B cell-activating factor (BAFF)-proliferation-inducing ligand (APRIL) dual inhibitor

Abstract

Variant B-cell maturation antigen (BCMA) cysteine-rich domain (CRD) molecules are provided that are capable of binding to the ligands B-cell activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL) with higher affinity compared to wild-type BCMA. In some embodiments, variant BCMA fusion proteins or constructs, dimers, conjugates, methods for making the molecules, and related methods and uses thereof, including immunoglobulin Fc domains are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 280,556, filed November 17, 2021; U.S. Provisional Patent Application No. 63 / 339,334, filed May 6, 2022; and U.S. Provisional Patent Application No. 63 / 350,392, filed June 8, 2022, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Field The present disclosure, in some aspects, relates to variant B-cell maturation antigen (BCMA) cysteine-rich domain (CRD) molecules capable of binding to the ligands B-cell Activating Factor of the TNF family (BAFF) and A Proliferation Inducing Ligand (APRIL) with higher affinity compared to wild-type BCMA. In some aspects, the disclosure further relates to methods for making variant BCMA fusion proteins or constructs, dimers, conjugates, molecules comprising an immunoglobulin Fc domain, and related methods and uses thereof. [Background technology]

[0003] background Human BCMA binds to BAFF and APRIL, TNF superfamily ligands implicated in immune cell functions such as B cell differentiation, survival, and proliferation. Improved strategies for preventing or treating B cell-mediated conditions are needed to provide effective molecules with a strong safety profile, e.g., molecules that provide dual blockade of BAFF and APRIL without inducing a highly potent cytotoxic response. Compositions, methods, and uses that meet such needs are provided. Summary of the Invention [Means for solving the problem]

[0004] overview Provided herein are variant B-cell maturation antigen (BCMA) polypeptides. In some of the provided embodiments, the variant BCMA polypeptides comprise a variant cysteine-rich domain (CRD) that includes at least one amino acid substitution selected from the following, relative to the amino acid positions of SEQ ID NO: 1: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A).

[0005] In some embodiments, the variant CRD comprises at least three amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD comprises at least three amino acid substitutions selected from among: (1) a histidine or arginine at position 12 (S12H or S12R); (2) an isoleucine or valine at position 14 (L14I or L14V); (3) an arginine at position 15 (H15R); and (4) a valine at position 22 (L22V), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD further comprises at least one modification selected from among: (1) a deletion of residue 38 (N38del) or an amino acid residue that is not asparagine at position 38 (N38X, where X is any amino acid residue that is not asparagine), and (2) an amino acid residue that is not serine or threonine at position 40 (S40X, where X is any amino acid residue that is not serine or threonine), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD further comprises a glycine at residue 40 (S40G), relative to the amino acid position of SEQ ID NO:1.

[0006] In some embodiments, the variant CRD comprises at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least about 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 1. In some embodiments, the variant CRD comprises at least 95%, or at least about 95%, sequence identity to SEQ ID NO: 1. In some embodiments, the variant CRD comprises at least 99%, or at least about 99% sequence identity to SEQ ID NO: 1.

[0007] In some embodiments, the variant CRD comprises at least 90%, or at least about 90%, sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-146. In some embodiments, the variant CRD comprises at least 90%, or at least about 90% sequence identity to the sequence set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19. In some embodiments, the variant CRD comprises the sequence set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19.

[0008] In some embodiments, the variant CRD comprises at least three amino acid substitutions selected from the group consisting of: (1) histidine at position 12 (S12H); (2) isoleucine at position 14 (L14I); (3) arginine at position 15 (H15R or H15N); and (4) a serine to glycine mutation at residue 40 (S40G), relative to the amino acid positions of SEQ ID NO:1, wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO:1. In some embodiments, the variant CRD comprises each of the following amino acid substitutions relative to the amino acid positions of SEQ ID NO:1: (1) histidine at position 12 (S12H); (2) isoleucine at position 14 (L14I); (3) arginine at position 15 (H15R); and (4) a serine to glycine mutation at residue 40 (S40G), wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO:1.

[0009] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 3. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:3.

[0010] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 4. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:4.

[0011] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 8. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:8.

[0012] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:9.

[0013] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 19. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:19.

[0014] Fusion polypeptides are also provided herein. In some of the provided embodiments, the fusion polypeptide comprises any of the variant BCMA polypeptides provided herein and an additional polypeptide. In some embodiments, the additional polypeptide is an immunoglobulin (Ig) Fc polypeptide.

[0015] Also provided herein are fusion polypeptides comprising any of the variant BCMA polypeptides provided herein and an immunoglobulin (Ig) Fc polypeptide.

[0016] Also provided herein is a fusion polypeptide comprising an immunoglobulin (Ig) Fc polypeptide and a variant B-cell maturation antigen (BCMA) polypeptide comprising a variant cysteine-rich domain (CRD) that includes at least one amino acid substitution selected from, based on the amino acid positions of SEQ ID NO: 1, among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A).

[0017] In some embodiments, the variant CRD comprises at least three amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD comprises at least three amino acid substitutions selected from among: (1) a histidine or arginine at position 12 (S12H or S12R); (2) an isoleucine or valine at position 14 (L14I or L14V); (3) an arginine at position 15 (H15R); and (4) a valine at position 22 (L22V), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD further comprises at least one modification selected from among: (1) a deletion of residue 38 (N38del) or an amino acid residue that is not asparagine at position 38 (N38X, where X is any amino acid residue that is not asparagine), and (2) an amino acid residue that is not serine or threonine at position 40 (S40X, where X is any amino acid residue that is not serine or threonine), relative to the amino acid positions of SEQ ID NO: 1. In some embodiments, the variant CRD further comprises a glycine at residue 40 (S40G), relative to the amino acid position of SEQ ID NO:1.

[0018] In some embodiments, the variant CRD comprises at least 90%, or at least about 90%, sequence identity to the sequence set forth in any one of SEQ ID NOs: 3-146. In some embodiments, the variant CRD comprises at least 90%, or at least about 90% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19.

[0019] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 3. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:3.

[0020] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 4. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:4.

[0021] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 8. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:8.

[0022] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 9. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:9.

[0023] In some embodiments, the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 19. In some embodiments, the variant CRD comprises the sequence set forth in SEQ ID NO:19.

[0024] In some embodiments, the variant BCMA polypeptide is fused directly or indirectly to the N-terminus or C-terminus of the Ig Fc polypeptide.

[0025] In some embodiments, the Ig Fc polypeptide is or is derived from an isotype G immunoglobulin (IgG) or a variant thereof.

[0026] In some embodiments, the Ig Fc polypeptide is or is derived from an IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc. In some embodiments, the Ig Fc polypeptide is or is derived from a human IgG Fc. In some embodiments, the Ig Fc polypeptide is or is derived from a human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0027] In some embodiments, the Ig Fc polypeptide is or is derived from an IgG1 Fc. In some embodiments, the Ig Fc polypeptide is or is derived from a human IgG1 Fc. In some embodiments, the Ig Fc polypeptide is a human IgG1 Fc and comprises the sequence set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, or SEQ ID NO: 230, or a sequence having at least 90% sequence identity thereto. In some embodiments, the Ig Fc polypeptide is a human IgG1 Fc and comprises the sequence set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, or SEQ ID NO: 230.

[0028] In some embodiments, the Ig Fc polypeptide is or is derived from an IgG2 Fc. In some embodiments, the Ig Fc polypeptide is or is derived from a human IgG2 Fc. In some embodiments, the Ig Fc polypeptide is a human IgG2 Fc and comprises the sequence set forth in SEQ ID NO: 171, SEQ ID NO: 235, or SEQ ID NO: 236, or a sequence having at least 90% sequence identity thereto. In some embodiments, the Ig Fc polypeptide is a human IgG2 Fc and comprises the sequence set forth in SEQ ID NO: 171, SEQ ID NO: 235, or SEQ ID NO: 236.

[0029] In some embodiments, the Ig Fc polypeptide is or is derived from an IgG4 Fc. In some embodiments, the Ig Fc polypeptide is or is derived from a human IgG4 Fc. In some embodiments, the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 172, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, or SEQ ID NO: 234, or a sequence with at least 90% sequence identity thereto. In some embodiments, the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 172, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, or SEQ ID NO: 234. In some embodiments, the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, or a sequence with at least 90% sequence identity thereto. In some embodiments, the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 163, or a sequence having at least 90% sequence identity thereto. In some embodiments, the Ig Fc polypeptide comprises SEQ ID NO: 163.

[0030] In some embodiments, the Ig Fc polypeptide comprises isoleucine or valine substituted for one, two, three, four, or more native methionine residues.

[0031] In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 3 and the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 3 and the Ig Fc polypeptide comprises SEQ ID NO: 163. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 4 and the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 4 and the Ig Fc polypeptide comprises SEQ ID NO: 163. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 8 and the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 8 and the Ig Fc polypeptide comprises SEQ ID NO: 163. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 9 and the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 9 and the Ig Fc polypeptide comprises SEQ ID NO: 163. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 19 and the Ig Fc polypeptide comprises SEQ ID NO: 161. In some embodiments, the variant BCMA polypeptide comprises SEQ ID NO: 19 and the Ig Fc polypeptide comprises SEQ ID NO: 163.

[0032] In some embodiments, the fusion polypeptide further comprises a peptide linker connecting the variant BCMA polypeptide and the Ig Fc polypeptide.

[0033] In some embodiments, the peptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the peptide linker is 4, 5, 6, or 7 amino acids in length.

[0034] In some embodiments, the peptide linker comprises a residue selected from the group consisting of glycine, serine, alanine, and threonine. In some embodiments, the peptide linker comprises a sequence set forth in any one of SEQ ID NOs: 156, 158, 175-186, 188-213, GS, GGS, and GSA. In some embodiments, the peptide linker comprises SEQ ID NO: 156. In some embodiments, the peptide linker comprises SEQ ID NO: 158.

[0035] In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, a variant BCMA polypeptide, a peptide linker, and an Ig Fc polypeptide.

[0036] In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19; a peptide linker comprising the sequence set forth in any one of SEQ ID NOs:156, 158, 175-186, 188-213, GS, GGS, and GSA; and an Ig Fc polypeptide comprising the sequence set forth in SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:172, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234.

[0037] In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:3, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:3, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:4, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:4, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:8, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:8, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163.

[0038] In some embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 167, or a sequence having at least 90% sequence identity thereto. In some embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 167.

[0039] In some embodiments, the fusion polypeptide comprises a first monomer comprising a first variant BCMA polypeptide fused directly or indirectly to a first Ig Fc polypeptide, and one or more second monomers comprising a second variant BCMA polypeptide fused directly or indirectly to a second Ig Fc polypeptide, wherein the first monomer and one or more second monomers are fused in tandem. In some embodiments, the first variant BCMA polypeptide and the second variant BCMA polypeptide are the same. In some embodiments, the first variant BCMA polypeptide and the second variant BCMA polypeptide are different. In some embodiments, the first monomer and the second monomer are the same. In some embodiments, the first monomer and the second monomer are different.

[0040] Also provided herein are dimers, e.g., dimers of any of the variant BCMA polypeptides or fusion polypeptides provided herein. In some of the provided embodiments, the dimers comprise a first monomer comprising any of the variant BCMA polypeptides or fusion polypeptides provided herein and a second monomer comprising any of the variant BCMA polypeptides or fusion polypeptides provided herein. In some embodiments, the first monomer comprises a first variant BCMA polypeptide fused directly or indirectly to a first Ig Fc polypeptide, and the second monomer comprises a second variant BCMA polypeptide fused directly or indirectly to a second Ig Fc polypeptide.

[0041] In some embodiments, the first variant BCMA polypeptide and the second variant BCMA polypeptide are the same. In some embodiments, the first variant BCMA polypeptide and the second variant BCMA polypeptide are different. In some embodiments, the first Ig Fc polypeptide and the second Ig Fc polypeptide are the same. In some embodiments, the first Ig Fc polypeptide and the second Ig Fc polypeptide are different. In some embodiments, the first monomer and the second monomer are the same. In some embodiments, the first monomer and the second monomer are different.

[0042] In some embodiments, the first monomer and the second monomer are linked by at least one disulfide bond between a cysteine ​​residue of the first monomer and a cysteine ​​residue of the second monomer, hi some embodiments, the disulfide bond is between a cysteine ​​residue of the Ig Fc polypeptide of the first monomer and a cysteine ​​residue of the Ig Fc polypeptide of the second monomer.

[0043] Conjugates are also provided herein. In some embodiments, the conjugate comprises any of the variant BCMA polypeptides, any of the fusion polypeptides provided herein, or any of the dimers provided herein, and an additional moiety covalently attached to the variant BCMA polypeptide, fusion polypeptide, or dimer.

[0044] In some embodiments, the additional moiety is selected from a therapeutic moiety, a polymer moiety, a sugar moiety, and a lipophilic moiety.

[0045] In some embodiments, the additional moiety is selected from one or more of polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), branched polyethylene glycol in which two or more polyethylene glycol chains are joined by a linker group, polyvinyl alcohol (PVA), polycarboxylate, poly(vinylpyrrolidone), polyethylene-co-maleic anhydride, and dextran.

[0046] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate binds to B-cell activating factor of the TNF family (BAFF) and / or proliferation-inducing ligand (APRIL) or variants thereof.

[0047] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate exhibits a higher binding affinity for BAFF and / or APRIL compared to the binding affinity of a reference BCMA polypeptide or reference binding molecule.

[0048] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate exhibits greater inhibition of BAFF and / or APRIL activity or function compared to inhibition of BAFF and / or APRIL activity or function by a reference BCMA polypeptide or reference binding molecule.

[0049] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate reduces B cell proliferation or reduces BAFF and / or APRIL-mediated B cell proliferation.

[0050] In some embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate reduces production of a pro-inflammatory cytokine, hi some embodiments, the pro-inflammatory cytokine is one or more of IFNγ or IL-17A.

[0051] In any of the provided embodiments, the BAFF is human BAFF and the APRIL is human APRIL.

[0052] In any of the provided embodiments, the BAFF is mouse BAFF and the APRIL is mouse APRIL.

[0053] In some of the provided embodiments, the reference BCMA polypeptide is a wild-type human BCMA CRD set forth in SEQ ID NO: 1. In some of the provided embodiments, the reference BCMA polypeptide is a human BCMA CRD comprising a serine to glycine substitution at position 40 (S40G), comprising the sequence set forth in SEQ ID NO: 237.

[0054] In any of the provided embodiments, the reference binding molecule is selected from among Atacicept, Telitaccept, Belimumab, or BION-1301.

[0055] In some of the provided embodiments, the binding affinity of the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate to human BAFF is at least or at least about 1-fold, at least 2-fold, at least or at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least 500-fold greater than the binding affinity of the reference BCMA polypeptide or reference binding molecule to human BAFF. In some of the provided embodiments, inhibition of human BAFF activity or function by the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate is at least 5-fold, or at least about 5-fold, at least 10-fold, or at least about 10-fold, at least 20-fold, or at least about 20-fold, at least 25-fold, or at least about 25-fold, at least 50-fold, or at least about 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least about 250-fold, or at least 500-fold, greater than inhibition of human BAFF activity or function by the reference BCMA polypeptide or reference binding molecule.

[0056] In some of the provided embodiments, the binding affinity of the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate to human APRIL is at least or at least about 1-fold, at least 2-fold, at least or at least about 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least 500-fold greater than the binding affinity of the reference BCMA polypeptide or reference binding molecule to human APRIL. In some of the provided embodiments, inhibition of human APRIL activity or function by the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate is at least 5-fold, or at least about 5-fold, at least 10-fold, or at least about 10-fold, at least 20-fold, or at least about 20-fold, at least 25-fold, or at least about 25-fold, at least 50-fold, or at least about 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least about 250-fold, or at least 500-fold, greater than inhibition of human APRIL activity or function by the reference BCMA polypeptide or reference binding molecule.

[0057] In some of the provided embodiments, the ratio of the binding selectivity of human BAFF to the binding selectivity of human APRIL (huBAFF K D / huAPRIL K D ) is greater than 5, greater than 4, greater than 3, greater than 2, or greater than 1. In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human BAFF is DIn some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human BAFF is less than 600 pM, less than 550 pM, less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, less than 200 pM, or less than 150 pM. D ) is in the picomolar (pM) range.

