cells (J558) and at moderate levels in the A20 B cell lymphoma line. Mouse BCMA mRNA transcripts were detected at lower levels in T cell lymphoma (EL4, BW5147) and dendritic cell (CB1D6, D2SC1) lines, in contrast to human cell lines of T cell and dendritic cell origin. The mouse BCMA cDNA sequence has 69.3% nucleotide identity to the human BCMA cDNA sequence, with slightly higher identity (73.7%) when comparing the coding regions between these two cDNA sequences. The mouse BCMA protein is 62% identical to the human BCMA protein and, like human BCMA, contains one hydrophobic region that may be an integral transmembrane segment. The N-terminal 40 amino acid domain of both mouse and human BCMA proteins has six conserved cysteine ​​residues consistent with the formation of a cysteine ​​repeat motif found in the extracellular domain of TNFRs. Similar to members of the TNFR superfamily, BCMA proteins contain conserved aromatic residues 4-6 residues C-terminal from the first cysteine.

[0059] BCMA is not expressed on the cell surface, but rather is located in the Golgi apparatus. The amount of BCMA expression is proportional to the stage of differentiation of the cell (highest in plasma cells).

[0060] It is involved in B cell development and homeostasis through interaction with its ligands BAFF (B cell activating factor, also called TALL-1 or TNFSF13B) and APRIL (A proliferation-inducing ligand).

[0061] BCMA, together with its family members TACI (transmembrane activator and cyclophilin ligand interactor) and BAFF-R (B cell-activating factor receptor, also known as tumor necrosis factor receptor superfamily member 13C), regulates various aspects of humoral immunity, B cell development and homeostasis. BCMA expression appears somewhat later in B cell differentiation and contributes to the long-term survival of plasmablasts and plasma cells in the bone marrow. BCMA further supports the growth and survival of multiple myeloma (MM) cells.

[0062] BCMA is primarily known for its functional activity in mediating the survival of plasma cells that maintain long-term humoral immunity.

[0063] There is a need to have treatment options for solid tumor diseases associated with overexpression of BCMA, such as cancer, multiple myeloma, leukemia, and lymphoma. The present disclosure provides, in certain embodiments, single domain proteins that specifically bind to BCMA on the surface of tumor target cells.

[0064] The design of the BCMA-targeting trispecific proteins described herein allows for the flexibility of the binding domain for the BCMA protein in that it can be any type of binding domain, including but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding domain for BCMA is a single chain variable fragment (scFv), a single domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single domain antibody derived from camelid. In other embodiments, the binding domain for BCMA is a non-Ig binding domain, i.e., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and antibody mimetics such as monobodies. In a further embodiment, the binding domain for BCMA is a ligand or peptide that binds to or associates with BCMA. In yet a further embodiment, the binding domain for BCMA is a knottin. In yet a further embodiment, the binding domain for BCMA is a small molecule entity.

[0065] In some embodiments the BCMA binding domain binds to a protein comprising the sequence of SEQ ID NO: 469, 470, or 471. In some embodiments the BCMA binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 469, 470, or 471.

[0066] In some embodiments, the BCMA binding domain is an anti-BCMA antibody or antibody variant. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies described herein. In certain embodiments, amino acid sequence variants of the anti-BCMA antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-BCMA antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody.

[0067] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., antigen-binding). In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into the antibody of interest, and the products are screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for the preparation of antibody variants.

[0068] In another example of substitutions to create variant anti-BCMA antibodies, one or more hypervariable region residues of a parent antibody are substituted. Generally, variants are selected based on the desired improved properties compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, increased pH-dependent binding.

[0069] In some embodiments, the BCMA-binding domain of the BCMA-targeting trispecific protein is a single domain antibody specific for BCMA, such as a heavy chain variable domain (VH), a variable domain of a llama-derived sdAb (VHH), a peptide, a ligand, or a small molecule entity. In some embodiments, the BCMA-binding domain of the BCMA-targeting trispecific protein described herein is any domain that binds to BCMA, including but not limited to a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In certain embodiments, the BCMA-binding domain is a single domain antibody. In other embodiments, the BCMA-binding domain is a peptide. In some embodiments, the BCMA-binding domain is a small molecule.

[0070] In general, it should be noted that the term single domain antibody, as used herein in its broadest sense, is not limited to a particular biological source or a particular preparation method. A single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be any in the art or future single domain antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. For example, in some embodiments, single domain antibodies of the disclosure are obtained by: (1) isolation of a VHH domain of a naturally occurring heavy chain antibody; (2) expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanization" of a naturally occurring VHH domain, or expression of a nucleic acid encoding such a humanized VHH domain; (4) "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species such as human, or expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" or "Dab", or expression of a nucleic acid encoding such a camelized VH domain; (6) use of synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) preparation of a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the art, followed by expression of the nucleic acid obtained as above; and / or (8) any combination of one or more of the foregoing.

[0071] In one embodiment, the single domain antibody corresponds to a VHH domain of a naturally occurring heavy chain antibody against BCMA. As further described herein, such a VHH sequence is typically generated or obtainable by appropriately immunizing a species of llama with BCMA (i.e. to generate an immune response and / or heavy chain antibodies against BCMA), by obtaining a suitable biological sample from said llama (such as a blood sample, a serum sample or a sample of B cells) and generating a VHH sequence against BCMA starting from said sample using any suitable technique known in the art.

[0072] In another embodiment, such naturally occurring VHH domains against BCMA are obtained from a naive library of camelid VHH sequences, for example by screening such a library with BCMA or with at least one part, fragment, antigenic determinant or epitope thereof using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020 and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example VHH libraries are obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0073] In a further embodiment, yet another technique for obtaining VHH sequences against BCMA comprises appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e. to generate an immune response and / or heavy chain antibodies against BCMA), obtaining a suitable biological sample from said transgenic mammal (a blood sample, a serum sample, or a sample of B cells), and generating VHH sequences against BCMA starting from said sample using any suitable technique known in the art. For example, for this purpose heavy chain antibody expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used.

[0074] In some embodiments, the anti-BCMA single domain antibodies of the BCMA-targeting trispecific protein include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain, but "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and in particular within the framework sequences) with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional 4-chain antibody of human origin (e.g. as shown above). This can be done in any manner known in the art that will be clear to the skilled artisan, for example on the basis of the further description below. Again, such humanized anti-BCMA single domain antibodies of the present disclosure can be obtained in any suitable manner known per se (i.e. as shown in points (1) to (8) above), and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material. In some further embodiments, the single domain anti-BCMA antibodies comprise single domain antibodies having an amino acid sequence which corresponds to the amino acid sequence of a naturally occurring VH domain as described herein, but which has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional 4-chain antibody with one or more of the amino acid residues occurring at the corresponding positions in the VHH domain of a heavy chain antibody. Such "camelized" substitutions are preferably inserted at amino acid positions which form and / or are present at the VH-VL interface and / or at residues prominent in the so-called Camelidae family (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as starting material or starting point for generating or designing the camelized single domain is a VH sequence, preferably from a mammal, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that in certain embodiments, such camelized anti-BCMA single domain antibodies of the present disclosure are obtained in a manner known in the art (i.e. as indicated in points (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using a naturally occurring VH domain-containing polypeptide as starting material. For example, as further described herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, and then altering one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed to provide the desired anti-BCMA single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of each of the desired humanized or camelized anti-BCMA single domain antibodies of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques of peptide synthesis. In some embodiments a nucleotide sequence encoding the desired humanized or camelized anti-BCMA single chain antibody of the disclosure is designed based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques for nucleic acid synthesis, and the nucleic acid so obtained is then expressed using known expression techniques to obtain the desired anti-BCMA single domain antibody of the disclosure.

[0075] Other suitable methods and techniques for obtaining an anti-BCMA single domain antibody of the disclosure and / or a nucleic acid encoding said anti-BCMA single domain antibody, starting from a naturally occurring VH or VHH sequence, include, for example, combining in a suitable manner one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences to provide an anti-BCMA single domain antibody of the disclosure or a nucleotide sequence or nucleic acid encoding same.

[0076] In some embodiments, the BCMA binding domain is an anti-BCMA specific antibody comprising heavy chain variable complementarity determining region CDR1, heavy chain variable CDR2, heavy chain variable CDR3, light chain variable CDR1, light chain variable CDR2, and light chain variable CDR3. In some embodiments, the BCMA binding domain comprises any domain that binds to BCMA, including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragments, fragments composed of one or more CDRs, single chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some embodiments, the BCMA binding domain is a single domain antibody. In some embodiments, the anti-BCMA single domain antibody comprises the heavy chain variable complementarity determining regions (CDRs), CDR1, CDR2, and CDR3.

[0077] In some embodiments, a BCMA binding protein of the disclosure is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The r1 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 26, 27, 28, 29, 30, 31, 32, 33, and 34; the r2 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63; and the r3 residues of BCMA binding proteins of the disclosure include, for example, amino acid residues 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, and 108. In some embodiments, the BCMA binding protein comprises an amino acid sequence selected from SEQ ID NOs: 346-460.

[0078] In one embodiment, CDR1 does not include the amino acid sequence of SEQ ID NO: 599. In one embodiment, CDR2 does not include the amino acid sequence of SEQ ID NO: 600. In one embodiment, CDR3 does not include the amino acid sequence of SEQ ID NO: 601.

[0079] In some embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. An exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:6. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:7. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:9. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:10. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:11. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:12. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:13. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:14. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:15. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:30.Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:31. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:32. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:33. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:34. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:35. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:36. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:37. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:38. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:39. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:40. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:41. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:42. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:43. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:44. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 47. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 49. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 50. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 51. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 52. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 55. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 56. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 57. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 58. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:59.Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:60. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:61. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:62. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:63. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:64. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:65. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:66. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:67. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:68. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:69. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:70. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:71. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:72. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:73. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 74. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 75. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 76. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 77. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 78. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 79. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 80. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 82. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 83. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 84. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 85. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 86. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 87. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:88.Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:89. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:90. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:91. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:92. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:93. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:94. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:95. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:96. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:97. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:98. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:99. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:100. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:101. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:102. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 103. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 104. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 105. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 106. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 107. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 108. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 109. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 110. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 111. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 112. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 113. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 114. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 115. Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:116.Another exemplary CDR1 comprises the amino acid sequence set forth in SEQ ID NO:117.

[0080] In some embodiments, the CDR2 comprises the sequence set forth in SEQ ID NO:2, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:2. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:118. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:119. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:120. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:121. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:122. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:123. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:124. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:125. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:126. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:127. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:128. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 129. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 130. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 131. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 132. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 133. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 134. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 135. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 136. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 137. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 138. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 139. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 140. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 141. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 142. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 143.Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 144. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 145. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 146. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 147. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 148. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 149. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 150. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 151. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 152. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 153. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 154. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 155. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 156. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 157. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 158. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 159. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 160. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 161. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 162. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 164. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 165. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 166. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 167. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 168. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 169. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 170. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171.Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 172. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 173. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 174. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 175. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 176. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 177. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 178. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 179. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 180. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 181. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 182. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 183. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 184. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 185. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 186. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 187. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 188. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 189. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 190. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 191. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 192. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 193. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 194. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 195. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 196. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 197. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 198. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 199.Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:200. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:201. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:202. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:203. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:204. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:205. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:206. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:207. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:208. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:209. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:210. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:211. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:212. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:213. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:214. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:215. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:216. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:217. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:218. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:219. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:220. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:221. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:222. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:223. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:224. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO:225. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 226. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 227.Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 228. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 229. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 230. Another exemplary CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 231.