[0058] In some of the provided embodiments, the equilibrium dissociation constant (K D In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human BAFF is in the sub-picomolar (pM) range. D ) is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human APRIL is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. D ) is less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, or less than 30 pM.

[0059] In some of the provided embodiments, the equilibrium dissociation constant (K D In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human APRIL is in the picomolar (pM) range. D In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to human APRIL is in the sub-picomolar (pM) range. D ) is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to both human BAFF and human APRIL is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. D In some of the provided embodiments, the equilibrium dissociation constant (K) for binding to both human BAFF and human APRIL is less than 120 pM.D In some of the provided embodiments, the equilibrium dissociation constant (K) is less than 0.3 pM. D ) is measured by kinetic exclusion assay or surface plasmon resonance (SPR).

[0060] In some provided embodiments, the fusion polypeptide, dimer, or conjugate does not substantially bind to heparan sulfate proteoglycans (HSPGs), wherein the HSPGs are selected from one or more of syndecan-1 and syndecan-2.

[0061] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate inhibits an activity or function of BAFF and / or APRIL or variants thereof. In some of the provided embodiments, the activity or function of BAFF and / or APRIL is selected from B cell survival, B cell proliferation, and / or immunoglobulin production.

[0062] Polynucleotides are also provided herein. In some of the provided embodiments, the polynucleotide comprises a nucleotide sequence that encodes a first monomer and / or a second monomer of any one of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, or any one of the dimers provided herein.

[0063] Vectors are also provided. In some of the provided embodiments, the vector comprises any of the polynucleotides provided herein, or a polynucleotide comprising a nucleotide sequence encoding any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, or the first monomer and / or second monomer of any one of the dimers provided herein.

[0064] Also provided herein are cells. In some of the provided embodiments, the cells comprise any of the polynucleotides or vectors provided herein.

[0065] Also provided herein are methods for producing variant BCMA polypeptides, fusion polypeptides, or dimers. In some embodiments, the methods involve introducing any of the polynucleotides or vectors provided herein into a cell; culturing the host cell under conditions suitable for expression of the polypeptide; and recovering or isolating the polypeptide. In some embodiments, the methods also involve purifying the polypeptide.

[0066] Pharmaceutical compositions are also provided herein. In some of the provided embodiments, the pharmaceutical composition comprises any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, any of the dimers provided herein, any of the conjugates provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, or any of the cells provided herein.

[0067] In some embodiments, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients. In some embodiments, the one or more excipients include a pharmaceutically acceptable liquid carrier. In some embodiments, the one or more excipients include a pharmaceutically acceptable processing agent. In some embodiments, the pharmaceutical composition is a liquid formulation, an intravenous formulation, a solid dosage form, or an inhalable preparation.

[0068] Also provided herein are any of the provided pharmaceutical compositions for treating a disease or disorder. In some embodiments, the pharmaceutical composition is administered to a subject having the disease or disorder.

[0069] Also provided herein are any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, any of the dimers provided herein, any of the conjugates provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, or any of the cells provided herein for treating a disease or disorder.

[0070] In some embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell is administered to a subject with a disease or disorder.

[0071] Also provided herein are methods of treatment. In some embodiments, the methods involve administering to a subject having a disease or disorder any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, any of the dimers provided herein, any of the conjugates provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the pharmaceutical compositions provided herein.

[0072] Also provided herein is the use of any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, any of the dimers provided herein, any of the conjugates provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the pharmaceutical compositions provided herein in the manufacture of a medicament for treating a disease or disorder.

[0073] Also provided herein is the use of any of the variant BCMA polypeptides provided herein, any of the fusion polypeptides provided herein, any of the dimers provided herein, any of the conjugates provided herein, any of the polynucleotides provided herein, any of the vectors provided herein, any of the cells provided herein, or any of the pharmaceutical compositions provided herein to treat a disease or disorder.

[0074] In some of the provided embodiments, the variant BCMA polypeptide, fusion polypeptide, dimer, conjugate or pharmaceutical composition is administered to a subject having a disease or disorder.

[0075] In some of the provided embodiments, the disease or disorder is a B-cell or antibody-mediated disease or disorder.

[0076] In some of the provided embodiments, the disease or disorder is an autoimmune disease or disorder. In some of the provided embodiments, the autoimmune disease or disorder is an immune-mediated disease or disorder of the subject's tissues, bones, joints, blood vessels, thyroid, kidneys, nervous system, brain, lungs, and / or skin. In some of the provided embodiments, the autoimmune disease or disorder is selected from among a kidney disease or disorder, lupus, arthritis, a spondyloarticular disorder, a vasculitis disorder, a hemolytic anemia disorder, a thrombocytopenic disorder, a thyroiditis disorder, a demyelinating disease of the central and / or peripheral nervous system, an inflammatory and / or fibrotic lung disorder, a skin disorder, or an allergic disorder.

[0077] In some of the provided embodiments, the disease or disorder is selected from the group consisting of systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berge's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), renal sarcoidosis, rheumatoid arthritis, juvenile chronic arthritis, arthritis associated with inflammatory bowel disease, ankylosing spondylitis, spondylitis associated with psoriasis, juvenile spondyloarthropathy, unclassifiable spondyloarthropathy, Reiter's syndrome, scleroderma, Sjögren's syndrome, systemic necrotizing vasculitis, polyarteritis nodosa, allergic vasculitis and granulomatosis, polyangiitis, Wegener's granulomatosis, lymphomatoid granulomatosis, mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease), isolated central nervous system vasculitis, Behcet's disease, and / or vasculitis associated with inflammatory bowel disease. disease), thromboangiitis obliterans (Buerger's disease), cutaneous necrotizing venulitis, sarcoidosis, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, thrombocytopenic purpura, immune-mediated thrombocytopenia, Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis, type 1 diabetes mellitus, glomerulonephritis and tubulointerstitial nephritis, multiple sclerosis (MS), idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, bullous dermatitis, erythema multiforme, contact dermatitis, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, or urticaria.

[0078] In some provided embodiments, the disease or disorder is selected from systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berge's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), or renal sarcoidosis. In some provided embodiments, the disease or disorder is selected from systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), or IgA nephropathy (Berge's disease).

[0079] In some of the provided embodiments, the disease or disorder is tissue or organ transplant rejection. In some of the provided embodiments, the tissue or organ transplant rejection is selected from among acute or chronic B-cell or antibody-mediated rejection of tissue allografts consisting of bone marrow, stem cells, skin, and solid organs, acute or chronic graft-versus-host disease (GVHD), antibody-mediated rejection (AMR) of solid organs, hyperacute organ transplant rejection, acute organ transplant rejection, and chronic organ transplant rejection.

[0080] In some of the provided embodiments, the disease or disorder is a B-cell malignancy. In some of the provided embodiments, the B-cell malignancy is selected from among non-Hodgkin's lymphoma, multiple myeloma (MM), B-cell chronic lymphocytic leukemia, plasmacytoma, macroglobulinemia, or Waldenstrom's macroglobulinemia (WM).

[0081] In some of the provided embodiments, a therapeutically effective amount of the variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector, cell or pharmaceutical composition is administered to the subject.

[0082] In some of the provided embodiments, the method or use is a therapeutic use or a prophylactic use. In some of the provided embodiments, the therapeutic use is for induction therapy. In some of the provided embodiments, the induction therapy lasts for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, or up to or about 4 weeks. In some of the provided embodiments, the therapeutic use is for maintenance therapy. In some of the provided embodiments, the maintenance therapy lasts for up to or about 1 week, up to or about 2 weeks, up to or about 3 weeks, or up to or about 4 weeks.

[0083] In some of the provided embodiments, the administration is selected from intravenous, oral, parenteral, sublingual, by inhalation, rectal, or topical. In some of the provided embodiments, the administration is intravenous. [Brief explanation of the drawings]

[0084] [Figure 1] Figure 1A shows the polynucleotide sequence (SEQ ID NO: 148) encoding full-length human BCMA. Figure 1B shows the amino acid sequence (SEQ ID NO: 149) encoded by the polynucleotide sequence of Figure 1A, corresponding to full-length human BCMA. The underlined portion indicates the BAFF (TALL-1) binding region (amino acid residues 8-46). The bolded portion indicates an exemplary TNFR (CRD) region (amino acid residues 7-45). The italicized portion indicates the transmembrane domain (amino acid residues 55-77).

[0085] [Figure 2] Figure 2A shows the polynucleotide sequence (SEQ ID NO: 2) corresponding to the extracellular domain of human BCMA. Figure 2B shows the amino acid sequence (SEQ ID NO: 152) corresponding to the extracellular domain of human BCMA (corresponding to amino acid residues 5-49 of SEQ ID NO: 150).

[0086] [Figure 3A] Figures 3A-3K show alignments of the amino acid sequences of the exemplary variant BCMA polypeptides described compared to the amino acid sequences of the extracellular domain of human BCMA (SEQ ID NO: 152) and the second cysteine-rich domain of TACI. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above.

[0087] [Figure 4] FIG. 4 is a schematic diagram of the vector CET1019-BCMA-PIg18 described in Example 1A.

[0088] [Figure 5] FIG. 5 provides a schematic diagram of the vector CET1019AS-Z-TACI-Ig, described in Example 1B.

[0089] [Figure 6] Figure 6 provides a schematic diagram of pre-BCMA ECD-Pig18 and pre-Z-TACI ECD-Ig.

[0090] [Figure 7] Figures 7A and 7B are sensorgram traces of the binding of an exemplary variant BCMA polypeptide 233622 (variant BCMA ECD component 233622, designated "233622-Ig"), a [S40G]huBCMA-Ig fusion polypeptide that is a fusion with an Ig polypeptide (designated "BCMA-Ig"), and a TACI-Ig fusion polypeptide (control designated "Z-TACI-Ig") to cynomolgus monkey APRIL (Figure 7A) and human BAFF (Figure 7B).

[0091] [Figure 8A]Figures 8A and 8B show the equilibrium dissociation constants (KD) of the indicated representative variant BCMA polypeptides and controls as a function of the molar concentration of huBAFF or huAPRIL, as determined by a kinetic exclusion binding assay (KINEXA3200). Vertical bars represent the 95% confidence interval of the measurements. The x-axis designations represent the variant BCMA-Ig or control Ig fusion polypeptide and ligand (huAPRIL with the suffix ".A" or huBAFF with the suffix ".B"). Figure 8A provides a comparison of the equilibrium dissociation constants of variant BCMA-Ig polypeptides with a control (Z-TACI-Ig. Figure 8B provides a comparison of various stalk and Ig versions of the BAv9-1 variant described in Example 17 with [S40G]BCMA ECD-pIg18 (referred to as "BCMA[S40G]-pIg18"). An "*" indicates that the value is the average of two or more assays. [Figure 8B] Same as above.

[0092] [Figure 9] Figure 9 shows inhibition curves for the representative variant BCMA polypeptides and controls described as percent maximal proliferation signal in response to soluble huBAFF as determined by a HEK293-huBCMA NFkappaB luciferase reporter cell assay.

[0093] [Figure 10] Figure 10 shows inhibition curves for the indicated representative variant BCMA polypeptides and controls as percent maximal proliferation signal in response to HEK-293 cells expressing membrane-form huBAFF, as determined in a HEK293-huBCMA NFkappaB luciferase reporter cell assay.

[0094] [Figure 11A]Figure 11A shows the reduction in the percentage of splenic marginal zone B lymphocytes ("MZB cells") in lupus-prone NZB / W-F1 mice staining positive for the CD19 B cell marker after 8 weeks of treatment with the described exemplary variant BCMA polypeptide (BAv9-1-Ig), TACI-Ig, mBR3-Ig, or a PBS control. Figure 11B shows the percentage of CD19-positive B cells in lupus-prone mice treated with the fusion proteins BAv9-1-Ig, TACI-Ig, mBR3-Ig, and a PBS control in a similar experiment. [Figure 11B] Same as above.

[0095] [Figure 12] Figure 12 shows the reduction in IgG-producing bone marrow plasma cells (antibody secreting cells, "IgG ASC") in ELISPOT forming NZB / W-F1 mice after 8 weeks of treatment with the exemplary variant BCMA polypeptide described (variant BAv9-1-Ig), TACI-Ig, mBR3-Ig, or PBS.

[0096] [Figure 13A] Figure 13A shows the reduction in total serum IgA levels in NZB / W-F1 mice after 8 weeks of treatment with the described exemplary variant BCMA fusion polypeptide (BAv9-1-Ig), TACI-Ig, mBR3-Ig, or PBS. Figure 13B shows the total serum IgA levels in lupus-prone mice after treatment with the described BCMA fusion polypeptide (BAv9-1-Ig), TACI-Ig, mBR3-Ig, or PBS. [Figure 13B] Same as above.

[0097] [Figure 14A]Figure 14A shows reduced anti-dsDNA IgM levels in NZB / W-F1 mice before (Pre) and after 4 weeks of treatment (W4) with the described exemplary variant BCMA polypeptide (BAv9-1-Ig), Z-TACI-Ig, mBR3-Ig, or PBS. Figure 14B shows nucleosomal ASC measured in NZB / W-F1 mice after 8 weeks of treatment with the described exemplary variant BCMA polypeptide (BAv9-1-Ig), TACI-Ig, mBR3-Ig, or PBS. [Figure 14B] Same as above.

[0098] [Figure 15] FIG. 15 shows the reduction in renal pathological deviation as measured by renal histology in NZB / W-F1 mice after 8 weeks of treatment with the described exemplary variant BCMA polypeptide (BAv9-1-Ig), TACI-Ig, mBR3-Ig, or PBS.

[0099] [Figure 16] FIG. 16 shows the mean proteinuria (mg / dL) in mice in response to treatment with the IgG1 fusion protein BAv9-1-Ig (CRD, SEQ ID NO: 3), TACI-Ig, mBR3-Ig, or PBS control over 16 weeks.

[0100] [Figure 17] FIG. 17 shows the reduction in the percentage of peripheral blood B lymphocytes in cynomolgus monkeys staining positive for the CD19 B cell marker over a 28 day period following a single intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.3 mg / kg and 1.0 mg / kg) and TACI-Ig (1.0 mg / kg).

[0101] [Figure 18]FIG. 18 shows the reduction in total serum IgG in cynomolgus monkeys over a 28-day course following intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and TACI-Ig (0.1 mg / kg and 1.0 mg / kg).

[0102] [Figure 19] FIG. 19 shows the reduction in total serum IgA in cynomolgus monkeys over a 28-day course following intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and TACI-Ig (0.1 mg / kg and 1.0 mg / kg).

[0103] [Figure 20] Figure 20 shows the reduction in total serum IgM in cynomolgus monkeys over a 14-day course following intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and TACI-Ig (0.1 mg / kg and 1.0 mg / kg).

[0104] [Figure 21A] Figure 21A shows the reduction in serum anti-tetanus toxoid (TTx) IgG antibodies in cynomolgus monkeys at day 7 after intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and TACI-Ig (0.1 mg / kg and 1.0 mg / kg). Figure 21B shows the reduction in serum anti-tetanus toxoid (TTx) IgG antibodies in cynomolgus monkeys at day 14 after intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and Z-TACI-Ig (0.1 mg / kg and 1.0 mg / kg). [Figure 21B] Same as above.

[0105] [Figure 22] Figure 22 shows the reduction in serum anti-tetanus toxoid (TTx) IgM antibodies in cynomolgus monkeys at day 7 following intravenous treatment with the described exemplary variant BCMA polypeptides (BAv9-1-Ig, 0.1 mg / kg, 0.3 mg / kg, and 1.0 mg / kg) and TACI-Ig (0.1 mg / kg and 1.0 mg / kg).

[0106] [Figure 23] Figure 23 shows the reduction in the percentage of B220 B cells of the marginal zone, transitional, and follicular B cell phenotype in the spleens of Balb / c mice analyzed by FACS staining after 1 and 3 weeks of "induction" treatment with a high dose (10 mg / kg) of the described exemplary BAFF-selective variant BCMA polypeptide (variant 238833-Ig, "238833-Ig") or PBS ("vehicle"). The graph on the right shows results for B220 B cells in transferred animals treated for an additional 3 weeks with mBR3-Ig (50 mg / kg) "maintenance" treatment (variant 238833-Ig / mBR3-Ig group) or continuous induction treatment (variant 238833-Ig / variant 238833-Ig group).

[0107] [Figure 24] Figure 24 shows the reduction in IgG-secreting bone marrow plasma cells after 1 and 3 weeks of "induction" treatment with a high dose (10 mg / kg) of the described exemplary BAFF-selective variant BCMA polypeptide (variant 238833-Ig) or PBS (left and center graphs). The right graph shows results for bone marrow plasma cells in induction animals treated for an additional 3 weeks with mBR3-Ig (50 mg / kg) "maintenance" treatment (variant 238833-Ig / mBR3-Ig group) or continued induction treatment (variant 238833-Ig / variant 238833-Ig group).

[0108] [Figure 25]Figure 25 shows the reduction in serum IgA after 1 and 3 weeks of "induction" treatment with a high dose (10 mg / kg) of the described exemplary BAFF-selective variant BCMA polypeptide (variant 238833-Ig) or PBS (left and center graphs). The right graph (labeled "6-week time point") shows results for serum IgA in induction animals treated for an additional 3 weeks with mBR3-Ig (50 mg / kg) "maintenance" treatment (variant 238833-Ig / mBR3-Ig group) or continued induction treatment (variant 238833-Ig / variant 238833-Ig group).

[0109] [Figure 26] Figure 26 shows a reduction in the percentage of B220 B cells of marginal zone, transitional, and follicular B cell phenotype in the spleen of Balb / c mice analyzed by FACS staining after 3 weeks of treatment with the described exemplary BAFF-selective variant BCMA polypeptide (variant BAv66-Ig) or PBS at set doses (150 μg or 500 μg / mouse).

[0110] [Figure 27] Figure 27 shows the reduction of IgG-secreting bone marrow plasma cells in Balb / c mice analyzed by FACS staining after 3 weeks of treatment with the described exemplary BAFF-selective variant BCMA polypeptide (variant BAv66-Ig) or PBS at set doses (150 μg or 500 μg / mouse).

[0111] [Figure 28] Figure 28 shows the reduction in serum IgA in Balb / c mice after 3 weeks of treatment with the described exemplary BAFF-selective variant BCMA polypeptide (variant BAv66-Ig) or PBS at set doses (150 μg or 500 μg / mouse).

[0112] [Figure 29]Figure 29 shows free drug concentrations in mice after a single injection with variant BCMA BAv9-1 fused to three different IgG2 Fc variants.

[0113] [Figure 30] Figure 30 shows the SDS-PAGE band patterns of two variant BCMAs, one in which the N-linked glycosylation site is retained (Gly+) or one in which the N-linked glycosylation site has been removed by an S40G mutation (Gly-).

[0114] [Figure 31] Figure 31 shows that protection of renal function was observed in NZB / W F1 lupus-prone mice treated with the exemplary variant BCMA polypeptide described (variant BAv9-1-5L-pIg22[K278]del, labeled "BAv9-1-Ig"), but not with a human BCMA surrogate control protein (labeled "BCMA-Ig") or a control Fc protein.

[0115] [Figure 32] Figure 32 shows that a reduction in splenic B cells was observed in NZB / W F1 lupus-prone mice treated with the exemplary variant BCMA polypeptide described (variant BAv9-1-5L-pIg22[K278]del, labeled "BAv9-1-Ig"), but not with a human BCMA surrogate control protein (labeled "BCMA-Ig") or a control Fc protein.

[0116] [Figure 33] Figures 33A and 33B show a comparison of the IC50 of RC18 (telitacicept), atacicept, and an exemplary variant BCMA CRD-IgG4 Fc fusion protein against human BAFF (Figure 33A) and human APRIL (Figure 33B).

[0117] [Figure 34] Figures 34A and 34B show a comparison of the IC50 of RC18 (telitacicept), atacicept, and an exemplary variant BCMA CRD-IgG4 Fc fusion protein against murine BAFF (Figure 34A) and murine APRIL (Figure 34B).

[0118] [Figure 35] Figures 35A and 35B show a comparison of the EC50 of RC18 (telitacicept), atacicept, and an exemplary variant BCMA CRD-IgG4 Fc fusion protein against the heparan sulfate proteoglycans syndecan-1 (Figure 35A) and syndecan-2 (Figure 35B).

[0119] [Figure 36] Figure 36 shows the results of flow cytometry to assess B cell enrichment efficiency by staining for CD19 and gating on lymphocytes. Pre-enriched PBMCs were used as a control. The percentage of CD19+ cells is shown.

[0120] [Figure 37] Figure 37 shows flow cytometry results assessing BAFF-R expression in enriched B cell populations for all three donors. Unstained cells were used as a control. The percentage of BAFF-R+ cells is shown.

[0121] [Figure 38] Figure 38 shows flow cytometry results assessing TACI expression in enriched B cell populations for all three donors. Unstained cells were used as a control. The percentage of TACI+ cells is shown.

[0122] [Figure 39] Figure 39 shows flow cytometry results assessing BCMA expression in enriched B cell populations for all three donors. Unstained cells were used as a control. The percentage of BCMA+ cells is shown.

[0123] [Figure 40] Figure 40 shows flow cytometry histogram overlays showing BAFF-R, TACI, and BCMA expression in enriched B cell populations for donors 1, 2, and 3. Mean fluorescence intensity (MFI) values ​​are shown near each peak.

[0124] [Figure 41] Figure 41 shows the stimulation and proliferation of enriched B cell populations in the presence of stimulatory ligands (BAFF, APRIL, or BAFF-60mer) with or without plate-bound anti-IgM antibody for all three donors: no stimulation ("No Stim"), anti-IgM antibody alone ("aIgM alone"), anti-IgM antibody and ligand ("aIgM+BAFF", "aIgM+APRIL", or "aIgM+BAFF-60mer"), and ligand alone ("BAFF", "APRIL", or "BAFF-60mer") for all three donors, expressed as relative luminescence units (RLU) using the CellTiter-Glo® Luminescent Cell Viability Assay.

[0125] [Figure 42A] Figures 42A-42C show inhibition curves for three inhibitors, BAv9-1 CRD-IgG4 Fc, atacicept, and RC18 (telitacicept), on the proliferation of enriched B cell populations stimulated with human BAFF, human APRIL, or human BAFF-60mer and anti-IgM antibody for donor 1 (Figure 42A), donor 2 (Figure 42B), and donor 3 (Figure 42C), represented by plotting RLU from the CellTiter-Glo® Luminescent Cell Viability Assay against serial dilutions of inhibitor. The determined IC50 values ​​for each are shown on the plots. [Figure 42B] Same as above. [Figure 42C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0126] Detailed Description Provided herein are variant B-cell maturation antigen (BCMA) polypeptides, e.g., variant BCMA fragments that bind to the ligands B-cell activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL), as well as related fusion proteins, compositions, nucleic acids, methods, and uses. The provided variant BCMA polypeptides contain a mutant cysteine-rich domain (CRD) and can bind, e.g., specifically bind, to the ligands BAFF and APRIL, thereby providing dual blockade of B-cell-stimulating cytokines. In some cases, the variant BCMA polypeptide is linked to an immunoglobulin Fc region. Such variant BCMA-Fc fusion proteins can further assemble to form dimers. Expression constructs encoding the variant BCMA and fusion proteins can be used to produce the polypeptide. The various provided polypeptides and expression constructs can be used in prophylactic or therapeutic methods, such as preventing or treating immune cell-mediated diseases or conditions through dual blockade of BAFF and APRIL.

[0127] Autoimmune disorders can result from a cycle of immune cell stimulation mediated in part by the interaction of stimulatory cytokines and immune cell receptors. Plasma B cells and plasmablasts secrete autoreactive antibodies, which then attack tissues. Tissue damage results in the release of autoantigens, which bind to autoreactive antibodies, thereby triggering the production of more autoantibody-secreting plasma B cells by T cell-dependent and T cell-independent mechanisms.

[0128] The tumor necrosis factor (TNF) family receptors B-cell maturation antigen (BCMA), transmembrane activator and interactor of CAML (TACI), and BAFF receptor (BR3) play complementary roles in regulating immune cell activity (see Pelletier et al., J. Biol. Chem. 2003; 278, 33127-33133; Treml et al., Cell Biochem Biophys. 2009; 53 (1): 1-16; and Bischof et al, Blood. 2006; 107(8):3235-42). All three receptors are expressed on the surface of normal B cells late in development and on malignant B cells. BCMA, TACI, and BR3 together regulate B cell survival, function, and differentiation (Cancro, D'Cruz, and Khamashta J. Clinical Invest. 2009; 119: 1066-1073. Dillon, S. et al. (2006) Nature Reviews 2006; 5:235-246). TACI is also expressed in activated T cells (Khare et al. Proc. Natl. Acad. Sci. USA 2000; 97 (7): 3370-3375).

[0129] Both BCMA and TACI bind to the ligands BAFF and APRIL, whereas BR3 binds only to BAFF. BAFF and APRIL are members of the TNF family of ligands that enhance the differentiation of B cells into pathogenic plasma cells and prolong the survival of such plasma cells. BAFF is thought to be a key costimulatory molecule for the proliferation and survival of mature B cells, including the survival of autoreactive B cells (Day et al., Biochemistry 2005; 44:1919-1931 and Liu, Trends Immunol. 2011; 32 (8): 388-394). In addition to its involvement in the autoimmune response cycle, BAFF has also been implicated in modulating the proliferative capacity and survival of multiple myeloma cells (Novak, et al., Blood 2004: 103 (2): 689-694).

[0130] BAFF and APRIL have been implicated in the establishment and / or maintenance of certain autoimmune diseases, such as systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis (RA), multiple sclerosis, and Sjogren's syndrome (see, e.g., Dillon, S. et al. (2006) Nature Reviews 2006; 5:235-246; Gross et al. Nature 2000; 404 (6782): 995-999; MacKay et al. Annu. Rev. Immunol. 1999; 21:231-2264 (2004); and Khare et al. Proc. Natl. Acad. Sci. USA 2000; 97 (7): 3370-3375). Preclinical and clinical studies have demonstrated that BAFF and / or APRIL antagonists can reduce autoantibody levels and control the activity of autoimmune diseases.Animal models support the conclusion that antagonism of both BAFF and APRIL is desirable to optimally block the B cell autoimmune response cycle by reducing or eliminating the production of pathogenic antibody-producing cells, thereby reducing tissue damage and the subsequent formation of autoimmune complexes, and thus blocking the further production of new pathogenic plasma cells and plasmablasts.Compared to the inhibition of either BAFF or APRIL alone, blocking BAFF and APRIL is a more effective way to rapidly deplete autoreactive plasmablasts and plasma cells.

[0131] Strategies for the prevention or treatment of B cell mediated conditions, such as autoimmune diseases, include administering molecules to interfere with the signaling of either ligand alone or both BAFF and APRIL. Molecules that have been reported to modulate B cell function by interfering with BAFF and / or APRIL signaling include antibodies such as the anti-BAFF antibody BENLYSTA® (belimumab; Baker KP et al. Arthritis Rheum. 2003; 48:3253-3265), formerly known as Lymphostat-B, and atacicept, a TACI-Ig fusion protein with specificity for BAFF and APRIL (Baker JA et al. Nature 2000; 404:995-999 and Gross, JA et al. (2000) Immunity 15: 289-302), BR3-Fc (Pelletier et al., J. Biol. Chem. 2003; 278, 33127-33133), and BCMA-Ig (Melchers Ann. Rheum. Dis. 2003; 62 (Suppl. 2): ii25-ii27; Patel, DR et al. J. Biol. Chem. 2004; 279:16727-16735; WO03 / 072713; and US2009 / 0297504). However, the efficacy and safety of existing molecules indicate the need for improved treatment options. For example, in clinical trials for rheumatoid arthritis, the dual BAFF / APRIL inhibitor atacicept was shown to offer no significant benefit over placebo (van Vollenhoven et al., Rheumatoid Arthritis 2011; 63 (7): 1782-1792; Genovese et al., Rheumatoid Arthritis 2011; 63 (7): 1793-1803; Nanda, Nature Reviews Rheumatology 2011; 7: 313).

[0132] Provided herein are variant BCMA polypeptides, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, cells, pharmaceutical compositions, and related methods and uses that meet these needs. In some embodiments, exemplary fusion proteins are provided that include a variant BCMA CRD and another polypeptide, such as an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is an IgG4 Fc region. In some embodiments, existing therapeutic antibodies and Fc fusion proteins often include an IgG1 Fc region (Santos et al., Braz. J. Pharm. Sci. 2018; 54 (Special): e01007 and Duivelshof et al., J. Sep. Sci 2021; 44 (1): 35-62). IgG1 Fc polypeptides generate potent cytotoxic responses due to their high-affinity interaction with IgG Fc receptors (FcγRs) (Kang, Exp. Mol. Medicine 2019; 51 :1-9). The resulting Fc-mediated effector functions include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). While the induction of potent cytotoxic responses can be advantageous for some applications, such as in the treatment of cancer or infectious diseases, the treatment of autoimmune diseases requires strategies of targeted immunosuppression (Kellner et al., Transfus Med Hemother 2017; 44:327-336).

[0133] Provided herein are variant BCMA polypeptides containing a mutant cysteine-rich domain (CRD) that bind to both BAFF and APRIL, resulting in dual blockade of B cell-stimulating cytokines and offering improved properties and benefits over existing molecules that target BAFF and / or APRIL. For example, as described herein, the provided variant BCMA polypeptides and related molecules (e.g., fusion proteins) and compositions offer improved binding, e.g., binding with substantially higher affinity, improved specificity, and reduced, limited, or eliminated binding to heparin sulfate proteoglycans (HSPGs). In some embodiments, the provided variant BCMA polypeptides include an engineered BCMA-binding pocket containing specific modifications that result in substantially enhanced binding and substantially higher binding affinity. In some embodiments, the structural modifications to the binding pocket result in a stronger conformational fit, which, as demonstrated herein, results in significantly higher binding affinity and potency. In some embodiments, improved affinity, improved specificity, and reduced HSPG binding may provide the benefits of improved efficacy and / or reduced dose levels or frequency, and may preserve T cell-independent responses to infection, which in turn may result in higher patient compliance and efficacy, and an improved safety profile. Additionally, provided variant BCMA polypeptides, related molecules, and compositions also exhibit reduced or minimal effector function, allowing for an improved safety profile.

[0134] Compared to existing molecules, the provided variant BCMA molecules bind BAFF and APRIL with higher affinity. In some aspects, fusion proteins comprising a variant BCMA CRD and an IgG4 Fc region are provided. In some aspects, provided embodiments offer the advantage of attenuating cytotoxic responses by selecting an IgG4 Fc, which is a weak inducer of Fc-mediated effector functions, such as ADCC or CDC (Kang, Exp. Mol. Medicine 2019; 51:1-9 and WO2021068752). In some cases, ADCC and CDC can cause undesirable side effects, such as toxicity, or shorten the duration of efficacy of the fusion protein (Kang, Exp. Mol. Medicine 2019; 51:1-9). In some aspects, provided embodiments are based on effective, high-affinity binding and inhibition of BAFF and APRIL, while reducing or minimizing effector functions. In some embodiments, the Fc region provides enhanced stability and uniform expression of the fusion protein, which is desirable for production and quality control.

[0135] Also provided are variant BCMA polypeptides or fusion polypeptides, polynucleotides encoding one or more monomers of a dimer, vectors comprising such polynucleotides, pharmaceutical compositions, e.g., therapeutic compositions, comprising any of such provided molecules, cells, and methods of treatment involving administering such variant BCMA polypeptides, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, cells, pharmaceutical compositions.