[0081] In some embodiments, the CDR3 comprises the sequence set forth in SEQ ID NO:3, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in SEQ ID NO:3. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:232. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:233. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:234. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:235. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:236. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:237. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:238. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:239. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:240. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:241. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:242. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 243. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 244. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 245. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 246. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 247. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 248. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 249. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 250. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 251. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 252. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 253. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 254. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 255. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 256. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 257.Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 258. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 259. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 260. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 261. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 262. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 263. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 264. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 265. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 266. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 267. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 268. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 269. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 270. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 271. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 272. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 273. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 274. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 275. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 276. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 277. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 278. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 279. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 280. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 281. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 282. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 283. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 284. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 285.Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:286. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:287. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:288. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:289. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:290. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:291. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:292. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:293. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:294. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:295. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:296. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:297. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO:298. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 299. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 300. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 301. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 302. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 303. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 304. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 305. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 306. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 307. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 308. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 309. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 310. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 311. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 312. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 313.Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 314. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 315. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 316. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 317. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 318. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 319. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 320. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 321. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 322. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 323. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 324. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 325. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 326. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 327. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 328. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 329. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 330. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 331. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 332. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 333. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 334. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 335. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 336. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 337. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 338. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 339. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 340. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 341.Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 342. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 343. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 344. Another exemplary CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 345.

[0082] In various embodiments, the BCMA binding proteins of the disclosure have a CDR1 having an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:4-117.

[0083] In various embodiments, the BCMA binding proteins of the disclosure have a CDR2 having an amino acid sequence that is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 118-231.

[0084] In various embodiments, the complementarity determining regions of the BCMA binding proteins of the disclosure have a CDR3 having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:232-345.

[0085] In various embodiments, the BCMA binding proteins of the disclosure have an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 346-460.

[0086] In various embodiments, the BCMA binding proteins of the disclosure have a framework 1 (f1) having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:461 or SEQ ID NO:462.

[0087] In various embodiments, the BCMA binding proteins of the disclosure have a framework 2 (f2) having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:463.

[0088] In various embodiments, the BCMA binding proteins of the disclosure have a framework 3 (f3) having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:464 or SEQ ID NO:465.

[0089] In various embodiments, the BCMA binding proteins of the disclosure have a framework 4 (f4) having an amino acid sequence that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:466 or SEQ ID NO:467.

[0090] In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 346. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 347. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 348. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 349. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 350. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 351. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 352. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 353. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 354. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 355. In some embodiments, the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 356. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 357. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 358. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 359.

[0091] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 360. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 361. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 362. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 363. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 364. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 365. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 366. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 367. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 368. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 369.

[0092] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 370. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 371. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 372. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 373. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 374. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 375. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 376. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 377. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 378. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 379.

[0093] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 380. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 381. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 382. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 383. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 384. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 385. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 386. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 387. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 388. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 389.

[0094] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 390. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 391. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 392. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 393. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 394. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 395. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 396. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 397. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 398. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 399.

[0095] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 400. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 401. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 402. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 403. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 404. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 405. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 406. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 407. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 408. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 409.

[0096] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 410. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 411. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 412. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 413. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 414. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 415. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 416. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 417. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 418. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 419.

[0097] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 420. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 421. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 422. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 423. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 424. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 425. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 426. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 427. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 428. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 429.

[0098] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 430. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 431. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 432. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 433. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 434. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 435. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 436. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 437. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 438. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 439.

[0099] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 440. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 441. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 442. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 443. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 444. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 445. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 446. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 447. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 448. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 449.

[0100] In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 450. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 451. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 452. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 453. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 454. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 455. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 456. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 457. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 458. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 459. In some embodiments the BCMA binding protein is a single domain antibody comprising the sequence of SEQ ID NO: 460.

[0101] The BCMA binding proteins described herein can bind to human BCMA with an hKd ranging from about 0.1 nM to about 500 nM. In some embodiments, the hKd range is from about 0.1 nM to about 450 nM. In some embodiments, the hKd range is from about 0.1 nM to about 400 nM. In some embodiments, the hKd range is from about 0.1 nM to about 350 nM. In some embodiments, the hKd range is from about 0.1 nM to about 300 nM. In some embodiments, the hKd range is from about 0.1 nM to about 250 nM. In some embodiments, the hKd range is from about 0.1 nM to about 200 nM. In some embodiments, the hKd range is from about 0.1 nM to about 150 nM. In some embodiments, the hKd range is from about 0.1 nM to about 100 nM. In some embodiments, the hKd range is from about 0.1 nM to about 90 nM. In some embodiments, the hKd range is about 0.2 nM to about 80 nM. In some embodiments, the hKd range is about 0.3 nM to about 70 nM. In some embodiments, the hKd range is about 0.4 nM to about 50 nM. In some embodiments, the hKd range is about 0.5 nM to about 30 nM. In some embodiments, the hKd range is about 0.6 nM to about 10 nM. In some embodiments, the hKd range is about 0.7 nM to about 8 nM. In some embodiments, the hKd range is about 0.8 nM to about 6 nM. In some embodiments, the hKd range is about 0.9 nM to about 4 nM. In some embodiments, the hKd range is about 1 nM to about 2 nM.

[0102] In some embodiments, any of the aforementioned BCMA binding domains are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is further six consecutive histidine residues, also referred to as a His tag or 6X-his (His-His-His-His-His-His; SEQ ID NO: 471).

[0103] In certain embodiments, the BCMA binding domain of the present disclosure preferentially binds membrane-bound BCMA over soluble BCMA. Membrane-bound BCMA refers to the presence of BCMA in or on the cell membrane surface of cells expressing BCMA. Soluble BCMA refers to BCMA that is no longer present in or on the cell membrane surface of cells that express or have expressed BCMA. In certain examples, soluble BCMA is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the BCMA binding domain binds membrane-bound BCMA at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more than soluble BCMA. In one embodiment, the BCMA-targeting trispecific antigen binding protein of the present disclosure preferentially binds membrane-bound BCMA 30-fold greater than soluble BCMA. Determination of preferential binding of an antigen binding protein to membrane-bound BCMA over soluble BCMA can be readily determined using assays well known in the art.

[0104] Trispecific Proteins The BCMA binding trispecific protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 483-597.

[0105] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 483. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 484. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 485. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 486. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 487. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 488. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 489. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 490. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 491. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 492. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 493. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 494. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 495. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 496. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 497. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 498. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 499.

[0106] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 500. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 501. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 502. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 503. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 504. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 505. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 506. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 507. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 508. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 509.

[0107] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 510. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 511. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 512. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 513. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 514. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 515. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 516. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 517. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 518. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 519.

[0108] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 520. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 521. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 522. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 523. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 524. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 525. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 526. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 527. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 528. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 529.

[0109] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 530. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 531. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 532. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 533. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 534. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 535. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 536. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 537. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 538. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 539. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO:540.

[0110] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 541. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 542. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 543. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 544. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 545. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 546. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 547. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 5048. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 549. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 550.

[0111] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 551. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 552. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 553. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 554. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 555. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 556. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 557. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 558. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 559.

[0112] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 560. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 561. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 562. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 563. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 564. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 565. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 566. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 567. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 568. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 569.

[0113] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 570. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 571. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 572. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 573. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 574. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 575. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 576. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 577. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 578. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 579.

[0114] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 580. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 581. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 582. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 583. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 584. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 585. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 586. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 587. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 588. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 589.

[0115] In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 590. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 591. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 592. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 593. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 594. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 595. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 596. In one example, the BCMA binding trispecific protein comprises the amino acid sequence of SEQ ID NO: 597.

[0116] Polynucleotides encoding BCMA-targeting trispecific proteins In some embodiments, polynucleotide molecules are further provided that encode the anti-BCMA trispecific binding proteins described herein. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0117] The polynucleotide molecule is constructed by known methods, such as by combining genes encoding the three binding domains, separated by peptide linkers or in other embodiments directly linked by peptide bonds, into one genetic construct operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems, such as CHO cells. In embodiments where the BCMA binding domain is a small molecule, the polynucleotide comprises genes encoding the CD3 binding domain and the half-life prolonging domain. In embodiments where the half-life prolonging domain is a small molecule, the polynucleotide comprises genes encoding the domains that bind to CD3 and BCMA. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. The promoter is selected to facilitate expression of the polynucleotide in the respective host cell.

[0118] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods. Among others, vectors of interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0119] A variety of expression vector / host systems can be utilized to contain and express the polynucleotides encoding the polypeptides of the described trispecific antigen binding proteins. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0120] Thus, the BCMA-targeting trispecific proteins described herein are produced, in some embodiments, by introducing vectors encoding such proteins into a host cell and culturing said host cell under conditions whereby the protein domains can be expressed, isolated and optionally further purified.

[0121] Incorporation into chimeric antigen receptors (CARs) The BCMA-targeting trispecific antigen binding proteins of the present disclosure can be incorporated into chimeric antigen receptors (CARs) in certain instances. Engineered immune effector cells, such as T cells or NK cells, can be used to express CARs comprising anti-BCMA-targeting trispecific proteins containing anti-BCMA single domain antibodies as described herein. In one embodiment, a CAR comprising an anti-BCMA-targeting trispecific protein as described herein is linked to a transmembrane domain via a hinge region and to a costimulatory domain, such as OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or a functional signaling domain obtained from 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain, such as 4-1BB and / or CD3 zeta.

[0122] Modification of BCMA trispecific proteins The BCMA trispecific targeting proteins described herein encompass derivatives or analogs in which (i) amino acids are replaced with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence, or a sequence for purification of the protein.

[0123] Exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, e.g., arginylation, and ubiquitination.

[0124] Modifications may be made anywhere in the BCMA-targeting trispecific proteins described herein, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Certain common peptide modifications useful for modifying BCMA-targeting trispecific proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups, or both, in the polypeptide by covalent modifications, and ADP-ribosylation.

[0125] Pharmaceutical Compositions In some embodiments, pharmaceutical compositions are also provided that include the anti-BCMA trispecific binding proteins described herein, vectors comprising a polynucleotide encoding a polypeptide of a BCMA trispecific protein, or a host cell transformed with this vector, and at least one pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable carrier" includes, but is not limited to, a carrier that does not interfere with the effectiveness of the biological activity of the components and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile liquids, and the like. Such carriers can be formulated by conventional methods and administered to a subject in an appropriate dosage. Preferably, the compositions are sterilized. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents. Further embodiments provide one or more of the above BCMA trispecific proteins packaged in lyophilized form or packaged in an aqueous medium.