[0136] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0137] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. BCMA variant molecules

[0138] Human BCMA (huBCMA), also known as TNFRS17, is a 184-amino acid type III membrane protein of the tumor necrosis factor receptor superfamily (SEQ ID NO:149 and SEQ ID NO:150). HuBCMA contains an extracellular domain (ECD) set forth in SEQ ID NO:152 (corresponding to amino acid residues 5-54 of SEQ ID NO:149), a transmembrane domain (amino acid residues 55-77 of SEQ ID NO:149, italicized in Figure 1B), and a cytoplasmic domain (amino acid residues 78-184 of SEQ ID NO:149). The ECD contains a cysteine-rich domain (CRD) spanning residues 7-41 of SEQ ID NO:149. The CRD is a motif of the TNF receptor. An exemplary huBCMA CRD is set forth in SEQ ID NO:1. The BCMA stalk region is set forth in SEQ ID NO:152 (amino acid residues 51-55).

[0139] In some aspects, the provided variant BCMA molecules comprise a variant BCMA polypeptide comprising a mutated CRD compared to SEQ ID NO: 1. In some examples, the variant BCMA molecule comprises a variant BCMA fusion polypeptide comprising a mutated CRD compared to SEQ ID NO: 1. Non-limiting examples of variant BCMA fusion polypeptides include, for example, bispecific fusion polypeptides and variant BCMA-Ig fusion polypeptides. Further non-limiting examples include dimers, such as dimers in which the variant BCMA-Ig fusion polypeptide is covalently linked by a disulfide bond through the Ig component of the fusion polypeptide, and conjugates thereof. In some embodiments, provided herein are variant BCMA polypeptides comprising a mutated CRD compared to the corresponding residues in wild-type huBCMA (SEQ ID NO: 1), and variant BCMA molecules comprising the provided variant BCMA polypeptides. A. BCMA variant polypeptides

[0140] Provided herein are variant BCMA polypeptides that comprise a mutated CRD relative to the wild-type huBCMA CRD (SEQ ID NO: 1).

[0141] In some embodiments, the mutant CRD of the variant BCMA polypeptide is huBCMA at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CRD (SEQ ID NO: 1). In some embodiments, the mutant CRD of the variant BCMA polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, the variant CRD comprises at least 85% or at least about 85% sequence identity to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0142] In some embodiments, the variant BCMA CRD comprises one or more amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0143] In some embodiments, the variant BCMA CRD comprises at least two sequence features selected from the group consisting of: (1) a histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) an isoleucine or valine at position 14 (L14I or L14V); (3) an arginine or asparagine at position 15 (H15R or H15N); (4) a serine at position 16 (A16S); and (5) a valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0144] In some embodiments, the variant BCMA CRD comprises at least three sequence features selected from the group consisting of: (1) histidine or arginine at position 12 (S12H or S12R); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine at position 15 (H15R); (4) serine at position 16 (A16S); and (5) valine at position 22 (L22V), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0145] In some embodiments, the variant BCMA comprises one or more amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); and (4) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0146] In some embodiments, the variant BCMA comprises at least two sequence features selected from the group consisting of: (1) a histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) an isoleucine or valine at position 14 (L14I or L14V); (3) an arginine or asparagine at position 15 (H15R or H15N); and (4) a valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0147] In some embodiments, the variant BCMA comprises at least three sequence features selected from the group consisting of: (1) histidine or arginine at position 12 (S12H or S12R); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine at position 15 (H15R); and (4) valine at position 22 (L22V), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0148] In some embodiments, the variant BCMA CRD comprises the following sequence features: histidine at position 12 (S12H), isoleucine at position 14 (L14I), and arginine at position 15 (H15R). In some embodiments, the variant BCMA CRD comprises the following sequence features: arginine at position 12 (S12R), isoleucine at position 14 (L14I), and arginine at position 15 (H15R). In some embodiments, the variant BCMA CRD comprises the following sequence features: histidine at position 12 (S12H), arginine at position 15 (H15R), and valine at position 22 (L22V). In some embodiments, the variant BCMA CRD comprises the following sequence features: histidine at position 12 (S12H) and arginine at position 15 (H15R). In some embodiments, the variant BCMA CRD comprises the following sequence features: arginine at position 12 (S12R), valine at position 14 (L14V), asparagine at position 15 (H15N). In some embodiments, the variant BCMA CRD comprises the following sequence features: arginine at position 12 (S12R), valine at position 14 (L14V), asparagine at position 15 (H15N), and serine at position 16 (A16S). In some aspects, the variant BCMA CRD comprises such amino acid substitutions and comprises at least 85% or at least about 85% sequence identity, for example at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO:1.

[0149] In some embodiments, the variant BCMA polypeptide also comprises at least one sequence feature selected from the group consisting of: (1) a deletion of residue 38 (N38del) or a non-asparagine amino acid residue at position 38 (N38X, where X is any amino acid residue that is not asparagine) and (2) a non-serine / threonine amino acid residue at position 40 (S40X, where X is any amino acid that is not serine / serine / threonine), relative to the amino acid positions of the human BCMA CRD sequence set forth in SEQ ID NO: 1. In some embodiments, the variant BCMA polypeptide further comprises a serine to glycine mutation at residue 40 (S40G). In some aspects, the variant BCMA CRD comprises such an amino acid substitution and comprises at least 85% or at least about 85% sequence identity, e.g., at least 95% sequence identity, to the human BCMA CRD sequence set forth in SEQ ID NO: 1.

[0150] In some embodiments, the variant BCMA is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 1 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 143. The present invention also includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99% or higher sequence identity to a sequence selected from: 31, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 143.

[0151] In some embodiments, the variant BCMA is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 136, 137, 138, 139, 140, 141, 142, and 143, or a sequence comprising at least 95% sequence identity thereto.In some embodiments, the variant BCMA is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 1 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 143.

[0152] In some embodiments, the variant BCMA comprises SEQ ID NO:3. In some embodiments, the variant BCMA comprises SEQ ID NO:4. In some embodiments, the variant BCMA comprises SEQ ID NO:5. In some embodiments, the variant BCMA comprises SEQ ID NO:6. In some embodiments, the variant BCMA comprises SEQ ID NO:7. In some embodiments, the variant BCMA comprises SEQ ID NO:8. In some embodiments, the variant BCMA comprises SEQ ID NO:9. In some embodiments, the variant BCMA comprises SEQ ID NO:10. In some embodiments, the variant BCMA comprises SEQ ID NO:11. In some embodiments, the variant BCMA comprises SEQ ID NO:12. In some embodiments, the variant BCMA comprises SEQ ID NO:13. In some embodiments, the variant BCMA comprises SEQ ID NO:14. In some embodiments, the variant BCMA comprises SEQ ID NO:15. In some embodiments, the variant BCMA comprises SEQ ID NO:16. In some embodiments, the variant BCMA comprises SEQ ID NO:17. In some embodiments, the variant BCMA comprises SEQ ID NO:18. In some embodiments, the variant BCMA comprises SEQ ID NO: 19. In some embodiments, the variant BCMA comprises SEQ ID NO: 20. In some embodiments, the variant BCMA comprises SEQ ID NO: 21. In some embodiments, the variant BCMA comprises SEQ ID NO: 22. In some embodiments, the variant BCMA comprises SEQ ID NO: 23. In some embodiments, the variant BCMA comprises SEQ ID NO: 24. In some embodiments, the variant BCMA comprises SEQ ID NO: 25. In some embodiments, the variant BCMA comprises SEQ ID NO: 26. In some embodiments, the variant BCMA comprises SEQ ID NO: 27. In some embodiments, the variant BCMA comprises SEQ ID NO: 28. In some embodiments, the variant BCMA comprises SEQ ID NO: 29. In some embodiments, the variant BCMA comprises SEQ ID NO: 30. In some embodiments, the variant BCMA comprises SEQ ID NO: 31. In some embodiments, the variant BCMA comprises SEQ ID NO: 32. In some embodiments, the variant BCMA comprises SEQ ID NO: 33. In some embodiments, the variant BCMA comprises SEQ ID NO: 34. In some embodiments, the variant BCMA comprises SEQ ID NO: 35. In some embodiments, the variant BCMA comprises SEQ ID NO: 36.In some embodiments, the variant BCMA comprises SEQ ID NO: 37. In some embodiments, the variant BCMA comprises SEQ ID NO: 38. In some embodiments, the variant BCMA comprises SEQ ID NO: 39. In some embodiments, the variant BCMA comprises SEQ ID NO: 40, 41. In some embodiments, the variant BCMA comprises SEQ ID NO: 42. In some embodiments, the variant BCMA comprises SEQ ID NO: 43. In some embodiments, the variant BCMA comprises SEQ ID NO: 44. In some embodiments, the variant BCMA comprises SEQ ID NO: 45. In some embodiments, the variant BCMA comprises SEQ ID NO: 46. In some embodiments, the variant BCMA comprises SEQ ID NO: 47. In some embodiments, the variant BCMA comprises SEQ ID NO: 48. In some embodiments, the variant BCMA comprises SEQ ID NO: 49. In some embodiments, the variant BCMA comprises SEQ ID NO: 50. In some embodiments, the variant BCMA comprises SEQ ID NO: 51. In some embodiments, the variant BCMA comprises SEQ ID NO: 52. In some embodiments, the variant BCMA comprises SEQ ID NO: 53. In some embodiments, the variant BCMA comprises SEQ ID NO: 54. In some embodiments, the variant BCMA comprises SEQ ID NO: 55. In some embodiments, the variant BCMA comprises SEQ ID NO: 56. In some embodiments, the variant BCMA comprises SEQ ID NO: 57. In some embodiments, the variant BCMA comprises SEQ ID NO: 58. In some embodiments, the variant BCMA comprises SEQ ID NO: 59. In some embodiments, the variant BCMA comprises SEQ ID NO: 60. In some embodiments, the variant BCMA comprises SEQ ID NO: 61. In some embodiments, the variant BCMA comprises SEQ ID NO: 62. In some embodiments, the variant BCMA comprises SEQ ID NO: 63. In some embodiments, the variant BCMA comprises SEQ ID NO: 64. In some embodiments, the variant BCMA comprises SEQ ID NO: 65. In some embodiments, the variant BCMA comprises SEQ ID NO: 66. In some embodiments, the variant BCMA comprises SEQ ID NO: 67. In some embodiments, the variant BCMA comprises SEQ ID NO: 68. In some embodiments, the variant BCMA comprises SEQ ID NO: 69. In some embodiments, the variant BCMA comprises SEQ ID NO: 70. In some embodiments, the variant BCMA comprises SEQ ID NO: 71.In some embodiments, the variant BCMA comprises SEQ ID NO: 72. In some embodiments, the variant BCMA comprises SEQ ID NO: 73. In some embodiments, the variant BCMA comprises SEQ ID NO: 74. In some embodiments, the variant BCMA comprises SEQ ID NO: 75. In some embodiments, the variant BCMA comprises SEQ ID NO: 76. In some embodiments, the variant BCMA comprises SEQ ID NO: 77. In some embodiments, the variant BCMA comprises SEQ ID NO: 78. In some embodiments, the variant BCMA comprises SEQ ID NO: 80. In some embodiments, the variant BCMA comprises SEQ ID NO: 81. In some embodiments, the variant BCMA comprises SEQ ID NO: 82. In some embodiments, the variant BCMA comprises SEQ ID NO: 83. In some embodiments, the variant BCMA comprises SEQ ID NO: 84. In some embodiments, the variant BCMA comprises SEQ ID NO: 85. In some embodiments, the variant BCMA comprises SEQ ID NO: 86. In some embodiments, the variant BCMA comprises SEQ ID NO: 87. In some embodiments, the variant BCMA comprises SEQ ID NO: 88. In some embodiments, the variant BCMA comprises SEQ ID NO: 89. In some embodiments, the variant BCMA comprises SEQ ID NO: 90. In some embodiments, the variant BCMA comprises SEQ ID NO: 91. In some embodiments, the variant BCMA comprises SEQ ID NO: 92. In some embodiments, the variant BCMA comprises SEQ ID NO: 93. In some embodiments, the variant BCMA comprises SEQ ID NO: 94. In some embodiments, the variant BCMA comprises SEQ ID NO: 95. In some embodiments, the variant BCMA comprises SEQ ID NO: 96. In some embodiments, the variant BCMA comprises SEQ ID NO: 97. In some embodiments, the variant BCMA comprises SEQ ID NO: 98. In some embodiments, the variant BCMA comprises SEQ ID NO: 100. In some embodiments, the variant BCMA comprises SEQ ID NO: 101. In some embodiments, the variant BCMA comprises SEQ ID NO: 102. In some embodiments, the variant BCMA comprises SEQ ID NO: 103. In some embodiments, the variant BCMA comprises SEQ ID NO: 104. In some embodiments, the variant BCMA comprises SEQ ID NO: 105. In some embodiments, the variant BCMA comprises SEQ ID NO: 106. In some embodiments, the variant BCMA comprises SEQ ID NO: 107.In some embodiments, the variant BCMA comprises SEQ ID NO: 108. In some embodiments, the variant BCMA comprises SEQ ID NO: 109. In some embodiments, the variant BCMA comprises SEQ ID NO: 110. In some embodiments, the variant BCMA comprises SEQ ID NO: 112. In some embodiments, the variant BCMA comprises SEQ ID NOs: 113, 114. In some embodiments, the variant BCMA comprises SEQ ID NO: 115. In some embodiments, the variant BCMA comprises SEQ ID NO: 116. In some embodiments, the variant BCMA comprises SEQ ID NO: 117. In some embodiments, the variant BCMA comprises SEQ ID NO: 118. In some embodiments, the variant BCMA comprises SEQ ID NO: 119. In some embodiments, the variant BCMA comprises SEQ ID NO: 120. In some embodiments, the variant BCMA comprises SEQ ID NO: 121. In some embodiments, the variant BCMA comprises SEQ ID NO: 122. In some embodiments, the variant BCMA comprises SEQ ID NO: 123. In some embodiments, the variant BCMA comprises SEQ ID NO: 124. In some embodiments, the variant BCMA comprises SEQ ID NO: 125. In some embodiments, the variant BCMA comprises SEQ ID NO: 126. In some embodiments, the variant BCMA comprises SEQ ID NO: 127. In some embodiments, the variant BCMA comprises SEQ ID NO: 128. In some embodiments, the variant BCMA comprises SEQ ID NO: 129. In some embodiments, the variant BCMA comprises SEQ ID NO: 130. In some embodiments, the variant BCMA comprises SEQ ID NO: 131. In some embodiments, the variant BCMA comprises SEQ ID NO: 132. In some embodiments, the variant BCMA comprises SEQ ID NO: 133. In some embodiments, the variant BCMA comprises SEQ ID NO: 134. In some embodiments, the variant BCMA comprises SEQ ID NO: 135. In some embodiments, the variant BCMA comprises SEQ ID NO: 136. In some embodiments, the variant BCMA comprises SEQ ID NO: 137. In some embodiments, the variant BCMA comprises SEQ ID NO: 138. In some embodiments, the variant BCMA comprises SEQ ID NO: 139. In some embodiments, the variant BCMA comprises SEQ ID NO: 140. In some embodiments, the variant BCMA comprises SEQ ID NO: 141. In some embodiments, the variant BCMA comprises SEQ ID NO: 142.In some embodiments, the variant BCMA comprises SEQ ID NO: 143.

[0153] Libraries of these BCMA variant polypeptides can be generated and screened to determine binding to huBAFF and huAPRIL, for example, using the BIACORE assay of Example 9 or the KINEXA assay of Example 11. Methods for generating variant libraries are known. For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides (e.g., polynucleotides encoding huBCMA ECD, such as SEQ ID NO: 3), polynucleotides of the present disclosure (described herein below), or portions thereof, to generate variant libraries that can be expressed, screened, and assayed using the methods described herein. Mutagenesis and directed evolution methods are known.See, e.g., Ling, et al., Anal. Biochem. 1997; 254 (2): 157-78; Dale et al., Methods Mol. Biol., 1996; 57:369-74; Smith, Ann. Rev. Genet., 1985; 19:423-462; Botstein et al., Science, 1985; 229:1193-1201; Carter, Biochem. J., 1986; 237:1-7; Kramer et al., Cell 1984: 38: 879-887; Wells, et al., Gene, 1999; 34:284-290; Christians, et al., Nature Biotechnology, 1999; 17, all of which are incorporated herein by reference. :259-264 (1999), Crameri, et al., Nature, 391: 288-291;Crameri et al., Nature Biotechnology, 1997; 15: 436-438;Zhang, et al., Proceedings of the National Academy of Sciences, USA, 94:4504-4509;Crameri, et al., Nature Biotechnology, 1996; 14 :315-319 (1996) ;Stemmer, Nature, 1994; 370-389-391;Stemmer, Proceedings of the National Academy of Sciences, USA,1994; 91 :10747-10751; WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, US2009 / 0312196 and WO2009 / 152336. B. BCMA Variant Fusion Polypeptides

[0154] Provided herein are variant BCMA fusion polypeptides comprising a variant CRD, e.g., a variant CRD comprising mutations relative to the huBCMA CRD (SEQ ID NO: 1), and an additional polypeptide or domain or region, such as an immunoglobulin Fc region. In some aspects, the variant BCMA fusion protein comprises any one or more of the variant BCMA CRDs provided herein. In some aspects, the Fc region can be selected depending on the desired immune response, e.g., Fc-mediated effector function. In some embodiments, the Fc region is or is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is an Fc region of human origin, an Fc region of murine origin, a chimeric Fc region, and / or a humanized Fc region.

[0155] In some aspects, the variant BCMA fusion polypeptide comprises a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1) and an immunoglobulin Fc region. In some embodiments, the Fc region is of the subclass IgG1, IgG2, or IgG4.

[0156] Certain therapeutic antibodies and Fc fusion proteins contain IgG1 Fc regions (Santos et al., Braz. J. Pharm. Sci. 2018; 54 (Special): e01007 and Duivelshof et al., J. Sep. Sci 2021; 44 (1): 35-62). IgG1 Fc polypeptides generate potent cytotoxic responses due to high-affinity interactions with IgG Fc receptors (FcγRs) (Kang, Exp. Mol. Medicine 2019; 51:1-9). The resulting Fc-mediated effector functions include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). While the induction of potent cytotoxic responses can be advantageous for some applications, such as in the treatment of cancer or infectious diseases, strategies of targeted immunosuppression are required for the treatment of autoimmune diseases (Kellner et al., Transfus Med Hemother 2017; 44 :327-336).

[0157] Compared to existing molecules, the provided variant BCMA molecules bind with higher affinity to BAFF and APRIL. Furthermore, the Fc region of the variant BCMA-Fc fusion protein can be replaced with an Fc of a different subclass without changing the binding affinity for BAFF and APRIL. Replacing an Fc region of a different subclass makes it possible to select a desired immune response. For example, Fc region replacement can facilitate a highly potent cytotoxic response by selecting, for example, an IgG1 Fc, or attenuate the cytotoxic response by selecting, for example, an IgG4 Fc, which is a weak inducer of Fc-mediated effector function (Kang, Exp. Mol. Medicine 2019; 51:1-9 and WO2021068752).

[0158] The efficacy of effector functions such as ADCC and CDC varies among the four subtypes of human IgG Fc regions. IgG1 and IgG3 subtypes exhibit potent effector functions, while IgG2 and IgG4 subtypes exhibit relatively weak ADCC and CDC effects. In some cases, ADCC and CDC may cause undesirable side effects such as toxicity or shorten the duration of efficacy of the fusion protein (Kang, Exp. Mol. Medicine 2019; 51:1-9). In some embodiments, the selected Fc region enhances the stability of the fusion protein. In some embodiments, the selected Fc region results in a uniform fusion protein, which is desirable for production and quality control.

[0159] In some embodiments, provided BCMA fusion proteins comprise an Ig Fc polypeptide that is or is derived from isotype G immunoglobulin (IgG) or a variant thereof. In some embodiments, provided BCMA fusion proteins comprise an Ig Fc polypeptide that is an isotype G immunoglobulin (IgG).

[0160] In some embodiments, provided BCMA fusion proteins comprise an IgG1, IgG2, and IgG3, or IgG4 Fc region. In some embodiments, provided BCMA fusion proteins comprise a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region.

[0161] In some embodiments, provided fusion proteins comprise a human IgG1 Fc region, e.g., an IgG1 Fc region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 168, 169, 170, 226, 227, 228, 229, and 230. In some embodiments, provided fusion proteins comprise an IgG1 Fc region set forth in any one of SEQ ID NOs: 168, 169, 170, 226, 227, 228, 229, and 230.

[0162] In some embodiments, the provided BCMA fusion proteins comprise a human IgG2 Fc region, e.g., an IgG2 Fc region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 171, 235, and 236. In some embodiments, the provided fusion proteins comprise an IgG2 Fc region set forth in any one of SEQ ID NOs: 171, 235, and 236.

[0163] In some embodiments, the provided BCMA fusion proteins comprise a human IgG4 Fc region, for example, an IgG4 Fc region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 161, 163, 72, 231, 232, 233, 234. In some embodiments, the provided fusion proteins comprise an IgG4 Fc region set forth in any one of SEQ ID NOs: 161, 163, 72, 231, 232, 233, 234.

[0164] In some embodiments, the provided BCMA fusion proteins comprise a variant of a human IgG4 Fc region. In some embodiments, the variant human IgG4 Fc region comprises one or more amino acid substitutions, deletions, and / or additions compared to a wild-type or unmodified human IgG4 Fc region, e.g., the wild-type or unmodified IgG4 Fc region sequence set forth in SEQ ID NO: 172. In some embodiments, exemplary substitutions, deletions, and / or additions of human IgG4 Fc regions include those described, for example, in US2015 / 0104410, US2022 / 0033476, Dumet et al., (2019) MAbs DOI: 10.1080 / 19420862.2019.1664365, and Xu et al., (2019) MAbs 10.1080 / 19420862.2019.1631116. In some aspects, exemplary substitutions, deletions, and / or additions of a human IgG4 Fc region include one or more substitutions selected from among S228P, F234A, L235A, and / or L445P, based on EU numbering. In some aspects, exemplary substitutions, deletions, and / or additions of a human IgG4 Fc region include S228P, F234A, L235A, and L445P, based on EU numbering. In some embodiments, provided BCMA fusion proteins comprise a human IgG4 Fc region, e.g., an IgG4 Fc region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 161. In some embodiments, provided fusion proteins comprise the IgG4 Fc region set forth in SEQ ID NO: 161. In some embodiments, the provided BCMA fusion proteins comprise a human IgG4 Fc region, e.g., an IgG4 Fc region having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 163. In some embodiments, the provided fusion proteins comprise an IgG4 Fc region set forth in SEQ ID NO: 163.

[0165] In some embodiments, the human Fc polypeptide comprises isoleucine or valine substituted for one, two, three, four, or more native methionine residues.

[0166] In some embodiments, the variant BCMA polypeptide and an additional polypeptide, such as an Fc region, within a variant BCMA fusion polypeptide described herein are indirectly linked. In some embodiments, the variant BCMA fusion polypeptide and an Fc region, e.g., an IgG1, IgG2, or IgG4 Fc region, are linked via a peptide linker. In some embodiments, the peptide linker comprises a sequence set forth in any one of SEQ ID NOs: 156, 158, 175-186, 188-213, GS, GGS, and GSA. In some embodiments, the peptide linker comprises SEQ ID NO: 156. In some embodiments, the peptide linker comprises SEQ ID NO: 158.

[0167] In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, a variant BCMA polypeptide, a peptide linker, and an Ig Fc polypeptide. In some embodiments, the fusion polypeptide comprises a variant BCMA polypeptide set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19; a peptide linker comprising the sequence set forth in any one of SEQ ID NOs:156, 158, 175-186, 188-213, GS, GGS, and GSA; and an Ig Fc polypeptide comprising the sequence set forth in SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:172, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234.

[0168] In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:3, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:3, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:4, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:4, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:8, a peptide linker comprising SEQ ID NO:156, and an Ig Fc polypeptide comprising SEQ ID NO:161. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO:8, a peptide linker comprising SEQ ID NO:158, and an Ig Fc polypeptide comprising SEQ ID NO:163. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. In some embodiments, a fusion polypeptide comprises a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163.

[0169] In some embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 167, or a sequence having at least 90% sequence identity thereto. In some embodiments, the fusion polypeptide comprises the sequence set forth in SEQ ID NO: 167. C. BCMA Variant Polypeptide Conjugates

[0170] Provided herein is a variant BCMA polypeptide or variant BCMA-Ig-Fc fusion polypeptide, or dimer thereof, comprising a CRD mutated relative to the huBCMA CRD (SEQ ID NO: 1), covalently linked to at least one non-polypeptide conjugation moiety. In some embodiments, the polypeptide variant conjugate comprises a variant BCMA polypeptide comprising a CRD mutated relative to the huBCMA CRD (SEQ ID NO: 1), covalently linked to one or more non-polypeptide conjugation moieties.

[0171] In some embodiments, the non-polypeptide conjugation moiety is or comprises a non-polypeptide polymer, a sugar moiety, and / or a non-polymeric lipophilic moiety. In some embodiments, the non-polypeptide polymer is or comprises a water-soluble polymer that is not a peptide, polypeptide, or protein and can be a natural or synthetic polymer (e.g., a homopolymer, copolymer, terpolymer). In some embodiments, the sugar moiety is or comprises a carbohydrate molecule attached by an in vivo or in vitro glycosylation process, e.g., an N- or O-glycosylation process.

[0172] In some embodiments, the non-polypeptide conjugation moiety is generally selected to alter a particular characteristic of the provided variant BCMA molecule. Non-limiting examples of such characteristics include in vivo serum half-life or functional in vivo half-life, stability, and immunogenicity. In some embodiments, in vivo serum half-life refers to the point at which 50% of the compound of interest circulates in the bloodstream of a human or non-human mammal, such as a rat, mouse, rabbit, or monkey. Serum refers to its conventional meaning, i.e., plasma free of fibrinogen and other clotting factors. The term "functional in vivo half-life" is used herein to refer to the point at which 50% of the biological activity of the compound of interest is still present in the body or target organ, or the point at which the activity of the compound of interest is 50% of its original value.

[0173] In some embodiments, the conjugation moiety may be covalently attached to the polypeptide directly or indirectly via a linker. In some embodiments, suitable linker moieties include, for example, peptides, non-peptide, non-polymeric aliphatic moieties, and oligonucleotides. In some embodiments, a linker peptide is covalently attached to the N-terminus of the variant BCMA polypeptide or its fusion polypeptide, and a non-polypeptide conjugation moiety is covalently attached to the N-terminus of the attached linker peptide. In some embodiments, a linker peptide is attached to the N-terminus or C-terminus of the variant BCMA polypeptide or its fusion polypeptide, and a non-polypeptide conjugation moiety is covalently attached to an attachment group (e.g., a Cys or Lys residue) within the linker peptide. In some embodiments, glycosylation sites may also be incorporated into the linker peptide sequence.

[0174] In some embodiments, exemplary polymers for use in accordance with provided embodiments may be branched (i.e., two or more linear polymer chains joined by a linker group) or linear, and generally have an average molecular weight ranging from at or about 300 Daltons (Da) to at or about 100,000 Daltons (Da), generally from at or about 1,000 Da to at or about 80,000 Da, or ranging from at or about 2,000 Da to at or about 60,000 Da, or 50,000 or about 50,000 Da, or 40,000 or about 40,000 Da, or 30,000 or about 30,000 Da, or 20,000 or about 20,000 Da, or 10,000 or about 10,000 Da, and in some embodiments from at or about 1,000 Da to 5,000 or about 5,000 Da. More specifically, the average molecular weight of the polymer molecules is generally about 2,000 Da, about 5,000 Da, about 10,000 Da, about 12,000 Da, about 15,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da, about 50,000 Da, about 60,000 Da or about 80,000 Da.

[0175] In some embodiments, exemplary polymers for use in accordance with provided embodiments include, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), polyalkylene oxides (PAOs) such as polyalkylene glycol (PAG), which can be a branched polyethylene glycol in which two or more polyethylene glycol chains are linked by a linker group (e.g., a linker such as lysine, glycerol, etc.), polyvinyl alcohol (PVA), polycarboxylates, poly(vinylpyrrolidone), polyethylene-co-maleic anhydride, dextran (e.g., carboxymethyldextran, etc.), and other similar polymers. Exemplary polymers and methods for covalently attaching them to protein molecules are known.

[0176] Provided herein are methods of making a variant BCMA molecular conjugate, the methods comprising: (a) providing a variant BCMA polypeptide, fusion polypeptide or dimer thereof provided herein; and (b) covalently attaching at least one polyethylene glycol (PEG) moiety to either the provided variant BCMA polypeptide, fusion polypeptide or dimer thereof that comprises a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1).

[0177] In some embodiments, a hydroxylated polymer is covalently attached to a variant BCMA molecule provided herein. In some embodiments, a hydroxylated polymer, such as polyethylene glycol, is covalently attached to a variant BCMA molecule provided herein. In some embodiments, at least one terminal hydroxyl group of the polymer molecule is provided in an activated form, i.e., derivatized with a functional group that is reactive toward a target attachment group within the polypeptide. Exemplary reactive functional groups include primary amino groups, hydrazide (HZ), thiol, succinic acid (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carbonate (NPC), maleimide (MAL), and tresylate (TRES).

[0178] In some embodiments, a variant BCMA conjugate may comprise two or more variant BCMA polypeptides comprising mutated CRDs relative to the huBCMA CRD (SEQ ID NO: 1), covalently attached to one or more bifunctional PEGs. In some embodiments, a variant BCMA conjugate may comprise two variant BCMA polypeptides comprising mutated CRDs relative to the huBCMA CRD (SEQ ID NO: 1), covalently attached to one bifunctional PEG. As used herein, the term "bifunctional PEG" refers to a polyethylene glycol moiety derivatized with two functional groups that are reactive to target attachment groups within the described polypeptide.

[0179] The following publications illustrate polymers and / or PEGylation chemistries that can be used in accordance with the provided embodiments: US 5,824,778, US 5,476,653, US 6,875,841, US 5,872,191, US 5,767,284, EP 0 839 850, WO 97 / 32607, EP 229,108, EP 402,378, US 4,902,502, US 5,281,698, US 5,122,614, US 5,219,564, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28024, WO 95 / 00162, WO95 / 11924, WO95 / 13090, WO95 / 33490, WO96 / 00080, WO97 / 18832, WO98 / 41562, WO98 / 48837, WO99 / 32134, WO99 / 32139, WO99 / 32140, WO96 / 40791, WO98 / 32466, WO95 / 06058, EP439 508, WO97 / 03106, WO96 / 21469, WO95 / 13312, EP921 131, US5,736,625, WO98 / 05363, EP809 996, US5,629,384, WO96 / 41813, WO96 / 07670, US5,473,034, US5,516,673, EP605 963, US5,382,657, EP510 356, EP400 472, EP183 503 and EP154 316, all of which are incorporated herein by reference.

[0180] In some embodiments, conjugation of the inventive polypeptide to the activated polymer molecule(s) is carried out according to any conventional method, for example, as described in the following references, which also describe suitable methods for activating the polymer molecules: Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC, Washington; RF Taylor, (1991), "Protein Immobilization: Fundamentals and Applications", Marcel Dekker, NY; SS Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; GT Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, NY, all of which are incorporated herein by reference.

[0181] In some embodiments, variant BCMA polypeptides, fusion polypeptides (and dimers thereof) comprising a mutated CRD compared to huBCMA (SEQ ID NO: 1) can be glycosylated in vivo by introducing a polynucleotide encoding a variant BCMA molecule provided herein having one or more N- or O-glycosylation sites into a glycosylating eukaryotic expression host cell. In some embodiments, the glycosylating eukaryotic expression host cell can be selected from cells such as fungal cells (e.g., filamentous fungal cells or yeast cells), insect cells, mammalian cells, plant cells, or any other glycosylating eukaryotic expression host cell.