[0126] In some embodiments of the pharmaceutical composition, the BCMA trispecific targeting protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the BCMA trispecific antigen binding protein is bound to a liposome. In some instances, the BCMA trispecific antigen binding protein is bound to the surface of a liposome. In some instances, the BCMA trispecific antigen binding protein is encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0127] The BCMA-targeting trispecific proteins described herein are intended for use as medicines. Administration is accomplished by different methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The administration regimen is determined by the attending physician and other clinical factors. The dosage for a patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of treatment, health status, and other drugs administered simultaneously. An "effective amount" refers to an amount of active ingredient sufficient to affect the course and severity of the disease, thereby causing a reduction or remission of such pathology, and can be determined using known methods.

[0128] In some embodiments, the BCMA-targeting trispecific proteins of the present disclosure are administered at a dosage of up to 10 mg / kg once a week. In some cases, the dosage ranges from about 1 ng / kg to about 10 mg / kg. In some embodiments, the dose ranges from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 25 μg / kg, about 25 μg / kg, about 35 μg / kg, about 45 μg / kg, about 50 μg / kg, about 65 μg / kg, about 85 μg / kg, about 80 μg / kg, about 1 ... g / kg to about 5 μg / kg, about 2 μg / kg to about 10 μg / kg, about 7 μg / kg to about 15 μg / kg, about 12 μg / kg to about 25 μg / kg, about 20 μg / kg to about 50 μg / kg, about 35 μg / kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg / kg to about 0.1 mg / kg, and about 0.095 mg / kg to about 10 mg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg; about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. The frequency of administration is, in some embodiments, about less than daily, every other day, less than once a day, twice a week, every week, once every 7 days, every other week, every 3 weeks, every 4 weeks, or about once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or longer.

[0129] In some embodiments, the BCMA-targeting trispecific protein of the disclosure may be administered in a concentration of about 1 μg to about 100 μg, about 1 μg to about 500 μg, about 1 μg to about 1 mg, about 1 μg to about 2 mg, about 1 μg to about 5 mg, about 1 μg to about 10 mg, about 1 μg to about 100 mg, about 100 μg to about 500 μg, about 100 μg to about 1 mg, about 100 μg to about 2 mg, about 100 μg to about 5 mg, about 100 μg to about 10 mg, about 100 μg to about 10 0 mg, about 500 μg to about 1 mg, about 500 μg to about 2 mg, about 500 μg to about 5 mg, about 500 μg to about 10 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 10 mg, about 2 mg to about 100 mg, about 5 mg to about 10 mg, about 5 mg to about 100 mg, or about 10 mg to about 100 mg.

[0130] In some embodiments, the BCMA-targeting trispecific proteins of the disclosure may be administered in a concentration of about 5 μg to about 15 μg, about 5 μg to about 30 μg, about 5 μg to about 90 μg, about 5 μg to about 270 μg, about 5 μg to about 810 μg, about 5 μg to about 1620 μg, about 5 μg to about 2150 μg, about 5 μg to about 2860 μg, about 15 μg to about 30 μg, about 15 μg to about 90 μg, about 15 μg to about 270 μg, about 15 μg to about 810 μg, about 15 μg to about 1620 μg, about 15 μg to about 2150 μg, about 15 μg to about 2860 μg, about 30 μg to about 90 μg, about 30 μg to about 270 μg, about 30 μg to about 810 μg. g, about 30μg to about 1620μg, about 30μg to about 2150μg, about 30μg to about 2860μg, about 90μg to about 270μg, about 90μg to about 8 10μg, about 90μg to about 1620μg, about 90μg to about 2150μg, about 90μg to about 2860μg, about 270μg to about 810μg, about 270 The therapeutic agent is administered in a dosage of about 810 μg to about 1620 μg, about 810 μg to about 2150 μg, about 270 μg to about 2860 μg, about 1620 μg to about 2150 μg, or about 1620 μg to about 2860 μg.

[0131] The BCMA-targeting trispecific proteins described herein can be administered using different dosages. In some embodiments, the BCMA-targeting trispecific proteins of the present disclosure are administered according to a schedule comprising the steps of (i) administering a first dose of the BCMA-targeting trispecific protein, and (ii) administering a second dose of the BCMA-targeting trispecific protein, the second dose being higher than the first dose. In some embodiments, the schedule further comprises a step (iii) of administering a third dose of the BCMA-targeting trispecific protein, the third dose being higher than the second dose. In some embodiments, the schedule further comprises a step (iv) of administering a fourth dose of the BCMA-targeting trispecific protein, the fourth dose being higher than the third dose. In some embodiments, the schedule further comprises a step (v) of administering a fifth dose of the BCMA-targeting trispecific protein, the fifth dose being higher than the fourth dose.

[0132] In some embodiments, the first dose is from about 1 μg to about 100 μg, from about 1 μg to about 500 μg, from about 1 μg to about 1 mg, from about 1 μg to about 2 mg, from about 1 μg to about 5 mg, from about 1 μg to about 5 mg, from about 1 μg to about 8 mg, from about 1 μg to about 10 mg, from about 1 μg to about 50 mg, from about 1 μg to about 100 mg, from about 100 μg to about 500 μg, or from about 100μg to about 1mg, about 100μg to about 2mg, about 100μg to about 5mg, about 100μg to about 5mg, about 100μg to about 8mg, about 100μg to about 10mg , about 100μg to about 50mg, about 100μg to about 100mg, about 500μg to about 1mg, about 500μg to about 2mg, about 500μg to about 5mg, about 500μg to about about 5 mg, about 500 μg to about 8 mg, about 500 μg to about 10 mg, about 500 μg to about 50 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 8 mg, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 8 mg, about 2 mg to about 10 mg, about 2 mg to about 50 mg, about 2 mg to about 100 mg, about 5 mg to about 8 mg, about 5 mg to about 10 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 8 mg to about 10 mg, about 8 mg to about 50 mg, about 8 mg to about 100 mg, about 10 mg to about 50 mg, or about 50 mg to about 100 mg. In some embodiments, the first dose is about 5 μg. In some embodiments, the first dose is about 15 μg. In some embodiments, the first dose is about 30 μg. In some embodiments, the first dose is about 90 μg. In some embodiments, the first dose is about 270 μg. In some embodiments, the first dose is about 810 μg. In some embodiments, the first dose is about 1500 μg. In some embodiments, the first dose is about 1620 μg. In some embodiments, the first dose is about 2150 μg. In some embodiments, the first dose is about 2860 μg. In some embodiments, the first dose is about 3240 μg.

[0133] In some embodiments, the first dose is from about 1 week to about 5 weeks, from about 1 week to about 10 weeks, from about 1 week to about 20 weeks, from about 1 week to about 50 weeks, from about 1 week to about 80 weeks, from about 1 week to about 100 weeks, from about 5 weeks to about 10 weeks, from about 5 weeks to about 20 weeks, from about 5 weeks to about 50 weeks, from about 5 weeks to about 80 weeks, from about 5 weeks to about 100 weeks, from about 10 weeks to about 20 weeks, from about 10 weeks to about 50 weeks, from about 10 weeks to about 80 weeks, from about 10 weeks to about 100 weeks, or from about 20 weeks. The compound is administered for about 1 to about 50 weeks, about 20 to about 80 weeks, about 20 to about 100 weeks, about 50 to about 80 weeks, about 50 to about 100 weeks, about 80 to about 100 weeks, about 1 to about 9 weeks, about 1 to about 18 weeks, about 1 to about 27 weeks, about 1 to about 36 weeks, about 9 to about 18 weeks, about 9 to about 27 weeks, about 9 to about 36 weeks, about 18 to about 27 weeks, about 18 to about 36 weeks, or about 27 to about 36 weeks.

[0134] In some embodiments, the first dose is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, or ten times a day. In some embodiments, the first dose is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every other week, once every three weeks, once every four weeks, or once every five weeks.

[0135] In some embodiments, the second dose is from about 1 μg to about 100 μg, from about 1 μg to about 500 μg, from about 1 μg to about 1 mg, from about 1 μg to about 2 mg, from about 1 μg to about 5 mg, from about 1 μg to about 5 mg, from about 1 μg to about 8 mg, from about 1 μg to about 10 mg, from about 1 μg to about 50 mg, from about 1 μg to about 100 mg, from about 100 μg to about 500 μg, Approximately 100μg to approximately 1mg, approximately 100μg to approximately 2mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 8mg, approximately 100μg to approximately 10m g, about 100μg to about 50mg, about 100μg to about 100mg, about 500μg to about 1mg, about 500μg to about 2mg, about 500μg to about 5mg, about 500μg about 5 mg, about 500 μg to about 8 mg, about 500 μg to about 10 mg, about 500 μg to about 50 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 8 mg, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 8 mg, about 2 mg to about 10 mg, about 2 mg to about 50 mg, about 2 mg to about 100 mg, about 5 mg to about 8 mg, about 5 mg to about 10 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 8 mg to about 10 mg, about 8 mg to about 50 mg, about 8 mg to about 100 mg, about 10 mg about 50 mg, or about 50 mg to about 100 mg. In some embodiments, the second dose is about 1 mg to about 6 mg, about 1 mg to about 12 mg, about 1 to about 24 mg, about 1 mg to about 36 mg, about 1 to about 48 mg, about 6 mg to about 12 mg, about 6 to about 24 mg, about 6 mg to about 36 mg, about 6 to about 48 mg, about 12 to about 24 mg, about 12 to about 36 mg, about 12 to about 48 mg, about 24 mg to about 36 mg, about 24 to about 48 mg, or about 36 to about 48 mg. In some embodiments, the second dose is about 5 μg. In some embodiments, the second dose is about 15 μg. In some embodiments, the second dose is about 30 μg. In some embodiments, the second dose is about 90 μg. In some embodiments, the second dose is about 270 μg. In some embodiments, the second dose is about 810 μg. In some embodiments, the second dose is about 1620 μg. In some embodiments, the second dose is about 2150 μg.In some embodiments, the second dose is about 2860 μg. In some embodiments, the second dose is about 3240 μg. In some embodiments, the second dose is about 5 mg. In some embodiments, the second dose is about 10 mg. In some embodiments, the second dose is about 12 mg. In some embodiments, the second dose is about 24 mg. In some embodiments, the second dose is about 36 mg. In some embodiments, the second dose is about 48 mg.

[0136] In some embodiments, the second dose is from about 1 week to about 5 weeks, from about 1 week to about 10 weeks, from about 1 week to about 20 weeks, from about 1 week to about 50 weeks, from about 1 week to about 80 weeks, from about 1 week to about 100 weeks, from about 5 weeks to about 10 weeks, from about 5 weeks to about 20 weeks, from about 5 weeks to about 50 weeks, from about 5 weeks to about 80 weeks, from about 5 weeks to about 100 weeks, from about 10 weeks to about 20 weeks, from about 10 weeks to about 50 weeks, from about 10 weeks to about 80 weeks, from about 10 weeks to about 100 weeks, or from about 20 weeks. The compound is administered for about 1 to about 50 weeks, about 20 to about 80 weeks, about 20 to about 100 weeks, about 50 to about 80 weeks, about 50 to about 100 weeks, about 80 to about 100 weeks, about 1 to about 9 weeks, about 1 to about 18 weeks, about 1 to about 27 weeks, about 1 to about 36 weeks, about 9 to about 18 weeks, about 9 to about 27 weeks, about 9 to about 36 weeks, about 18 to about 27 weeks, about 18 to about 36 weeks, or about 27 to about 36 weeks.

[0137] In some embodiments, the second dose is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, or ten times a day. In some embodiments, the first dose is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every other week, once every three weeks, once every four weeks, or once every five weeks.

[0138] Treatment Method In certain embodiments, the BCMA-targeting trispecific proteins of the present disclosure inhibit tumor cell proliferation when administered in vivo to a subject having tumor cells expressing BCMA. Measurement of inhibition of tumor cell proliferation can be determined by several different methods well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of resected tumor masses and comparison to control subjects, measurement by imaging techniques (e.g., CT or MRI) with or without the use of isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, etc. In certain embodiments, administration of the trispecific proteins of the present disclosure results in inhibition of tumor cell proliferation in vivo by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control antigen binding agent, with about 100% inhibition of tumor proliferation indicating complete remission or disappearance of the tumor. In further embodiments, administration of a trispecific protein of the disclosure results in about 50-100%, about 75-100%, or about 90-100% inhibition of in vivo tumor cell proliferation compared to a control antigen binding agent, hi further embodiments, administration of an antigen binding agent of the disclosure results in about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% inhibition of in vivo tumor cell proliferation compared to a control antigen binding agent.

[0139] Also provided herein, in some embodiments, are methods and uses for stimulating the immune system of an individual in need thereof, comprising administration of an anti-BCMA targeted trispecific protein described herein, in some instances, administration of an anti-BCMA targeted trispecific protein described herein induces and / or maintains cytotoxicity against cells expressing the target antigen.

[0140] Also provided herein, in some embodiments, are methods and uses for stimulating the immune system of an individual in need thereof, comprising administration of a BCMA binding protein described herein. In some examples, administration of a BCMA binding protein described herein induces and / or maintains cytotoxicity against a cell expressing a target antigen. In some examples, the cell expressing a target antigen is a terminally differentiated B cell that is a cancer or tumor cell, or a metastatic cancer or tumor cell.

[0141] Further provided herein are methods and uses for the treatment of a BCMA associated disease, disorder or condition comprising administering a BCMA binding protein, or a multispecific binding protein comprising a BCMA binding protein as described herein, to an individual in need of such treatment.

[0142] BCMA-associated diseases, disorders, or conditions include, but are not limited to, cancers or metastases of the B-cell lineage.

[0143] Cancers that can be treated, prevented, or managed by the BCMA binding proteins of the disclosure, or methods of using same, include, but are not limited to, primary or metastatic cancers.

[0144] Examples of such leukemia include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), as well as many less common types, such as hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia. Treated acute lymphoblastic leukemia (ALL) subtypes include, but are not limited to, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia, and acute biphenotypic leukemia. Treated chronic lymphocytic leukemia (CLL) subtypes include, but are not limited to, B-cell prolymphocytic leukemia. Acute myeloid leukemia (AML) subtypes that may be treated include, but are not limited to, acute promyelocytic leukemia, acute myeloblastic leukemia, and acute megakaryoblastic leukemia.Chronic myeloid leukemia (CML) subtypes that may be treated include, but are not limited to, chronic myelomonocytic leukemia.

[0145] Examples of lymphomas treated by the subject method include, but are not limited to, Hodgkin's disease, non-Hodgkin's disease, or any subtype of lymphoma.

[0146] Examples of such multiple myeloma include, but are not limited to, multiple myeloma of bone or other tissues, including, for example, smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, and the like.

[0147] For a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America.

[0148] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to a therapeutic treatment aimed at delaying (alleviating) an undesirable physiological disease, disorder, or condition, or obtaining a beneficial or desired clinical outcome. For purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the disease, disorder, or disease; stabilization (i.e., not worsening) of the disease, disorder, or disease state; delaying the onset or slowing the progression of the disease, disorder, or disease; amelioration of the disease, disorder, or disease state; and remission (whether partial or total), or enhancement or amelioration of the disease, disorder, or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment further includes extending survival time compared to the expected survival time in the absence of treatment. In other embodiments, "treatment" or "treating" or "treated" refers to a preventative measure, the purpose of which is to delay the onset of or reduce the severity of an unwanted physiological disease, disorder, or condition, e.g., in an individual predisposed to the condition (e.g., an individual bearing genetic markers for a condition such as breast cancer).

[0149] In some embodiments of the methods described herein, a BCMA targeting trispecific protein as described herein is administered in combination with an agent for the treatment of a particular disease, disorder, or condition. Agents include, but are not limited to, therapeutic agents including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapeutic agents (gamma rays, X-rays, and / or directed delivery of radioisotopes, microwave, UV radiation, etc.), gene therapy agents (e.g., antisense, retroviral therapy, etc.), and other immunotherapeutic agents. In some embodiments, an anti-BCMA targeting trispecific protein as described herein is administered in combination with an antidiarrheal, antiemetic, analgesic, opioid, and / or nonsteroidal anti-inflammatory agent. In some embodiments, an anti-BCMA targeting trispecific protein as described herein is administered in combination with an anti-cancer agent.

[0150] Non-limiting examples of anti-cancer drugs that can be used in various embodiments of the present disclosure, including the pharmaceutical compositions and dosage forms and kits of the present disclosure, include acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, aztomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bianthrene hydrochloride, and bianthamin. Bisnafide dimesylate, biceresin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingal, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dextromaplatin, dezagamine, dezagamine mesylate , diazicon, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, phosphate Fludarabine, fluorouracil, fluoroocitabine, foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole,Leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengesrolol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitodilline, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone hydrochloride , mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxislan, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, sa Fingol, safingol hydrochloride, semustine, simtrazene, sparphosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenal, tallysomycin, tecogalan sodium, tegafur, teroxantrone hydrochloride, temoporfin, teniposide, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifecitrate Other examples of anti-cancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecipenol, adzelesin, aldesleukin, ALL-TK antagonist, altretamine, ambamustine, amidox, amidox,Amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogen, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, balanol, batimastat, BCR / ABL antagonist agonists, benzochlorines, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, biansafide, bisstraten A, biceresin, breflate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canaripox IL-2, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analog, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentane tyramine, cycloplatam, sipemycin, cytarabine ocfosfate, cytotoxic factor, cytostatin, daclizumab, decitabine, dehydrodidemin B, deslorelin, dexamethasone, dexphosphamide, dexrazoxane, dexverapamil,Diazicon, Didemmin B, Didox, Diethylnorspermine, Dihydro-5-azacytidine, Dihydrotaxol, 9-, Dioxamycin, Diphenylspiromustine, Docetaxel, Docosanol, Dolasetron, Doxifluridine, Droloxifene, Dronabinol, Duocarmycin SA, Ebselen, Ecomustine, Edelfosine, Edrecolomab, Eflornithine, Elemene, Emitefur, Epirubicin, Epristeride, Estramustine Analogs, Estrogen Agonists, Estrogen Antagonists, Etanidazole, Etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texapyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin cin, idoxifene, idramantone, ilmofosine, ilmostat, imidazoacridone, imiquimod, immunostimulant peptides, insulin-like growth factor I receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, ilopract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenogra stim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, HMG-CoA reductase inhibitors (including but not limited to lovastatin, pravastatin, fluvastatin, statins, simvastatin,and atorvastatin), loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, cytolytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, metalelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, millimostim, mismatched double-stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast proliferation Factors-Saporin, Mitoxantrone, Mofalotene, Molgramostim, Monoclonal antibodies (human placental gonadotropin), Monophosphoryl lipid A + Myobacterial cell wall sk, Mopidamol, Multidrug resistance gene inhibitors, Multiple tumor suppressor gene 1 based therapy, Mustard anticancer drugs, Mycaloxid B, Mycobacterial cell wall extract, Myriaporone, N-acetyldinaline, N-substituted benzamides, Nafarelin, Nagressip, Naloxone + Pentazocine, Napavine, Nafterpine, Nartograstim, Nedaplatin, Nemorubicin, Neridronic acid , neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidants, nitrulline, O6-benzylguamine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogs, paclitaxel derivatives, parauamine, palmitoyl rhizoxin, pamidronate, panaxytriol, panomyphen, parabactin , pazelliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillic alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2,Proteasome inhibitors, Protein A-based immunomodulators, Protein kinase C inhibitors, Protein kinase C inhibitors, Microalgae, Protein tyrosine phosphatase inhibitors, Purine nucleoside phosphorylase inhibitors, Purpurins, Pyrazoloacridines, Pyridoxylated hemoglobin, globin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reterliptin, rhenium Re 186 etidronate, rhizoxin, ribozymes, RII retinamide, logretimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxil, safingol, saintpin, SarCNU, sarcophytol A, sargramostim, Sdi1 mimics, semustine, senescence-derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single-stranded antigen binding proteins, sizofiran, sobuzoxane, borocaptate sodium, phenylacetate sodium sulphonate, sorberol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongiostatin 1, squalamine, stem cell inhibitors, stem cell division inhibitors, stipiamid, stromelysin inhibitors, sulfinodine, superactive vasoactive intestinal peptide antagonists, slajista, suramin, swainsonine, synthetic glycosaminoglycans, talimustine, tamoxifen methiodide, tauromustine, Tazarotene, tecogalan sodium, tegafur, telluropyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thalicarpine, thiocoraline, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etioproprine, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, Tretinoin, triacetyluridine, tricibirine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verdins, verteporfin, vinorelbine, vinxartin, Vitaxin®, vorozole,The anticancer agents include zanoterone, zeniplatin, zilascorub, and zinostatin stimalamer. Additional anticancer agents are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and topoisomerase inhibitors. In some embodiments, the BCMA-targeting trispecific proteins of the present disclosure are used in combination with gemcitabine.

[0151] In some embodiments, the anti-BCMA targeted trispecific proteins described herein are administered before, during, or after surgery.

[0152] In some embodiments, the anti-cancer agent is conjugated to the trispecific protein by any suitable means.

[0153] Methods for detecting BCMA expression and diagnosing BCMA-associated cancers According to another embodiment of the present disclosure, a kit is provided for detecting BCMA expression in vitro or in vivo. The kit comprises a BCMA-targeting trispecific protein as described above (e.g., a trispecific protein comprising a labeled anti-BCMA single domain antibody or antigen-binding fragment thereof) and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzymatic label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0154] In some cases, BCMA expression is detected in biological samples.Samples can be any samples, including but not limited to tissue from biopsy, autopsy, and pathological specimens.Biological samples also include tissue sections, such as frozen sections taken for histological purposes.Biological samples also include bodily fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine.Biological samples are usually obtained from mammals, such as humans and non-human primates.

[0155] The samples obtained for use in the assays described herein include tissues and body fluids and may be processed using conventional means in the art (e.g., homogenization, serum isolation, etc.).Accordingly, the samples obtained from patients are transformed prior to use in the assays described herein.If BCMA is in the sample, it is further transformed, for example, by binding to an antibody, in the methods described herein.