[0182] In some embodiments, non-polypeptide lipophilic moieties suitable for conjugation to variant BCMA molecules comprising a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1) include naturally occurring compounds such as saturated or unsaturated fatty acids, fatty acid diketones, terpenes, prostaglandins, vitamins, carotenoids or steroids, phospholipids, or alternatively, synthetic compounds such as straight-chain or branched aliphatic, aryl, alkaryl acids (e.g., carboxylic acids, sulfonic acids), alcohols, amines, etc. Conjugation to the non-polypeptide lipophilic moiety can be at any one of the following exemplary attachment sites: the N- or C-terminus of the provided variant BCMA molecule, the hydroxyl group of amino acid residues Ser, Thr, or Tyr, the epsilon-amino group of Lys, the SH group of Cys, or the carboxyl groups of Asp and Glu. In some embodiments, the provided variant BCMA molecule and non-polypeptide lipophilic moiety can be conjugated to each other directly or indirectly via a linker according to known methods such as those described in Bodanszky, "Peptide Synthesis", John Wiley, New York (1976) and WO 96 / 12505, both of which are incorporated herein by reference. D. Characteristics of BCMA Variant Polypeptides

[0183] Provided herein are variant BCMA polypeptides comprising a mutated CRD relative to the wild-type human BCMA CRD (SEQ ID NO: 1). In some embodiments, the variant BCMA polypeptides provided herein and molecules comprising such variant BCMA polypeptides, e.g., fusion polypeptides and dimers, exhibit particular characteristics, including but not limited to, specific affinity for the ligands BAFF and / or APRIL, inhibition of BAFF and / or APRIL activity or function, and / or reduced binding to heparan sulfate proteoglycans (HSPGs). In some embodiments, the variant BCMA polypeptides provided herein and molecules comprising such variant BCMA polypeptides, e.g., fusion polypeptides and dimers, exhibit reduced glycosylation and / or enhanced molecular weight uniformity. In some embodiments, the variant BCMA polypeptides provided herein exhibit reduced glycosylation, such as by a glycosylating host cell, resulting in enhanced molecular weight uniformity. In some embodiments, the variant BCMA polypeptides provided herein exhibit reduced binding to heparan sulfate proteoglycans (HSPGs) or do not bind, e.g., do not specifically bind, to HSPGs. 1. Binding affinity to BAFF and APRIL

[0184] Provided herein are BCMA variant BCMA polypeptides comprising a mutated CRD relative to the wild-type human BCMA CRD (SEQ ID NO: 1), which bind, e.g., specifically bind, BAFF and APRIL. In some embodiments, the provided variant BCMA polypeptides bind, e.g., specifically bind, mammalian BAFF and APRIL. In some aspects, the variant BCMA polypeptides, fusion polypeptides, or dimers exhibit higher binding affinity for BAFF and / or APRIL compared to the binding affinity of a reference BCMA polypeptide or reference binding molecule. In some embodiments, the provided variant BCMA polypeptides bind, e.g., specifically bind, human BAFF and APRIL (huBAFF and huAPRIL, respectively). In some embodiments, the provided variant BCMA polypeptides bind, e.g., specifically bind, mouse BAFF and APRIL.

[0185] In some embodiments, binding affinity or binding avidity depends on the assay molecule and assay format used to assess binding activity. Exemplary assays are described in the Examples section herein. For example, binding activity to huBAFF and huAPRIL or to mouse BAFF and mouse APRIL can be assessed using phage display, surface plasmon resonance (SPR), BIACORE™, Kinetic Exclusion Assay (KINEXA™), and cell-based binding assays, as described in the Examples. Other binding assays are also known. In some embodiments, surface plasmon resonance (SPR) can be used to assess K D In some embodiments, a Kinetic Exclusion Assay (KINEXA™) is used to measure K D Measure.

[0186] BAFF (B-cell activating factor of the TNF family) is also known as B-lymphocyte stimulator (BLyS), TALL-1, THANK, and zTNF4. In some embodiments, huBAFF comprises the amino acid sequence of a mature human BAFF protein or an isoform thereof, such as that set forth in SEQ ID NO: 214. APRIL (proliferation-inducing ligand) is also known as TALL-2. In some embodiments, huAPRIL comprises the amino acid sequence of mature huAPRIL or an isoform thereof. In some embodiments, the mature huAPRIL protein lacks a signal peptide or propeptide. In some embodiments, huAPRIL comprises amino acid residues 105-250 of SEQ ID NO: 215.

[0187] In some embodiments, a variant BCMA polypeptide comprising a mutated CRD relative to wild-type huBCMA (SEQ ID NO: 1) binds with higher affinity, e.g., binds specifically, to BAFF than a protein comprising the wild-type huBCMA CRD (SEQ ID NO: 1). In some embodiments, provided variant BCMA polypeptides bind to BAFF, e.g., huBAFF, with at least 2-fold or at least about 2-fold, at least 3-fold or at least about 3-fold, at least 4-fold or at least about 4-fold, at least 5-fold or at least about 5-fold, at least 10-fold or at least about 10-fold, at least 20-fold or at least about 20-fold, at least 30-fold or at least about 30-fold, at least 40-fold or at least about 40-fold, at least 50-fold or at least about 50-fold, at least 60-fold or at least about 60-fold, at least 70-fold or at least about 70-fold, at least 80-fold or at least about 80-fold, at least 90-fold or at least about 90-fold, or at least 100-fold greater affinity than a reference BCMA polypeptide or reference binding molecule, e.g., a protein comprising a wild-type huBCMA CRD (SEQ ID NO: 1), such as the huBCMA ECD (SEQ ID NO: 152).

[0188] In some embodiments, a variant BCMA polypeptide that comprises a mutated CRD relative to wild-type huBCMA (SEQ ID NO: 1) binds with higher affinity, e.g., specifically, to APRIL than a protein comprising the wild-type huBCMA CRD (SEQ ID NO: 1). In some embodiments, provided variant BCMA polypeptides bind to APRIL, e.g., huAPRIL, with at least 2-fold, or at least about 2-fold, at least 3-fold, or at least about 3-fold, at least 4-fold, or at least about 4-fold, at least 5-fold, or at least about 5-fold, at least 10-fold, or at least about 10-fold, at least 20-fold, or at least about 20-fold, at least 30-fold, or at least about 30-fold, at least 40-fold, or at least about 40-fold, or at least 50-fold, more affinity than a reference BCMA polypeptide or reference binding molecule, e.g., a protein comprising the wild-type huBCMA CRD (SEQ ID NO: 1), such as the huBCMA ECD (SEQ ID NO: 152).

[0189] In some embodiments, the provided variant BCMA molecules exhibit a binding activity to huAPRIL that is lower than the binding activity of a reference BCMA polypeptide or reference binding molecule, e.g., a corresponding control protein. In these embodiments, this will generally be about 0.1% or less, about 0.2% or less, about 0.5% or less, about 1% or less, about 5% or less, or 10% or less, or about 80% or less, or about 90% or less, or about 95% or less, or about 99% or less of the binding activity of the corresponding control protein, e.g., huAPRIL, as measured, for example, in the BIACORE assay of Example 9 or the KINEXA assay of Example 11. The binding activity ranges from 0.05% or about 0.05% to 99% or about 99%, or from 0.5% or about 0.5% to 95% or about 95% of the binding activity to RIL, more typically from 0.05% or about 0.05%, or from 0.1% or about 0.1%, or from 1% or about 1%, to 70% or about 70%, or 60% or about 60%, or 50% or about 50%, or 40% or about 40% of the binding activity of the corresponding control protein to huAPRIL. 2. Glycosylation and molecular weight uniformity

[0190] Glycosylation is a post-translational modification that can affect protein structure and function. Wild-type huBCMA is a glycoprotein that contains N-glycosylation sites (Huang et al., PNAS 2013; 110 (27): 10928-10933). Glycosylating cells, such as glycosylating host cells expressing huBCMA or variants thereof, can produce various glycoforms of a protein, such as huBCMA or its variants. Glycoform variability can result in heterogeneity in protein characteristics, such as molecular weight, structure, and function. In some embodiments, glycosylation of a variant BCMA polypeptide comprising a serine 40 to glycine 40 mutation (S40G) is reduced compared to a variant BCMA polypeptide comprising a serine at residue 40 and wild-type huBCMA (determined by comparison with SEQ ID NO: 1). In some embodiments, the provided variant BCMA polypeptides, for example variant BCMA polypeptides comprising an S40G mutation, are more homogeneous in terms of molecular weight than a reference BCMA polypeptide, such as a protein comprising the wild-type serine 40 residue, for example the huBCMA CRD (SEQ ID NO: 1).

[0191] In some embodiments, sequence features that promote greater protein molecular weight uniformity can be combined with any one or more of the provided sequence features that are believed to correlate with a phenotype of improved binding activity to huBAFF. In some embodiments, sequence features that promote greater protein molecular weight uniformity can be combined with any one or more of the provided sequence features that are believed to correlate with a phenotype of improved binding activity to APRIL. In some embodiments, sequence features that promote greater protein molecular weight uniformity can be combined with any one or more of the provided sequence features that are believed to correlate with a phenotype of improved binding activity to huBAFF and / or huAPRIL. In some embodiments, sequence features that promote greater protein molecular weight uniformity can be combined with any one or more of the provided sequence features that are believed to correlate with a phenotype of higher binding affinity to huBAFF compared to huAPRIL. In some embodiments, sequence features that promote greater protein molecular weight uniformity can be combined with any one or more of the provided sequence features that are believed to correlate with higher binding affinity to huAPRIL compared to huBAFF. 3. Binding affinity to heparan sulfate proteoglycans (HSPGs)

[0192] Heparan sulfate proteoglycans (HSPGs), which consist of a core protein covalently linked to glycosaminoglycan (GAG) chains formed by unbranched sulfated anionic polysaccharides known as heparan sulfate, are widely expressed and mediate a variety of biological activities (Cagno et al., Viruses. 2019 Jul; 11 (7): 596) and Sarrazin et al., Cold Spring Harb Perspect Biol. 2011; 3 (7): a004952). It may be advantageous to reduce or minimize HSPG binding, thereby preserving HSPG-mediated activities that exert effects at the cellular, tissue, and organismal levels.

[0193] In some embodiments, provided herein are variant BCMA polypeptides that exhibit reduced binding to HSPGs or that do not bind to HSPGs. In some embodiments, provided herein are variant BCMA polypeptides that exhibit reduced binding to or lack affinity for HSPGs, such as syndecan-1 and syndecan-2. In some embodiments, provided herein are variant BCMA polypeptides that exhibit reduced binding to HSPGs compared to the binding of a reference BCMA polypeptide or reference binding molecule to HSPGs. 4. Inhibition of BAFF and / or APRIL

[0194] In some embodiments, the provided variant BCMA polypeptides, fusion polypeptides, or dimers inhibit the activity or function of BAFF and / or APRIL. In some embodiments, the provided variant BCMA polypeptides, fusion polypeptides, or dimers exhibit greater inhibition of BAFF and / or APRIL activity or function compared to inhibition of BAFF and / or APRIL activity or function by a reference BCMA polypeptide or reference binding molecule. In some embodiments, the reference BCMA polypeptide includes a corresponding control protein or a corresponding wild-type BCMA polypeptide. In some embodiments, the reference binding molecule is selected from among atacicept, telitacicept, belimumab, or BION-1301. In some embodiments, the activity or function of BAFF and / or APRIL is selected from B cell survival, B cell proliferation, and / or immunoglobulin production.

[0195] In some embodiments, inhibition of BAFF and / or APRIL activity or function is assessed using any known assay for measuring BAFF and / or APRIL activity or function, such as a binding assay, a cell-based expression assay, a cell proliferation assay, a cell-based effector assay, an immunoglobulin production assay, and / or a reporter assay, including, for example, those described in the Examples herein. In some embodiments, the assessed BAFF and / or APRIL activity or function includes binding to cells, e.g., B cells, that express their corresponding receptors, e.g., BAFF-R, TACI, and BCMA. In some embodiments, inhibition of BAFF and / or APRIL activity or function is assessed by measuring blocking of BAFF and / or APRIL binding to cells that express such receptors.

[0196] In some aspects, the BAFF and / or APRIL activity or function that is assessed includes stimulation of the growth and / or proliferation of cells that express the corresponding receptors, e.g., BAFF-R, TACI, and BCMA, e.g., B cells. In some embodiments, B cell proliferation in the presence of BAFF and / or APRIL and inhibition of B cell proliferation by a provided variant BCMA polypeptide, fusion polypeptide, or dimer is assessed.

[0197] In some embodiments, inhibition of human BAFF activity or function by the variant BCMA polypeptide, fusion polypeptide, or dimer is at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 20-fold, at least or at least about 25-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 200-fold, at least or at least about 250-fold, or at least or at least about 500-fold greater than inhibition of human BAFF activity or function by a reference BCMA polypeptide or reference binding molecule. In some embodiments, inhibition of human APRIL activity or function by the variant BCMA polypeptide, fusion polypeptide, or dimer is at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 20-fold, at least or at least about 25-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 200-fold, at least or at least about 250-fold, or at least or at least about 500-fold greater than inhibition of human APRIL activity or function by the reference BCMA polypeptide or reference binding molecule. II. Methods of generating BCMA variant molecules

[0198] Also provided are methods, polynucleotides, compositions, and kits for expressing variant BCMA polypeptides and fusion proteins, and for generating cells that express such molecules. In some embodiments, cells, such as host cells, can be genetically engineered with one or more variant BCMA polypeptides and fusion proteins. Genetic engineering generally involves introducing nucleic acids encoding recombinant or engineered components into cells, for example, by retroviral transduction, transfection, or transformation. A. Polynucleotides encoding BCMA variant molecules

[0199] Provided herein are polynucleotides encoding any of the variant BCMA polypeptides, fusion proteins, conjugates, dimers, multimers, or components thereof described herein. In some embodiments, the provided polynucleotides encoding variant BCMA molecules can be engineered to target polypeptide expression to a desired cellular compartment, cell membrane, or organelle, or to direct polypeptide secretion into the periplasmic space or cell culture medium. In some embodiments, the polynucleotides encoding the variant BCMA molecules can be fused in-frame with a nucleic acid encoding a signal sequence, such as a secretory or localization sequence. Exemplary signal sequences are known. Non-limiting examples of signal sequences include secretory leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, endoplasmic reticulum (ER) retention signals, mitochondrial import sequences, chloroplast import sequences), and membrane localization or anchoring sequences (e.g., stop transport sequences, GPI anchor sequences).

[0200] In some aspects, provided herein are polynucleotides encoding any of the variant BCMA polypeptides or fusion proteins provided herein. In some embodiments, the polynucleotides described herein encode variant BCMA polypeptides comprising a mutated CRD compared to the wild-type human BCMA CRD (SEQ ID NO: 1) encoded by SEQ ID NO: 2. Full-length huBCMA (SEQ ID NO: 149) is encoded by the polynucleotide sequence set forth in SEQ ID NO: 148. HuBCMA ECD (SEQ ID NO: 152) is encoded by the polynucleotide sequence set forth in SEQ ID NO: 151. Due to the degeneracy of the genetic code, it will be understood that there are numerous polynucleotide sequences encoding the provided variant BCMA polypeptides. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at any position where arginine is specified by a codon in the polynucleotides of the invention, that codon can be altered to any of the corresponding codons listed above without altering the encoded polypeptide. It will be understood that U in an RNA sequence corresponds to T in a DNA sequence.

[0201] Such "silent variations" are a species of "conservative" variations. It will be understood that each codon within a polynucleotide sequence (other than AUG, which is normally the only codon for methionine, and UGG, which is normally the only codon for tryptophan) can be altered by standard techniques to encode a functionally identical polypeptide. Thus, with any described sequence, each silent variation of the polynucleotide encoding the polypeptide is implicit. All possible variations of the polynucleotide sequences encoding the polypeptides of the present invention that can be created by selecting combinations based on possible codon choices are contemplated and provided herein. These combinations are created according to the standard triplet genetic code applied to the polynucleotide sequences encoding the variant BCMA polypeptides provided.

[0202] A group of two or more different codons that all encode the same amino acid under the same translational context is referred to herein as a "synonymous codon." The described variant BCMA polypeptide can be codon-optimized for expression in a specific host organism by modifying the polynucleotide to match the optimal codon usage of the desired host organism. It can be understood that tables and other references providing preference information for a wide range of organisms are readily available. For example, see Henaut and Danchin in "Escherichia coli and Salmonella," Neidhardt, et al. Eds., ASM Press, Washington DC (1996), pp. 2047-2066, which is incorporated herein by reference.