[0156] In one embodiment, a method is provided for determining whether a subject has cancer by contacting a sample from the subject with an anti-BCMA single domain antibody disclosed herein and detecting binding of the single domain antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0157] In another embodiment, there is provided a method of confirming the diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti-BCMA single domain antibody disclosed herein and detecting binding of the sample to the antibody. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0158] In some examples of the disclosed methods, the BCMA single domain antibody is directly labeled.

[0159] In some examples, the method further comprises contacting the sample with a second antibody that specifically binds the anti-BCMA single domain antibody and detecting binding of the second antibody. Increased binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects or confirms a diagnosis of cancer in the subject.

[0160] In some cases, the cancer is leukemia, lymphoma, multiple myeloma, or any other type of cancer that expresses BCMA.

[0161] In some examples, the control sample is a sample from a subject who does not have cancer. In particular examples, the sample is a blood or tissue sample.

[0162] In some cases, the antibody that binds (e.g., specifically binds) BCMA is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) BCMA (first antibody) is unlabeled, and a second antibody or other molecule that can bind to the antibody that specifically binds BCMA is labeled. The second antibody is selected to be capable of specifically binding to the first antibody of a particular species and class. For example, if the first antibody is a llama IgG, the second antibody may be an anti-llama IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for antibodies or second antibodies are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary fluorescent material is luminol, a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 131I, 35S, or 3H.

[0163] In an alternative embodiment, BCMA can be assayed in biological samples by a competitive immunoassay that utilizes a BCMA standard labeled with a detectable substance and an unlabeled antibody that specifically binds to BCMA. In this assay, the biological sample, the labeled BCMA standard, and the antibody that specifically binds to BCMA are combined to determine the amount of labeled BCMA standard bound to the unlabeled antibody. The amount of BCMA in the biological sample is inversely proportional to the amount of labeled BCMA standard bound to the antibody that specifically binds to BCMA.

[0164] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, an antibody that specifically binds to BCMA can be used to detect the production of BCMA in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of BCMA in a tissue sample or a biological sample such as a blood or serum sample. In some examples, the BCMA is cell surface BCMA. In other examples, the BCMA is soluble BCMA (e.g., BCMA in a cell culture supernatant, or soluble BCMA in a body fluid sample such as a blood or serum sample).

[0165] In one embodiment, a kit is provided for detecting BCMA in a biological sample, such as a blood sample or a tissue sample. For example, a biopsy can be performed to obtain a tissue sample for histological examination to confirm a cancer diagnosis in a subject. Alternatively, a blood sample can be obtained to detect the presence of soluble BCMA protein or fragments. A kit for detecting a polypeptide typically includes a single domain antibody of the present disclosure that specifically binds to BCMA. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab, is included in the kit. In a further embodiment, the antibody is labeled (e.g., with a fluorescent label, a radioactive label, or an enzymatic label).

[0166] In one embodiment, the kit includes instructional materials disclosing the means of using the antibody that binds BCMA. The instructional materials may be written, in electronic form (such as a computer diskette or compact disk), visual (such as a video file), or provided via an electronic network, such as the Internet, the World Wide Web, an intranet, or other network. The kit may further include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally include a means for detecting the label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents routinely used in the practice of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0167] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, a method for detecting BCMA in a biological sample generally involves contacting the biological sample with an antibody that specifically reacts with a BCMA polypeptide under immunologically reactive conditions. The antibody specifically binds under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0168] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies can be conjugated to other compounds, including but not limited to enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can further be utilized in immunoassays, such as but not limited to radioimmunoassays (RIA), ELISA, or immunohistochemistry assays. Antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS employs multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels, among other advanced levels of detection, to separate or sort cells (see U.S. Pat. No. 5,061,620). Any of the single domain antibodies that bind to BCMA as disclosed herein can be used in these assays. Thus, antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation. EXAMPLES

[0169] The present application will be better understood by reference to the following non-limiting examples, which are provided as illustrative embodiments of the present application. The following examples are presented to more fully illustrate the embodiments, but should not be construed as limiting the broad scope of the present application.

[0170] Example 1 Protein production

[0171] The sequence of the BCMA-targeting trispecific molecule encompassing the BCMA-binding protein according to the present disclosure was cloned into the mammalian expression vector pcDNA3.4 (Invitrogen) preceded by a leader sequence and followed by a 6x histidine tag (SEQ ID NO: 471). Expi293 cells (Life Technologies A14527) were maintained in suspension in Expi293 medium at between 0.2 and 8x1e6 cells / mL in Optimum Growth Flasks (Thomson). Purified plasmid DNA was transfected into Expi293 cells according to the Expi293 Expression System Kit (Life Technologies A14635) protocol and maintained for 4-6 days post-transfection. The amount of the exemplary trispecific protein to be tested in the conditioned medium from the transfected Expi293 cells was quantified using an Octet instrument equipped with a Protein A chip and using a control trispecific protein as a standard curve.

[0172] T cell-dependent cytotoxicity assay

[0173] To evaluate whether anti-BCMA single domain antibodies can form synapses between T cells and BCMA expressing cell lines to allow T cells to kill BCMA expressing cell lines, titrations of conditioned media were added to a TDCC assay (T cell dependent cytotoxicity assay). In this assay (Nazarian et al., 2015. J.Biomol.Screen et., 20:519-27), T cells and target cancer cell line cells were mixed in a 10:1 ratio in a 384-well plate and various amounts of the trispecific protein to be tested were added. The tumor cell lines were engineered to express luciferase protein. After 48 hours, the remaining viable tumor cells were quantified using the STEADY-GLO® luminescence assay (Promega).

[0174] In this example, EJM cells were used, a cell line that serves as an in vitro model for multiple myeloma and plasma cell leukemia. After 48 hours, the viability of EJM cells is measured. The trispecific proteins were found to mediate T cell killing. Figure 2 shows an exemplary cell viability assay using test proteins 01H08, 01F07, 02F02, and BH253, compared to a negative control. TDCC activity of several other test trispecific proteins was measured using EC 50 are listed in Table 1 below.

[0175] binding affinity

[0176] In this study, the binding affinity of BCMA-targeted trispecific proteins containing BCMA binding proteins according to the present disclosure to human BCMA protein was determined. The affinity measurements are listed in Table 1.

[0177] [Table 1-1]

[0178] [Table 1-2]

[0179] [Table 1-3]

[0180] [Table 1-4]

[0181] ND=undefined

[0182] Molecule 01H08, 01F07, 01H06, 02G02, 02B05, 01C01, 02F02, 02E05, 01E08, 02 C01, 02E06, 02B06, 02F04, 01G08, 02C06, 01H09, 01F04, 01D02, 02D11, 0 1A07, 02C03, 02F07, 01E04, 02H09, 01E03, 02F05, 01B05, 01C05, 02F12 , 01H11, 02G06, 01E06, 01G11, 02A05, 01A08, 02G05, 01B09, 01G01, 01B0 6, 01F10, 01E05, 02G01, 01A06, 02B04, 01D06, 02B07, 02B11, 01H04, 01D03, 01A05, 02F11, 01D04, 01B04, 02C05, 02E03, 01D05, 01C04, 01E07, 01G06, 02F06, 01B01, 01D07, 02A08, 01A02, 02G11, 01G04, 02F03, 01C06, 01A01 had at least a 2-fold increase in TDCC potency compared to the parent CDR (253BH10) molecule and also showed increased affinity.

[0183] Molecule 01H08, 01F07, 01H06, 02G02, 02B05, 01C01, 02F02, 02E05, 01E08, 02C01, 02E06, 0 2B06, 02F04, 01G08, 02C06, 01H09, 01F04, 01D02, 02D11, 01A07, 02C03, 02F07, 01E04 , 02H09, 01E03, 02F05, 01B05, 01C05, 02F12, 01H11, 02G06, 01E06, 01G11, 02A05, 01A08, 02G05, 01B09 had at least 10-fold increased TDCC potency compared to the parent CDR-bearing molecule 253BH10 and also showed increased affinity.

[0184] An anti-GFP trispecific molecule, included in these assays as a negative control, had no detectable BCMA binding and no effect on cell viability in the TDCC assay (data not shown).

[0185] Example 2 Methods for assessing the binding and cytotoxic activity of exemplary BCMA-targeting trispecific proteins of the present disclosure against Jeko1, MOLP8, and OPM2 cells

[0186] Protein production The sequence of the BCMA-targeting trispecific molecule containing the BCMA binding protein according to the present disclosure, preceded by a leader sequence and followed by a 6x Histidine tag (SEQ ID NO: 471), was expressed using vectors and methods previously described (Running Deer and Allison, 2004. Biotechnol Prog. 20:880-9), except that lipid-based reagents and non-linearized plasmid DNA were used for cell transfection. The recombinant trispecific protein was purified using affinity chromatography, ion exchange, and / or size exclusion chromatography. The purified protein was quantified using theoretical extinction coefficients and absorption spectroscopy. An image of a Coomassie stained SDS-PAGE demonstrates the purity of the protein (Figure 3).

[0187] Cytotoxicity assay

[0188] A human T cell-dependent cytotoxicity (TDCC) assay was used to measure the ability of T cell engagers containing trispecific molecules to instruct T cells to kill tumor cells (Nazarian et al., 2015. J. Biomol. Screen., 20:519-27). In this assay, T cells and cells of the target cancer cell line are mixed together in a 10:1 ratio in a 384-well plate and various amounts of the trispecific protein to be tested are added. The tumor cell line is engineered to express luciferase protein. After 48 hours, the amount of viable tumor cells remaining was quantified using the Steady-GloR Luminescent Assay (Promega).

[0189] In this study, we added titrations of purified proteins to a TDCC assay (T cell-dependent cytotoxicity assay) to assess whether anti-BCMA single domain antibodies can form synapses between T cells and the BCMA-expressing Jeko1, MOLP8, and OPM2 cancer cell lines. Jeko1 is a B cell lymphoma cell line. MOLP-8 is a myeloma cell line. OPM-2 is a human myeloma cell line.

[0190] Cell viability was measured after 48 hours. It was observed that the trispecific proteins mediated T cell killing. Figure 4 shows an example of a cell viability assay comparing the test proteins with a negative control. The EC of TDCC activity of several other trispecific proteins tested 50 are listed below in Table 2. An anti-GFP trispecific molecule included in these assays as a negative control had no effect on cell viability (data not shown).

[0191] [Table 2]

[0192] binding affinity

[0193] In this study, the binding affinity of BCMA-targeted trispecific proteins containing a BCMA binding protein according to the present disclosure to human BCMA protein was determined.

[0194] [Table 3]

[0195] The data in Figure 3, Figure 4, Table 2, and Table 3 show that the BCMA-targeted trispecific proteins can be expressed and purified to greater than 90% purity. The purified proteins exhibit TDCC activity that is approximately 13-213-fold greater in potency compared to the trispecific proteins with the parent BCMA-targeting sequence. The purified trispecific proteins bind BCMA with an affinity of approximately 3-7 nM.

[0196] Example 3 Xenograft tumor models The exemplary BCMA-targeted trispecific proteins described herein were evaluated in a xenograft model.

[0197] On day 0, NCG mice were subcutaneously inoculated with RPMI-8226 cells and also implanted intraperitoneally with normal human peripheral blood mononuclear cells (PBMCs). Treatment with exemplary BCMA-targeting trispecific protein (02B05) (SEQ ID NO: 520) was also initiated on day 0 (qdx10) (once daily for 10 days). The doses of administration were 5 μg / kg, 50 μg / kg, or 500 μg / kg of BCMA-targeting trispecific protein 02B05 or control vehicle. Tumor volumes were determined for 25 days. As shown in FIG. 30, the mean tumor volume was significantly lower in mice treated with exemplary BCMA-targeting trispecific protein (02B05) (50 μg / kg, or 500 μg / kg) compared to mice treated with vehicle or a lower dose of BCMA-targeting trispecific protein (02B05) (5 μg / kg).

[0198] On day 0, NCG mice were subcutaneously inoculated with Jeko1 cells and also implanted intraperitoneally with normal human peripheral blood mononuclear cells (PBMCs). Treatment with exemplary BCMA-targeting trispecific protein (02B05) (SEQ ID NO: 520) was also initiated on day 3 (qdx10) (once daily for 10 days). The doses of administration were 5 μg / kg, 50 μg / kg, or 500 μg / kg of BCMA-targeting trispecific protein 02B05 or control vehicle. Tumor volumes were determined for 25 days. As shown in FIG. 31, the mean tumor volume was significantly lower in mice treated with exemplary BCMA-targeting trispecific protein (02B05) (500 μg / kg) compared to mice treated with vehicle or lower doses of BCMA-targeting trispecific protein (02B05) (5 μg / kg or 50 μg / kg).

[0199] Example 4 Proof-of-concept clinical trial protocol for administration of the disclosed BCMA trispecific antigen binding proteins to multiple myeloma patients This is a Phase I / II clinical trial to study the BCMA trispecific antigen binding protein of Example 1 as a treatment for multiple myeloma.

[0200] Research purpose

[0201] Primary Objective. Maximum tolerated dose of the BCMA-targeted trispecific protein of the previous examples.

[0202] Secondary Objective. To determine whether the in vitro responses of the BCMA-targeted trispecific proteins of the previous examples correlate with clinical responses.

[0203] Phase I

[0204] The maximum tolerated dose (MTD) will be determined in the Phase I section of the study.

[0205] 1.1 The maximum tolerated dose (MTD) will be determined in the Phase I section of the study.

[0206] 1.2 Patients who meet the eligibility criteria will be enrolled in the study for the BCMA-targeted trispecific protein of the previous example.

[0207] 1.3 The goal is to identify the maximum dose of the BCMA-targeted trispecific protein of the above example that can be safely administered without severe or unmanageable side effects in participants. The dose given will depend on the number of participants pre-enrolled in the study and how well the dose is tolerated. Not all participants will receive the same dose.

[0208] Phase II

[0209] 2.1 In the Phase II section that follows, treatment will be performed at the MTD with the goal of determining whether treatment with the BCMA-targeted trispecific proteins of the previous examples results in a response rate of at least 20%.

[0210] Primary Outcomes for Phase II---determine whether treatment with the BCMA-targeted trispecific proteins of the previous examples results in at least 20% of patients achieving a clinical response (blow-up response, minor response, partial response, or complete response).

[0211] Eligibility

[0212] The eligibility criteria for participation in the study were:

[0213] Previously untreated patients with multiple myeloma and no serious or impending complications (e.g., impending pathologic fracture, hypercalcemia, renal failure). All asymptomatic patients with low or intermediate tumor burden are eligible.

[0214] Patients were ineligible if they had a large tumor burden, symptomatic or threatening fracture, hypercalcemia (corrected calcium >11.5 mg%), anemia (Hgb <8.5 gm / dl), renal failure (creatinine >2.0 mg / dl), high serum lactate dehydrogenase (>300 U / L), or plasma cell leukemia (>1000 / ul).

[0215] Overt infection or unexplained fever should be resolved prior to treatment. Adequate liver function (including SGPT, bilirubin, and LDH) is required.

[0216] Patients must have a Zubrod performance of 1 or less.

[0217] Patients must provide written informed consent indicating that they understand the investigational nature of this study.

[0218] Life expectancy should be > 1 year.

[0219] Patients with idiopathic monoclonal hypergammopathy and nonsecretory multiple myeloma are ineligible. Patients with prior therapy with localized radiation therapy, α-IFN, or ATRA only are eligible. Patients with previous exposure to high-dose glucocorticoids or alkylating agents are not eligible.

[0220] Example 5 Affinity measurements of human and cynomolgus monkey BCMA, CD3ε, and albumin using exemplary BCMA-targeted trispecific proteins of the present disclosure The purpose of this study was to evaluate the affinity of an exemplary BCMA-targeting trispecific protein (02B05) (SEQ ID NO: 520) of the present disclosure for human BCMA, cynomolgus BCMA, human CD3ε, cynomolgus CD3ε, human albumin, cynomolgus albumin, and mouse albumin. Affinity was measured using an Octet instrument. For these measurements, streptavidin chips were first loaded with 2.5 nM human BCMA-FC, 2.5 nM cynomolgus BCMA-FC, 2.5 nM human CD3ε-FC, 2.5 nM cynomolgus CD3ε-FC, 50 nM human serum albumin (HSA), 50 nM cynomolgus serum albumin, or 50 nM mouse serum albumin. The exemplary BCMA-targeting trispecific protein 02B05 was then incubated with the chip, and after an association period, the chip was transferred to a buffer to dissociate the exemplary BCMA-targeting trispecific protein (02B05). The affinity of binding to human and cynomolgus BCMA and CD3ε was measured in the presence of 15mg / ml human serum albumin. The average calculated KD values ​​from these studies are shown in Table 4 (n indicates the number of independent measurements, n / d indicates no binding was detected under the conditions tested). Binding was detected to human BCMA, human CD3ε, cynomolgus CD3ε, human serum albumin, cynomolgus serum albumin, and mouse serum albumin. No binding was detected to cynomolgus BCMA under the conditions tested.

[0221] [Table 4]

[0222] Example 6 Ability of Exemplary BCMA-Targeted Trispecific Proteins of the Present Disclosure to Bind to Human T Cells Exemplary BCMA-targeting trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to bind purified T cells. Briefly, BCMA trispecific protein or phosphate buffered saline (PBS) was incubated with purified T cells from four different anonymous human donors. After washing away unbound proteins, T cells were incubated with Alexa Fluor 647-conjugated antibodies that recognize the anti-albumin domain in BCMA trispecific antigen binding protein 02B05. T cells were then analyzed by flow cytometry. It was observed that human T cells incubated with BCMA trispecific antigen binding protein 02B05 had a significant shift associated with Alexa Fluor 647 dye compared to cells incubated with PBS. The results are shown in Figures 5A, 5B, 5C, and 5D. In conclusion, this study demonstrated that exemplary BCMA-targeting trispecific protein can bind human T cells.

[0223] Example 7 Ability of Exemplary BCMA-Targeting Trispecific Proteins of the Disclosure to Bind to BCMA-Expressing Cells An exemplary BCMA-targeting trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to bind to BCMA-expressing cells. Briefly, BCMA trispecific antigen binding protein 02B05 was incubated with cell lines expressing BCMA (NCI-H929; EJM; RPMI-8226; OPM2) or lacking BCMA (NCI-H510A; DMS-153). BCMA RNA expression in these cells is indicated by the FPKM values ​​(fragments per kilobase million) listed in Figure 6A-F: RNA FPKM values ​​are from Cancer Cell Line Encyclopedia (Broad Institute, Cambridge, MA, USA). After washing away unbound proteins, cells were incubated with an Alexa Fluor 647-conjugated antibody that recognizes the anti-albumin domain in BCMA trispecific antigen binding protein 02B05. Cells were then analyzed by flow cytometry. As a negative control, cells were incubated with a trispecific protein targeting GFP. Cells expressing BCMA RNA and incubated with BCMA trispecific protein had a significant shift associated with Alexa Fluor 647 staining compared to cells incubated with GFP trispecific protein (shown in Figures 6A, 6B, 6D, and 6E). Meanwhile, cells lacking BCMA RNA produced comparable Alexa Fluor 647 staining to BCMA trispecific protein and GFP trispecific protein (seen in Figures 6C and 6F). Thus, this study demonstrated that the exemplary BCMA trispecific antigen binding can selectively bind to cells expressing BCMA.

[0224] Example 8 Ability of Exemplary BCMA-Targeted Trispecific Proteins to Mediate T-Cell Killing of BCMA-Expressing Cancer Cells Using a standard TDCC assay as described in Example 1, the exemplary BCMA trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to induce T cells to kill BCMA expressing cells in the presence and absence of human serum albumin (HSA). Because the exemplary BCMA trispecific protein contains an anti-albumin domain, this experiment was performed to ensure that binding to albumin does not prevent the BCMA trispecific antigen binding protein from inducing T cells to kill BCMA expressing cells. Five BCMA expressing cell lines were tested: EJM, Jeko, OPM2, MOLP8, and NCI-H929. Representative data from an experiment with EJM cells is shown in FIG. 7. It was observed that in the presence or absence of human serum albumin (HSA), the viability of EJM cells decreased with increasing amounts of the exemplary 02B05 BCMA trispecific antigen binding protein, while the control GFP-targeted trispecific protein had no effect on cell viability. In the presence of albumin, higher concentrations of BCMA trispecific protein were required to reduce the viability of EJM cells. Cell killing by BCMA trispecific protein in EJM cells and Jeko, OPM2, MOL8, and NCI-H929 cells in the absence or presence of HSA was investigated using EC 50 The values ​​are shown in Table 5. For all five cell lines, the exemplary 02B05 BCMA trispecific antigen binding protein induced T cells to kill target cells in the presence of HSA.

[0225] [Table 5]

[0226] Example 9 Ability of Exemplary BCMA-Targeted Trispecific Proteins to Mediate T-Cell Killing of BCMA-Expressing Cancer Cells In a standard TDCC assay (as described in Example 1), a ratio of 1 target cell (EJM or OPM2 cell) to 10 effector cells (T cell) is used in a 48 hour assay. In this experiment, the ability of the exemplary 02B05 BCMA trispecific protein (SEQ ID NO:520) to induce T cells to kill target cells at a smaller target cell to effector cell ratio was tested. The expectation was that less killing would be observed when fewer effector cells were used. Two BCMA expressing cell lines, EJM and OPM2, were tested at target cell to effector cell ratios of 1:1, 1:3, and 1:10, and the experiment was performed in the presence of 15 mg / ml HSA. A GFP-targeted trispecific protein was used as a negative control. Data from this experiment are shown in Figure 8 (TDCC assay with EJM cells) and Figure 9 (TDCC assay with OPM2 cells). As expected, nearly complete killing of target cells was observed at a target to effector cell ratio of 1:10. The amount of killing decreased with decreasing effector cells. EC 50 The values ​​are listed in Table 6 (n / d is the EC 50 (Indicates that there is insufficient data to calculate the EC value.) 50 The values ​​increased when fewer effector cells were present, and therefore, as expected, a reduction in the number of effector cells relative to the target cells reduced the TDCC activity of the BCMA trispecific protein.