[0203] In some embodiments, the nucleotide sequence encoding the variant BCMA CRD has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 159. In some embodiments, the nucleotide sequence encoding the variant BCMA CRD comprises SEQ ID NO: 159.

[0204] In some embodiments, the nucleotide sequence encoding the peptide linker has at least 80%, or at least about 80%, at least 81%, or at least about 81%, at least 82%, or at least about 82%, at least 83%, or at least about 83%, at least 84%, or at least about 84%, at least 85%, or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95% or at least about 95% sequence identity to the sequence set forth in SEQ ID NO:155. In some embodiments, the nucleotide sequence encoding the peptide linker has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 157. In some embodiments, the nucleotide sequence encoding the linker comprises SEQ ID NO: 155. In some embodiments, the nucleotide sequence encoding the linker comprises SEQ ID NO: 157.

[0205] In some embodiments, the nucleotide sequence encoding the IgG4 Fc region has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 238. In some embodiments, the nucleotide sequence encoding the IgG4 Fc region comprises SEQ ID NO:238. In some embodiments, the nucleotide sequence encoding the IgG4 Fc region has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 160. In some embodiments, the nucleotide sequence encoding the IgG4 Fc region comprises SEQ ID NO:160.In some embodiments, the nucleotide sequence encoding the IgG4 Fc region has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 162. In some embodiments, the nucleotide sequence encoding the IgG4 Fc region comprises SEQ ID NO:162.

[0206] In some embodiments, provided herein are polynucleotides encoding variant BCMA polypeptides, such as variant BCMA fusion polypeptides, which can be designed to include a signal sequence that directs secretion of the mature polypeptide through a prokaryotic or eukaryotic cell membrane.

[0207] In some embodiments, the nucleotide sequence encoding the signal peptide has at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least about 94%, or at least 95%, at least 96%, at least 97%, or at least about 97%, or at least 98% or at least about 98% sequence identity to SEQ ID NO: 153. In some embodiments, the nucleotide sequence encoding the signal peptide comprises SEQ ID NO:153. In some embodiments, the nucleotide sequence encoding the signal peptide has at least 85%, or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, or at least about 98% or at least about 98% sequence identity to SEQ ID NO: 249. In some embodiments, the nucleotide sequence encoding the signal peptide comprises SEQ ID NO: 249. It is understood that the mature sequence lacking the signal peptide after the signal peptide is cleaved when expressed and produced in a cell is also encompassed.In some embodiments, the signal peptide has at least or at least about 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least about 94%, or at least 95%, at least 96%, at least 97%, or at least about 97%, or at least 98% or at least about 98% sequence identity to SEQ ID NO: 154. In some embodiments, the signal peptide comprises SEQ ID NO: 154. In some embodiments, the signal peptide has at least 85%, or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, or at least about 98% or at least about 98% sequence identity to SEQ ID NO: 250. In some embodiments, the signal peptide comprises SEQ ID NO: 250. It is understood that the mature sequence lacking the signal peptide after the signal peptide is cleaved when expressed and produced in a cell is also encompassed.

[0208] In some embodiments, the nucleotide sequence encoding the BCMA variant fusion polypeptide comprising a signal sequence has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, or at least 98% or at least about 98% sequence identity to SEQ ID NO: 164. In some embodiments, the polynucleotide sequence encoding the BCMA variant fusion polypeptide comprising a signal sequence comprises SEQ ID NO: 164. In some embodiments, the nucleotide sequence encoding the BCMA variant fusion polypeptide comprising a signal sequence has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, or at least 98% or at least about 98% sequence identity to SEQ ID NO: 239. In some embodiments, the polynucleotide sequence encoding the BCMA variant fusion polypeptide comprising a signal sequence comprises SEQ ID NO: 239. It is understood that the mature sequence lacking the signal peptide after the signal peptide is cleaved when expressed and produced in a cell is also encompassed.

[0209] In some embodiments, the nucleotide sequence encoding the BCMA variant fusion polypeptide lacking a signal sequence has at least or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, or at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, or at least 98% or at least about 98% sequence identity to SEQ ID NO: 166. In some embodiments, the polynucleotide sequence encoding the BCMA variant fusion polypeptide lacking a signal sequence comprises SEQ ID NO: 166. B. Expression Vectors

[0210] Provided herein are polynucleotides encoding any of the variant BCMA polypeptides, fusion proteins, conjugates, dimers, multimers or components thereof described herein, or vectors, e.g., expression vectors, comprising any of the polynucleotides described herein.

[0211] Polynucleotides encoding the provided variant BCMA molecules comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1) can be incorporated into any one of a variety of known expression vectors. The vector can be used to transform a host, e.g., a host cell, to facilitate expression of the provided polynucleotides encoding the provided variant BCMA molecules comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, expression vectors compatible with prokaryotic host cells, such as prokaryotic expression vectors, can be used. Non-limiting examples of expression vectors compatible with prokaryotic host cells include pUC vectors (e.g., New England BioLabs), BLUESCRIPT vectors (e.g., Stratagene), T7 expression vectors (e.g., Invitrogen), pET vectors (e.g., Novagen), and multifunctional E. coli cloning and expression vectors.

[0212] In some embodiments, expression vectors compatible with eukaryotic host cells can be used, such as known eukaryotic expression vectors.Non-limiting examples of expression vectors compatible with eukaryotic host cells include pCMV vectors (e.g., Invitrogen), pIRES vectors (e.g., Clontech), pSG5 vectors (e.g., Stratagene), pCDNA3.1 (e.g., Invitrogen Life Technologies), pCDNA3 (e.g., Invitrogen Life Technologies), and ubiquitous chromatin opening element (UCOE) expression vectors (e.g., Millipore).

[0213] In some embodiments, suitable expression vectors include chromosomal DNA sequences, non-chromosomal DNA sequences, and synthetic DNA sequences. In some embodiments, suitable expression vectors include bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), plasmids such as bacterial or yeast plasmids, cosmids, or phages. In some embodiments, suitable expression vectors are vectors derived from viral DNA, such as vaccinia, adenovirus, fowlpox, pseudorabies, adenovirus, adeno-associated virus, and retrovirus, as well as vectors derived from a combination of plasmid DNA and phage DNA. Any vector can be used that transduces genetic material into cells and, if replication is desired, is replicable and viable in the relevant host. In some embodiments, the expression vectors provided herein include pUC vector sequences. In some embodiments, the expression vector comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 219. In some embodiments, the expression vector is a pUC vector. In some embodiments, the expression vector comprises the sequence set forth in SEQ ID NO: 219.

[0214] In some embodiments, the expression vector contains one or more selectable marker genes that provide a phenotypic trait for selecting transformed host cells. In some embodiments, the selectable marker genes include genes encoding resistance to the antibiotics spectinomycin or streptomycin (e.g., the aadA gene), the streptomycin phosphotransferase (SPT) gene encoding resistance to streptomycin, the neomycin phosphotransferase (NPTII) gene encoding resistance to kanamycin or geneticin, and the hygromycin phosphotransferase (HPT) gene encoding resistance to hygromycin. In some embodiments, the selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli.

[0215] In some embodiments, the expression vectors provided herein comprise a puromycin resistance gene. In some embodiments, the puromycin resistance gene comprises a nucleotide sequence having at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 220. In some embodiments, the puromycin resistance gene comprises the nucleotide sequence set forth in SEQ ID NO: 220. In some embodiments, the puromycin resistance gene comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 221. In some embodiments, the puromycin resistance gene comprises the amino acid sequence set forth in SEQ ID NO: 221. In some embodiments, the expression vectors provided herein comprise an ampicillin resistance gene. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 222. In some embodiments, the ampicillin resistance gene comprises the nucleotide sequence set forth in SEQ ID NO: 222. In some embodiments, the ampicillin resistance gene comprises an amino acid sequence having at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 223. In some embodiments, the ampicillin resistance gene comprises the amino acid sequence set forth in SEQ ID NO: 223. In some embodiments, the expression vectors provided herein comprise a puromycin resistance gene and an ampicillin resistance gene. C. Regulatory Elements

[0216] In some embodiments, the expression vectors or constructs provided herein comprise one or more regulatory elements or sequences that direct expression of one or more polynucleotides described herein. Such regulatory elements or sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. In some embodiments, the regulatory element or sequence comprises a promoter and transcriptional and translational stop signals. In some embodiments, the regulatory element or sequence comprises an additional sequence that introduces a specific restriction site. In some embodiments, the introduced specific restriction site can facilitate ligation of the element or sequence with the coding sequence(s) of a nucleotide sequence encoding a BCMA variant polypeptide described herein.

[0217] In some embodiments, expression of the encoded variant BCMA molecule is regulated by one or more of a promoter, enhancer, and regulatory element. In some embodiments, the promoter and / or enhancer or regulatory element may be a conditional promoter, enhancer, and / or regulatory element. In some embodiments, these elements drive expression of a transgene. In some embodiments, an expression vector includes sequences for amplifying expression, such as an enhancer. In some embodiments, when a polynucleotide encoding a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1) is incorporated into an expression vector, the polynucleotide is operably linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Methods for achieving this operably linkage either before or after insertion of a DNA molecule into a vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in regulating transcription or translation. 1. Promoter

[0218] The polynucleotides described herein can be driven by a promoter or enhancer to control or regulate expression. In some embodiments, a promoter is operably linked to the coding region of the nucleic acid whose expression is desired. In some embodiments, other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or viruses therefor can be used. Non-limiting examples of promoters that can be operably linked to a provided polynucleotide, such as a polynucleotide encoding a variant BCMA molecule, in an expression construct include, for example, the EF1 alpha promoter with the HTLV1 enhancer, the Caviid herpesvirus 2 promoter A, the SV40 promoter, the E. coli lac or trp promoter, the phage lambda PL promoter, the tac promoter, or the T7 promoter. In some embodiments, a polynucleotide encoding a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1) is operably linked to a T5 promoter.

[0219] In some embodiments, the promoter may be tissue-specific. A tissue-specific promoter allows a protein to be produced in a particular cell population that has the appropriate transcription factors to activate the promoter. Numerous promoters are commercially available and widely known in the art. In some embodiments, the promoter is selected from the group consisting of cytomegalovirus promoter (CMV), bla promoter, Caviid herpesvirus 2-derived promoter A, phosphoglycerate kinase (PGK) promoter, Simian virus 40 early promoter (SV40), or Rous sarcoma virus long terminal repeat (LTR) promoter (RSV). The promoter may be a constitutive promoter, such as a CMV promoter, a tissue-specific promoter, or an inducible or regulatable promoter. In some embodiments, the polynucleotide contains an inducible promoter operably linked to the coding region, such that expression of the nucleic acid can be controlled by controlling the presence or absence of an appropriate inducer of transcription. Exemplary promoters, such as the SV40 promoter, include the sequence set forth in SEQ ID NO:247, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 95%, or at least 99%, or at least 99% sequence identity to SEQ ID NO:247.

[0220] In some embodiments, the promoter is a constitutive promoter. Exemplary promoters include, but are not limited to, a CMV promoter, a truncated CMV promoter, a PGK promoter such as a mouse PGK promoter, a bla promoter, a human serum albumin promoter, or a C-1-antitrypsin promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a truncated CMV promoter lacking known transcriptional repressor binding sites. The CMV-derived promoter may be of human or monkey origin. In some embodiments, the promoter is an inducible promoter. For example, the promoter is an inducible ecdysone promoter. Other examples of promoters include steroid promoters such as estrogen promoters and androgen promoters, and metallothionein promoters. In some embodiments, the enhancer may be a tissue-specific or non-specific enhancer. For example, the enhancer is a liver-specific enhancer element. Exemplary enhancer elements include, but are not limited to, the human serum albumin (HSA) enhancer, the human prothrombin (HPrT) enhancer, the C-1-microglobulin enhancer, the intronic aldolase enhancer, and the apolipoprotein E liver control region.

[0221] In some embodiments, a vector or construct may contain a single promoter driving the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, may be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, a transcription unit may be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), thereby allowing co-expression of gene products (e.g., encoding a first chimeric receptor and a second chimeric receptor) by messages from a single promoter. Exemplary IRES sequences include the sequence set forth in SEQ ID NO:244, or a sequence having at least 80%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 95%, or at least 99% sequence identity to SEQ ID NO:244.

[0222] Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (e.g., encoding first and second binding molecules, such as antibody recombinant receptors) in a single open reading frame (ORF) separated from each other by sequences encoding self-cleaving peptides (e.g., 2A cleavage sequences) or protease recognition sites (e.g., furin). Thus, the ORF encodes a single polypeptide, which is cleaved either during translation (in the case of T2A) or after translation to generate individual proteins. In some cases, a peptide such as T2A can cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), thereby separating the end of the 2A sequence from the next downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2: 13 (2004) and de Felipe et al. Traffic 5: 616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides disclosed herein include, but are not limited to, the 2A sequence from foot and mouth disease virus (F2A), the 2A sequence from equine rhinitis A virus (E2A), the 2A sequence from Thosea asigna virus (T2A), and the 2A sequence from porcine teschovirus-1 (P2A), as described in U.S. Patent Application Publication No. 20070116690. In some embodiments, one or more different or separate promoters drive the expression of one or more nucleic acid molecules encoding one or more BCMA variant molecules.

[0223] In some embodiments, the expression vectors provided herein comprise a human eukaryotic translation elongation factor 1 promoter. In some embodiments, the hEF1 promoter is a human eukaryotic translation elongation factor 1 alpha (hEF1 alpha or hEF1α) promoter. In some embodiments, the hEF1α promoter comprises a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 225. In some embodiments, the hEF1α promoter comprises the sequence set forth in SEQ ID NO: 225. 2. Bicistronic or IRES elements

[0224] In some embodiments, the expression cassette containing the encoding polynucleotide can be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). In some embodiments, a transcription unit can be engineered as a bicistronic unit containing bicistronic elements, thereby allowing co-expression of gene products with messages from a single promoter. In some embodiments, the bicistronic element is an IRES (internal ribosome entry site). In some embodiments, the bicistronic element can be a self-cleaving sequence such as a 2A sequence (e.g., P2A, FTA, or T2A). Exemplary IRES sequences include the sequence set forth in SEQ ID NO:244, or a sequence having at least 80%, at least about 80%, at least 85%, at least 90%, at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO:244.

[0225] An internal ribosome entry site (IRES) is a sequence that initiates translation from an internal start codon (usually AUG) within a bicistronic or multicistronic RNA transcript followed by multiple protein-coding regions. IRESs have been characterized in encephalomyocarditis virus and related picornaviruses (e.g., Jackson et al., RNA 1995; 1:985-1000 and Herman, Trends in Biochemical Sciences 1989; 14(6):219-222). IRES sequences have also been detected in mRNAs from other viruses, such as cardioviruses, rhinoviruses, aphthoviruses, hepatitis C virus (HCV), Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). The presence of IRESs in cellular RNAs has also been described. Expression vectors containing IRES elements have been described. See, e.g., PCT / US98 / 03699 and PCT / EP98 / 07380. In some embodiments, the expression vector comprising a provided polynucleotide encoding a variant BCMA molecule further comprises a ribosome binding site for translation initiation. 3.3' untranslated region (UTR)

[0226] The 3' untranslated region (3'UTR) is generally a part of mRNA, located between the protein coding region (i.e., open reading frame) and poly(A) sequence of the mRNA. The 3'UTR of an mRNA is not translated into an amino acid sequence. The 3'UTR sequence is generally encoded by a gene that is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into a premature mRNA containing optional introns. The premature mRNA is then further processed into a mature mRNA in a maturation process. This maturation process includes steps such as 5' cap formation, splicing of the premature mRNA to remove optional introns, and 3' end modification steps such as polyadenylation of the 3' end of the premature mRNA, and optional endonuclease or exonuclease cleavage.