[0227] [Table 6]

[0228] Example 10 Ability of Exemplary BCMA-Targeted Trispecific Proteins to Mediate T-Cell Killing of BCMA-Expressing Cancer Cells in a Time Course Study Using Smaller Target Cell to Effector Cell Ratios In a standard TDCC assay (Example 1), a ratio of 1 target cell to 10 effector cells (T cells) is used in a 48 hour assay. In this experiment, a time course was performed using a 1:1 target cell (EJM cells) to effector cell (T cells) ratio. With increasing time, a 1:1 ratio was expected to result in target cell killing. The experiment was performed in the presence of 15mg / ml HSA. GFP-targeted trispecific protein was used as a negative control. Target cell viability was measured on days 1, 2, 3, and 4 after incubation of target and effector cells at a 1:1 ratio in the presence of the exemplary 02B05 BCMA trispecific antigen binding protein and 15mg / ml HSA, or GFP-targeted trispecific protein and 15mg / ml HSA. In the presence of the BCMA trispecific antigen binding protein, no target cell killing was observed on day 1, but killing was observed at all other time points, with the amount of killing increasing with time (Figure 10). No killing was observed with the GFP-targeted trispecific protein. EC 50 The values ​​are shown in Table 7 (n / d is EC 50 (The figures show insufficient killing to determine a value.) From this study, it was concluded that the exemplary 02B05 BCMA trispecific protein is able to induce T cell killing with a lower number of effector cells, but more time is required to achieve more complete killing.

[0229] [Table 7]

[0230] Example 11 Ability of Exemplary BCMA-Targeted Trispecific Proteins to Induce Human T Cells to Kill BCMA-Expressing Cells The exemplary BCMA trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to induce T cells from four different anonymous human donors to kill four different BCMA expressing cells in the presence of 15 mg / ml human serum albumin (HSA) using a standard TDCC assay as described in Example 1. The BCMA expressing cell lines were EJM, NCI-H929, OPM2, and RPMI8226. As negative controls, two cell lines lacking BCMA expression, OVCAR8 and NCI-H510A, were also tested in the TDCC assay. A control GFP-targeted trispecific protein was also used as a negative control. In all four BCMA expressing cell lines and four T cell donors, cell viability decreased with increasing amounts of BCMA trispecific protein, but not with GFP trispecific protein (Figures 11, 12, 13, and 14). EC for cell killing 50 Values ​​are provided in Table 8. The exemplary 02B05 BCMA trispecific antigen binding protein did not induce killing of cell lines lacking BCMA expression (Figures 15 and 16). It was therefore speculated that the exemplary 02B05 BCMA trispecific antigen binding protein may be able to induce T cells from multiple donors to kill a range of BCMA expressing cell lines.

[0231] [Table 8]

[0232] Example 12 Ability of Exemplary BCMA-Targeted Trispecific Proteins to Induce Cynomolgus T Cells to Kill BCMA-Expressing Cells The exemplary BCMA-targeting trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to induce T cells from cynomolgus monkeys to kill BCMA-expressing cells in the presence of 15 mg / ml human serum albumin (HSA). The experimental conditions were the same as described in Example 1, except that peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys were used as the source of T cells. Two BCMA-expressing cell lines, RPMI8226 and NCI-H929, were tested. As shown in Figures 17 and 18, the BCMA trispecific protein was able to induce T cells present in cynomolgus monkey PBMCs to kill the two BCMA-expressing cell lines. EC for cell killing 50 The values ​​are listed in Table 9. The GFP trispecific protein did not affect the viability of BCMA expressing cells. Thus (as shown in Example 5), BCMA expressing trispecific proteins capable of binding to cyno CD3ε can induce cyno T cells to kill cells expressing human BCMA.

[0233] [Table 9]

[0234] Example 13 Exemplary BCMA Trispecific Antigen Binding Proteins and Target Tumor Cell-Mediated T Cell Activation Induction Exemplary BCMA-targeting trispecific protein 02B05 (SEQ ID NO: 520) was tested for its ability to activate T cells in the presence of BCMA-expressing cells. The BCMA-expressing cell lines were EJM, OPM2, and RPMI8226. Two cell lines lacking BCMA expression, OVCAR8 and NCI-H510A, were also included as negative controls. T cells were obtained from four different anonymous human donors. The assay was set up using the conditions of the standard TDCC assay described in Example 1, except that the assay was adapted to a 96-well format and performed in the presence of 15 mg / ml HSA. After 48 hours of assay, T cell activation was assessed by measuring the expression of T cell activation biomarkers CD25 and CD69 on the T cell surface using flow cytometry. When co-cultured with BCMA-expressing cells, increasing expression of CD69 and CD25 was observed on T cells as the concentration of exemplary 02B05 BCMA trispecific antigen binding protein increased (shown in Figures 19-24). Thus, the observed increase in expression was dependent on the interaction of the BCMA binding sequence in the exemplary 02B05 BCMA trispecific antigen binding protein with BCMA, since little to no activation was observed with the control GFP trispecific protein (shown in Figures 19-24) or with target cells without BCMA expression (shown in Figures 25-28). Thus, the exemplary 02B05 BCMA trispecific antigen binding protein activated T cells in co-cultures containing BCMA-expressing cells. This conclusion is supported by additional data. For example, expression of the cytokine TNFα was measured in media collected from co-cultures of T cells and BCMA-expressing target cells treated with increasing concentrations of the exemplary 02B05 BCMA trispecific antigen binding protein or the negative control GFP trispecific protein. Co-cultures were set up using the conditions of the standard TDCC assay (described in Example 1) supplemented with 15 mg / ml HSA. TNFα was measured using an electrochemiluminescence assay (Meso Scale Discovery). Robust induction of TNFα expression was observed with the exemplary BCMA-targeted trispecific protein of 02B05, but not with the GFP trispecific protein (FIG. 29).This result further supports that the exemplary BCMA-targeted trispecific protein of 02B05 activated T cells in co-cultures containing BCMA-expressing cells.

[0235] Example 14 Pharmacokinetics of exemplary BCMA-targeted trispecific proteins of the present disclosure Cynomolgus monkeys were administered a single intravenous dose of the exemplary BCMA-targeted trispecific protein (02B05) (SEQ ID NO: 520) at 0.01 mg / kg, 0.1 mg / kg, or 1 mg / kg. Two animals were included per dose group. After administration, serum samples were collected and analyzed by two different electrochemiluminescence assays. One assay used biotinylated CD3ε as the capture reagent and detected with sulfo-tagged BCMA (referred to as the functional assay). Another assay used a biotinylated antibody that recognizes the anti-albumin domain in the exemplary BCMA-targeted trispecific protein as the capture reagent and a sulfo-tagged antibody (i.e., an anti-idiotypic antibody) that recognizes the anti-CD3 binding domain in the exemplary BCMA-targeted trispecific protein as the detection reagent. The results of the electrochemiluminescence assay are plotted in FIG. 32. As can be seen in FIG. 32, the exemplary BCMA-targeted trispecific protein was detected in the cynomolgus serum sample even 504 hours after administration. An exemplary BCMA-targeting trispecific protein was identified using both sulfo-tagged BCMA (line labeled with the term "functional" in FIG. 32) and an anti-idiotypic antibody (line labeled with the term "anti-idiotypic" in FIG. 32).

[0236] To confirm that the exemplary BCMA-targeted trispecific protein retained the ability to induce T cells to kill BCMA-expressing EJM cells, serum samples at 168 hours after in vivo administration were tested in a TDCC assay (described in Example 1) in the presence of 16.7% serum from cynomolgus monkeys not exposed to the BCMA-targeted trispecific protein, and the exemplary BCMA-targeted trispecific protein was titrated using protein concentrations determined using an electrochemiluminescence assay (shown in FIG. 33). Freshly diluted exemplary 02B05 BCMA trispecific protein was compared to BCMA trispecific protein collected from test cynomolgus monkeys at 168 hours. GFP trispecific protein was included as a negative control. This study demonstrated that the exemplary BCMA-targeted trispecific protein collected from serum of test cynomolgus monkeys had comparable activity to the freshly diluted protein, and that the protein in the serum sample retained the ability to induce T cells to kill BCMA-expressing target cells.

[0237] Example 15 BCMA Trispecific Antigen Binding Protein Phase 1 / 2a Dose Escalation, Expansion, Safety and Pharmacokinetic Study The target population is patients with relapsed / refractory multiple myeloma (R / R MM), disease progression on a prior systemic regimen, and at least three prior therapies including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody.

[0238] Test Design

[0239] The BCMA trispecific antigen binding protein (SEQ ID NO:520) Phase 1 / 2 study design is shown in Figure 34. Study objectives are safety, PK, immunogenicity, and pharmacokinetic characterization, identification of the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D), and tumor assessment based on IMWG response criteria (International Myeloma Working Group Unified Response Criteria for Multiple Myeloma and Minimal Residual Disease Assessment in Multiple Myeloma).

[0240] The study will also test the clinical activity of the BCMA trispecific antigen binding protein in both BCMA-exposed (treated with an agent targeted against BCMA) and BCMA-naive (no prior exposure to an agent targeted against BCMA) patients.

[0241] Medication, Administration: BCMA trispecific antigen binding protein was administered weekly via 1 hour IV infusion. Premedication was used to manage cytokine release syndrome (CRS). Table 10 shows the dosing cohorts and number of subjects.

[0242] [Table 10]

[0243] Baseline characteristics

[0244] Table 11 shows the baseline characteristics and demographics of the subjects. Table 12 shows the prior systemic therapy of the subjects.

[0245] [Table 11]

[0246] [Table 12]

[0247] Adverse events

[0248] Based on available preliminary data, the most common treatment-emergent adverse events (TEAEs) were CRS, fatigue, increased alanine aminotransferase (ALT), and increased aspartate aminotransferase (AST). TEAEs occurring in >10% of patients are summarized in Table 13. Relevant TEAEs are summarized in Table 14.

[0249] TEAEs assessed by the investigator as >grade 3 in severity (per Common Terminology Criteria for Adverse Events [CTCAE] v.5.0) and occurring in >5% of patients included anemia (38%), increased AST (12%), neutropenia (12%), neutropenia (10%), hypophosphatasia (10%), thrombocytopenia (7%), increased ALT (7%), decreased thrombocytopenia (7%), and hypertension (7%).

[0250] Serious adverse events (SAEs) regardless of cause were reported in 16 (38%) patients. The most common SAEs (>2 patients) included increased ALT (7%), increased AST (7%), CRS (7%), pneumonia (5%), general health deterioration (5%), and dyspnea (5%). A summary of investigator-assessed SAEs related to the study drug is shown in Table 15. As of the most recent data cutoff date, the most common SAEs (>1 treated patient) assessed by investigators as related to the study drug were increased ALT (7%), increased AST (7%), and CRS (7%).

[0251] One patient experienced an SAE with a fatal outcome; this was assessed by the investigator as not related to study drug: Patient 102–120 (2150 μg / week fixed-dose cohort) experienced generalized physical health decline and died 7 days after receiving the first dose of study drug. The patient died of multiple organ failure upon documented disease progression.

[0252] [Table 13]

[0253] [Table 14-1]

[0254] [Table 14-2]

[0255] [Table 14-3]

[0256] [Table 15]

[0257] Treatment time

[0258] FIG. 35 shows the time of treatment for all treated patients.

[0259] Response evaluation

[0260] Figure 36 shows the overall response rate. Table 16 shows the overall response rate and disease control rate. In 8 disease-evaluable patients enrolled at 2150 μg / week, including patients with prior BCMA-targeted therapy exposure, one stringent CR, one VGPR, and three PRs were observed. 29% of all responders were MRD-negative (as assessed by next generation flow cytometry) and in stringent CR. All responders continue to receive study treatment.

[0261] Activity was seen at higher dose levels: seven of eight disease-evaluable patients in the 2150 μg / week cohort showed clinical benefit, with an ORR of 63% and a DCR of 88%. Responders included one patient with prior orbacabutagen autoleucel (JCARH125) treatment. A decrease in serum B-cell maturation antigen (sBCMA) from baseline was observed in patients with PR, VGPR, or sCR.

[0262] [Table 16]

[0263] Pharmacokinetics

[0264] [Table 17]

[0265] Figure 37A shows the pharmacokinetic data of the BCMA trispecific antigen binding protein for different dosing cohorts. The linear pharmacokinetic (PK) profile shows a dose-proportional increase in Cmax and AUC, dose-independent clearance and volume of distribution. The median half-life (T 1 / 2 ) at 74 hours. Evidence of BCMA trispecific accumulation is shown by comparing C1D1 and C2D15: a ∼1.5-2 fold increase in Cmax (Figure 37B) and AUC (Figure 37C) as well as a ∼2-3 fold increase in Clast (Figure 37D).

[0266] FIG. 38 shows serum cytokine concentrations 5 hours after the first (C1D1) and second (C1D8) dose for serum IL-6 (FIG. 38A) and serum TNFα (FIG. 38B).

[0267] serum BCMA

[0268] Measurements were available from 23 subjects who received fixed doses of 5, 15, 30, 90, 270, 810, 1620, or 2150 μg / week of BCMA trispecific antigen binding protein. Serum specimens for BCMA analysis (sBCMA) were collected pre- and post-infusion and samples were analyzed for sBCMA concentration using a validated BCMA enzyme-linked immunoassay kit (hBCMA / TNFRSF17 Duo Set, RnD Systems).

[0269] In the 23 patients, baseline sBCMA concentrations ranged from 176 ng / mL (90 μg / week dose group, n=1) to 1213 ng / mL (15 μg / week dose group, n=1). In the higher dose groups, in which more than one patient was enrolled, mean sBCMA concentrations were 540 ng / mL, 588 ng / mL, and 609 ng / mL for the 810 μg / week, 1620 μg / week, and 2150 μg / week dose groups, respectively, indicating comparable levels of baseline sBCMA between these cohorts.

[0270] Concentrations of sBCMA (change from C1D15 to baseline) are shown in Figure 39. Following BCMA trispecific binding protein administration, 11 of 21 patients evaluated (52.4%) showed an increase in sBCMA ranging from 3.72 to 268%. These patients were in the 1620μg / week or lower dose group. In 10 patients (47.6%), sBCMA concentrations showed a decrease of 1.08-85.6% at day 15 pre-dose cycle 1. All patients in the 2150μg / week dose group showed a decrease in sBCMA concentrations on treatment. The four patients who showed a response (PR or better) to treatment (2150μg / week dose group) were also patients who had a decrease in sBCMA concentrations on treatment.

[0271] Pharmacokinetics

[0272] Figure 40 shows the concentration-time profile of a BCMA trispecific antigen binding protein. Figure 40A shows the concentration-time profile after the 6th dose and Figure 40B shows the concentration-time profile after the 1st dose.

[0273] Serum cytokine levels

[0274] Measurements were available from 29 patients who received 5, 15, 30, 90, 270, 810, 1620, 2150, or 2860 μg / week of BCMA trispecific antigen binding protein, as well as 3 subjects who received 1620 μg / week as the priming dose and 3240 μg / week as the target dose. Dexamethasone was given prior to infusion in cycle 1 and, if needed, in subsequent cycles at the discretion of each investigator. Serum samples were taken pre-infusion (baseline) and 5 hours post-infusion (5-hour EOI). The Myriad RBM (Austin, TX) HMPCORE1 human multiplex cytokine panel was used to evaluate serum changes in 12 cytokines (GM-CSF, IFNγ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-18, TNFα, and TNFβ) and 4 chemokines (IL-8, MIP-1α, MIP-1β, and MCP-1). In five patients treated with 5-90 μg of BCMA trispecific antigen binding protein, 5-hour EOI concentrations of all 16 cytokines were below the assay LLOQ or had no significant change from baseline (Figures 41A and 41B). Among patients treated with 270-2860 μg, there was at least one patient in each cohort who similarly had no significant change. Other patients had increases to various degrees in IL-6, IL-10, TNFα, IFNγ, IL-8, MIP-1β, and MCP-1. Variability in response to BCMA trispecific antigen binding protein is evident across individual patients within each cohort. However, there is good agreement among the seven cytokines in any given individual, with concentrations tending to increase and decrease as a group, suggesting a common mechanism of induction.

[0275] The number of patients with a cytokine spike at 5 hours EOI increased with dose escalation. For example, none of the patients in the 5-90 μg cohorts showed an increase in cytokines. The 270 and 810 μg dose cohorts each had 50% of patients with a cytokine spike. The majority of patients receiving 1620, 2150, or 2860 μg had a cytokine spike. Nevertheless, median cytokine concentrations did not appear to increase in a dose-dependent manner from 270 to 2860 μg / week (Figures 41A and 41B).

[0276] The kinetics of change in cytokine levels across dosing cycles was characterized by peak concentrations at 5-hour EOI on day 1 of cycle 1, followed by a trend back to baseline over the next 24-48 hours. Equal or lower magnitude cytokine spikes were observed at 5-hour EOI of subsequent weekly infusions. The 2150 μg cohort is given as an example of a fixed dosing cohort (Figures 42A and 42B, left panels). In the 1620-3240 μg step-dosing cohorts, peak concentrations of IL-6, TNFα, IFNγ, IL-8, MIP-1β, and MCP-1 were recorded at 5-hour EOI after the priming dose (1620 μg) on ​​day 1 of cycle 1. Subsequent infusions of the target dose (3240 μg) induced much lower levels of these cytokines (Figures 42A and 42B, right panels).

[0277] Preliminary analysis of a limited number of patients in the two highest fixed dose cohorts (2150 and 2860 μg / week) suggested that robust cytokine and chemokine spikes at the 5 hour EOI in C1D1 correlated with clinical response (FIG. 43).

[0278] Changes in circulating T cells

[0279] Whole blood samples collected before and after BCMA trispecific antigen binding protein infusion were analyzed by flow cytometry. T cells were monitored based on the expression of the following markers: CD45+CD3+CD4+ (helper T cells), CD45+CD3+CD8+ (cytotoxic T cells). A significant but transient decrease in CD4+ and CD8+ T cell counts was observed in a dose-dependent manner from 15 μg to 2860 μg after the first infusion. T cells reached their lowest numbers at 5 h EOI on day 1 of cycle 1 and gradually recovered over the next 24 to 48 h. There was a general trend of weaker recovery of T cell counts by 48 h EOI (cycle 1, day 3) among patients receiving higher doses. The decline in T cell counts at 5 h EOI on day 8 of cycle 1, day 15 of cycle 1, and day 1 of cycle 3 was less pronounced than with C1D1 (Figure 44). This also appeared to be true in the 1620-3240 μg step-dose cohort, although data were available for only three subjects in this cohort at the time of analysis (Figure 45).

[0280] With regard to T cell activation in response to BCMA trispecific antigen binding protein, CD4+ and CD8+ T cells in subjects across all fixed dose cohorts (5-2860 μg) upregulated cell surface expression of CD69, a marker of T cell activation. This is consistent with the CD3 binding properties of the TriTAC platform. However, there is no evidence of a dose-dependent increase in CD69 across cohorts. The percentage of CD69+ T cells peaked 5-24 hours after the first infusion and remained higher than baseline at 48 hours. The magnitude of CD69 upregulation in response to subsequent BCMA trispecific antigen binding protein infusions in C1D8, C1D15, and C3D1 was modest compared to the first dose (Figure 46). This appeared to be true even with the 1620-3240 μg step-dose regimen, although data were available for only three subjects in this cohort at the time of analysis (data not shown).

[0281] Preliminary analysis of the 2150 and 2860 μg / week dose cohorts suggested a correlation between higher expression of CD69 on CD8 T cells and clinical response (Figure 47). This result was supported by similar findings in cytokine spikes (Figure 43), suggesting that the BCMA trispecific antigen binding protein was highly effective in engaging the target molecule (BCMA) on myeloma cells and activating T cells.

[0282] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in the practice of the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of the claims, and equivalents thereof, be covered thereby.

[0283] [Table 18-1]

[0284] [Table 18-2]

[0285] [Table 18-3]

[0286] [Table 18-4]

[0287] [Table 18-5]

[0288]

Table 18-6

[0289]

Table 18-7

[0290]

Table 18-8

[0291]

Table 19-1

[0292]

Table 19-2

[0293]

Table 19-3

[0294]

Table 19-4

[0295]

Table 19-5

[0296]

Table 19-6

[0297]

Table 19-7

[0298]

Table 19-8

[0299]

Table 20-1

[0300]

Table 20-2

[0301]

Table 20-3

[0302]

Table 20-4

[0303]

Table 20-5

[0304]

Table 20-6

[0305]

Table 20-7

[0306]

Table 20-8

[0307]

Table 20-9

[0308]

Table 20-10

[0309]

Table 20-11

[0310]

Table 20-12

[0311]

Table 20-13

[0312]

Table 20-14

[0313]

Table 20-15

[0314]

Table 20-16

[0315]

Table 20-17

[0316]

Table 20-18

[0317]

Table 20-19

[0318]

Table 20-20

[0319]

Table 20-21

[0320]

Table 20-22

[0321]

Table 20-23

[0322]

Table 20-24

[0323]

Table 20-25

[0324]

Table 20-26

[0325]

Table 20-27

[0326]

Table 20-28

[0327]

Table 20-29

[0328]

Table 20-30

[0329]

Table 20-31

[0330]

Table 20-32

[0331]

Table 20-33

[0332]

Table 20-34

Claims

**Claim 1** A method of treating cancer, comprising the step of administering to a subject an effective amount of a BCMA-targeted trispecific protein, wherein the BCMA-targeted trispecific protein comprises (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extension domain, and (c) a third domain (C) that specifically binds to BCMA. These domains are in the order of H ２ N-(C)-(B)-(A)-COOH, or are linked by linker L1 and linker L2, and the BCMA-targeted trispecific protein is administered at a dosage of about 1 μg to about 100 mg, a method.

Images