[0227] In some embodiments, the 3'UTR corresponds to the sequence of a mature mRNA that is located 3' to the stop codon of the protein-coding region, e.g., immediately 3' to the stop codon of the protein-coding region, and extends 5' to the poly(A) sequence, e.g., to the nucleotide immediately 5' to the poly(A) sequence. The term "corresponding to" indicates that the 3'UTR sequence can be an RNA sequence, e.g., within the mRNA sequence used to define the 3'UTR sequence, or a DNA sequence that corresponds to such an RNA sequence. In some embodiments, the term "3'UTR" refers to a sequence that corresponds to the 3'UTR of a mature mRNA derived from this gene, i.e., an mRNA obtained by transcription of the gene and maturation of the immature mRNA. In some embodiments, the term "3'UTR of a gene" encompasses both DNA and RNA sequences of the 3'UTR.

[0228] In some embodiments, the 3'UTR sequence comprises a transcription termination sequence. In some embodiments, the transcription termination sequence comprises a polyA tail. A polyA tail is also referred to as a 3' poly(A) tail or poly(A) sequence. A polyA tail is a long sequence of adenosine nucleotides added to the 3' end of an RNA molecule. Polyadenylation is the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, for example, a premature mRNA. Polyadenylation can be induced by a polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3' end of the nucleic acid molecule, such as an RNA molecule, to be polyadenylated. A polyadenylation signal generally comprises a hexamer of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also contemplated. Polyadenylation generally occurs during the processing of pre-mRNA (also referred to as premature mRNA). Generally, RNA maturation (pre-mRNA to mature mRNA) includes a polyadenylation step.

[0229] In some embodiments, the provided expression vectors comprise a transcription termination sequence comprising an SV40 polyadenylation signal sequence. In some embodiments, exemplary SV40 polyadenylation signal sequences comprise a sequence having at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 248. In some embodiments, exemplary SV40 polyadenylation signal sequences comprise the sequence set forth in SEQ ID NO: 248. In some embodiments, the provided expression vectors comprise a transcription termination sequence comprising the potato proteinase inhibitor II (Pin II) gene (Xing et al., Plant Biotechnol J. 2010; 8 (7): 772-82). In some embodiments, the provided expression vectors comprise a transcription termination sequence comprising the bovine growth hormone (bGH) polyadenylation signal sequence. In some embodiments, the bGH polyadenylation signal sequence comprises a sequence having at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 218. In some embodiments, the bGH polyadenylation signal sequence comprises the sequence set forth in SEQ ID NO: 218. In some embodiments, the bGH polyadenylation signal sequence comprises a sequence having at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 240. In some embodiments, the bGH polyadenylation signal sequence comprises the sequence set forth in SEQ ID NO: 240. D. Production and Recovery of BCMA Variant Polypeptides

[0230] Provided herein are methods of making any of the provided variant BCMA polypeptides, fusion proteins, and / or dimers. In some embodiments, the methods include the steps of: (1) selecting an expression host, e.g., a host cell; (2) introducing a recombinant polynucleotide encoding a provided variant BCMA molecule into the host cell; (3) culturing the host cell containing the recombinant polynucleotide in a culture medium under conditions suitable for expression of the encoded variant BCMA molecule; and (4) recovering the variant BCMA molecule from the culture medium or from the cultured host cell. 1. Expression Hosts and Methods for Introducing Recombinant Polynucleotides

[0231] An expression host, e.g., a host cell, is any cell type amenable to introduction of a vector or construct comprising a recombinant polynucleotide described herein, e.g., a polynucleotide encoding any of the variant BCMA polypeptides, fusion proteins, and / or dimers provided herein. In some embodiments, the host cell is an isolated host cell. In some embodiments, the isolated host cell is a eukaryotic cell. In some embodiments, the isolated host cell is a mammalian cell, e.g., a Chinese hamster ovary (CHO) cell, a yeast cell, or a plant cell. In some embodiments, the isolated host cell is a prokaryotic cell. In some embodiments, the isolated host cell is a bacterial cell, e.g., an E. coli cell, a Bacillus spp. cell, a Streptomyces spp. cell).

[0232] In some embodiments, host cell lines are selected for their ability to modulate expression of inserted sequences or process expressed proteins, e.g., post-translationally modify them. Such protein modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational modifications (PTMs) can affect correct insertion, folding, and / or protein function. In one example of a PTM, a mature protein is formed by cleavage of a preprotein, and thus the mature protein lacks amino acid residues encoded by the signal sequence. Various host cells, such as E. coli, Bacillus spp., yeast, or mammalian cells, e.g., CHO, HeLa, BHK, MDCK, HEK293, WI38, have specific cellular machinery and mechanisms characteristic of such post-translational activities. In some embodiments, host cells can be selected to ensure correct modification and processing of the encoded recombinant protein, including a variant BCMA molecule provided herein. In some aspects, the host cell is a CHO cell.

[0233] Provided herein are expression hosts, e.g., host cells, comprising any of the polynucleotides encoding a variant BCMA molecule comprising a mutated CRD compared to the provided huBCMA CRD (SEQ ID NO: 1), such as any provided herein. In some embodiments, host cells are transduced with a vector or construct comprising a polynucleotide encoding a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1), such as any provided herein. In some embodiments, host cells are transformed with a vector or construct comprising a polynucleotide encoding a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1), such as any provided herein. In some embodiments, host cells are transfected with a vector or construct comprising a polynucleotide encoding a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1), such as any provided herein.

[0234] Introduction of nucleic acid constructs into host cells can be carried out by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, gene or vaccine gun, injection, or other common techniques (see, e.g., Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology, incorporated herein by reference, for in vivo, ex vivo, or in vitro methods). 2. Cultivation of Expression Host and Polypeptide Production

[0235] Provided herein are methods of culturing an expression host, e.g., a host cell, comprising a recombinant polynucleotide encoding a variant BCMA molecule comprising a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1), such as any provided herein, to facilitate production of the provided variant BCMA molecules.

[0236] The culture and / or production of expression hosts, e.g., host cells, of bacterial, plant, animal (especially mammalian), and archaeal origin is well known. The culture and / or production of cells of bacterial, plant, animal (especially mammalian), and archaeal origin is described, for example, in Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001; Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York (and references cited therein); Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques, John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition, W.H. Freeman and Company; and Ricciardelli, et al. In vitro Cell Dev. Biol. 1989; 25:1016-1024), all of which are incorporated herein by reference.References describing plant cell culture and regeneration include Payne, et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (Eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg, New York); Jones, Ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, New Jersey, and Plant Molecular Biology (1993) RRD Croy, Ed. Bios Scientific Publishers, Oxford, UK ISBN 0 12 198370 6, all of which are incorporated herein by reference.

[0237] Cell culture media are generally described in Atlas and Parks (Eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., which is incorporated herein by reference. Further information regarding cell culture can be found in advertising materials such as Sigma-Aldrich, Inc. (St. Louis, Mo.)'s Life Science Research Cell Culture Catalogue (1998) and Sigma-Aldrich, Inc. (St. Louis, Mo.)'s The Plant Culture Catalogue and supplement (1997), both of which are incorporated herein by reference.

[0238] In some embodiments, the variant BCMA molecules produced by the host cells, such as any of the variant BCMA polypeptides, fusion proteins and / or dimers described herein, may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and / or vector used. As will be appreciated by those of skill in the art, expression vectors comprising recombinant polynucleotides encoding the provided variant BCMA molecules can be designed to include a signal sequence that directs secretion of the mature polypeptide through a prokaryotic or eukaryotic cell membrane.

[0239] In some embodiments, stable expression can be used for long-term, high-yield production of recombinant proteins. In some embodiments, host cell lines stably expressing the polypeptides and fusion proteins described herein are transduced with expression vectors or constructs containing a viral origin of replication, endogenous expression elements, and / or a selectable marker gene. In some embodiments, after introduction of the vector or construct, the host cells can be grown in an enriched medium for 1-2 days before being switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection; its presence allows for the growth and recovery of cells that successfully express the introduced recombinant polynucleotide. In some embodiments, resistant clumps of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type. 3. Recovery of Polypeptides

[0240] Provided herein are methods for recovering variant BCMA molecules comprising a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1), such as any provided herein. In some embodiments, host cells comprising a recombinant polynucleotide encoding a variant BCMA molecule provided herein are cultured under conditions that facilitate recovery of the encoded variant BCMA protein. In some embodiments, the variant BCMA molecule is recovered from the host cell culture medium. In some embodiments, the variant BCMA molecule is recovered from the host cell. In some embodiments, the variant BCMA molecule is recovered from the host cell culture medium and the host cell.

[0241] In another embodiment, a method for producing a variant BCMA fusion polypeptide dimer is provided herein. In some aspects, the method includes culturing a host cell transformed or transfected with a vector comprising a polynucleotide encoding a variant BCMA fusion polypeptide described herein in a culture medium under conditions suitable for expression of the encoded fusion polypeptide and dimer formation to produce a fusion polypeptide dimer; and recovering the fusion polypeptide dimer from the culture medium or from the cultured host cell. Typically, the polynucleotide also encodes a secretory or signal peptide operably linked to the encoded fusion polypeptide. In this embodiment, the fusion polypeptide is typically secreted from the host cell as a disulfide-bonded fusion polypeptide dimer.

[0242] In some embodiments, the host cell is a mammalian cell, such as a CHO cell. In some embodiments, the host cell used to produce and recover the provided polypeptides is an isolated host cell, as compared to a higher organism, such as a plant or animal. In some methods, the BCMA variant polypeptides, fusion polypeptides, and fusion polypeptide dimers are recovered from the culture medium, the host cell, or the host cell periplasm.

[0243] In some embodiments, after transformation into a suitable host strain and growing the host strain to an appropriate cell density, the selected promoter is induced by appropriate means, for example, by temperature shift or chemical induction, and the cells are cultured for an additional period. In some embodiments, the cells are collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. In some embodiments, microbial cells used for protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other known or exemplary methods.

[0244] In some embodiments, the described BCMA variant polypeptides (variant polypeptides, fusion polypeptides (and fusion polypeptide dimers)) can be recovered / isolated from recombinant cell culture and, if desired, purified by any of several methods, such as, for example, ammonium sulfate or solvent precipitation (such as by use of solvents such as ethanol, acetone, etc.), acid extraction, ion (anion or cation) exchange chromatography, high performance liquid chromatography (HPLC), phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, and size exclusion chromatography.Suitable protein purification methods are described in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice, 3rd Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, Humana Press, NJ In some embodiments, the purification step comprises chromatographic purification using an eluent mixture that may include arginine, glycine, citric acid, or a mixture thereof.

[0245] In some embodiments, bacterially produced polypeptides may form inclusion bodies (IBs), which require further processing steps to produce an active polypeptide. In some embodiments, this further processing may involve isolating and solubilizing the inclusion bodies, unfolding the polypeptide, and then refolding the polypeptide into the correct biologically active tertiary structure. In some embodiments, methods for producing the polypeptides described herein are provided. In some aspects, the methods involve culturing host cells transformed with a polynucleotide encoding a described polypeptide under conditions suitable for expression of the polypeptide as inclusion bodies; recovering inclusion bodies containing the encoded polypeptide from the transformed and cultured host cells; solubilizing (denaturing) the recovered inclusion bodies containing the polypeptide with a solubilizing agent; purifying the solubilized polypeptide; refolding the polypeptide; and purifying the refolded polypeptide. In some embodiments, the inclusion bodies can be solubilized in a solvent such as urea. In some embodiments, refolding can be achieved, for example, by incubating the solubilized polypeptide in a dilute solution of urea and glutathione. III. Composition of BCMA variant molecules

[0246] Provided herein are compositions, e.g., pharmaceutical compositions, of variant BCMA molecules, including any of the variant BCMA polypeptides, fusion proteins, dimers, conjugates, polynucleotides, vectors, and cells described herein, including variant BCMA molecules that comprise a CRD that is mutated compared to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, the provided compositions, e.g., pharmaceutical compositions, comprise a variant BCMA polypeptide, a BCMA-Ig-Fc fusion polypeptide, a polynucleotide, a vector, or a cell. In some embodiments, the provided compositions, e.g., pharmaceutical compositions, comprise a BCMA-Ig-Fc fusion polypeptide dimer, a BCMA-Ig-Fc fusion polypeptide conjugate, a polynucleotide, a vector, and / or a cell. In some embodiments, provided herein are compositions, e.g., pharmaceutical compositions, comprising any of the provided variant BCMA molecules, in any combination. A. Dosage Forms and Administration

[0247] In some embodiments, compositions comprising variant BCMA molecules, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, or cells, e.g., pharmaceutical compositions comprising a variant BCMA CRD polypeptide mutated relative to the huBCMA CRD (SEQ ID NO: 1), may be in any form suitable for the intended method of administration. In some embodiments, compositions, e.g., pharmaceutical compositions, comprising provided variant BCMA molecules are formulated for administration by inhalation, parenteral administration, sublingual administration, rectal administration, or topical administration. In some embodiments, topical administration may also involve the use of transdermal administration, such as a transdermal patch or iontophoresis device. In some embodiments, compositions, e.g., pharmaceutical compositions, e.g., pharmaceutical compositions comprising provided variant BCMA molecules, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, or cells, are formulated to provide a unit dosage form, e.g., a single unit dosage form or a plurality of unit dosage forms. 1. Liquid dosage form

[0248] In some embodiments, provided compositions, e.g., pharmaceutical compositions, comprising variant BCMA molecules, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, or cells may be in the form of a solution, suspension, or emulsion. In some embodiments, non-limiting examples of liquid carriers include water, saline, pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils and fats, and mixtures of any two or more thereof. In some embodiments, the liquid carrier comprises other suitable pharmaceutically acceptable excipients, such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, viscosity adjusters, stabilizers, etc. Non-limiting examples of suitable organic solvents include monohydric alcohols such as ethanol and polyhydric alcohols such as glycols. Non-limiting examples of suitable oils include soybean oil, coconut oil, olive oil, safflower oil, and cottonseed oil. In some embodiments, particularly when intended for parenteral administration, the carrier may be an oily ester, such as ethyl oleate or isopropyl myristate. Parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, intrasternal injection, transdermal or transmucosal administration, or infusion techniques. In some embodiments, any of the provided pharmaceutical compositions, such as any provided herein, including a variant BCMA molecule, fusion polypeptide, dimer, conjugate, polynucleotide, vector, or cell, is formulated for intravenous administration.

[0249] Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, transdermal or transmucosal administration, or infusion techniques.

[0250] In some embodiments, provided compositions, e.g., pharmaceutical compositions, comprising provided variant BCMA molecules are in the form of microparticles, microcapsules, liposomal encapsulations, and combinations of any two or more of these. In some embodiments, provided compositions, e.g., pharmaceutical compositions, comprising provided variant BCMA molecules comprise liposomes. In some aspects, liposomes are generally derived from phospholipids or other lipid substances. In some embodiments, liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. In some embodiments, any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used in liposomal formulations of variant BCMA molecules comprising a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, provided compositions, e.g., pharmaceutical compositions, comprising lipids or liposomes further comprise stabilizers, preservatives, and excipients. Exemplary lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art and are described in Prescott, Ed., "Methods in Cell Biology," Volume XIV, Academic Press, New York, NY, p. 33 et seq. (1976), which is incorporated herein by reference.

[0251] Injectable preparations (such as sterile injectable aqueous or oily suspensions) can be formulated using exemplary methods and materials, for example, suitable dispersants, wetting agents, and suspending agents. Sterile injectable preparations can be solvents, for example, solutions in 1,3-propanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic saline. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any non-irritating fixed oil, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables. 2.Solid dosage forms

[0252] The solid dosage form for oral administration can include capsules, tablets, pills, powders and granules.In such solid dosage forms, active compound can be mixed with at least one inert diluent, such as sucrose, lactose or starch.Such dosage forms can also contain additional materials other than inert diluents, such as lubricants (e.g., magnesium stearate).In the case of capsules, tablets and pills, dosage forms can also contain buffering agents.Tablets and pills can also be prepared with enteric coating. B. Carriers, Vehicles, and Excipients

[0253] In some embodiments, the provided compositions, e.g., pharmaceutical compositions, further comprise a carrier or excipient, e.g., a pharmaceutically acceptable carrier (or vehicle) or excipient. In some embodiments, the pharmaceutically acceptable carrier (or vehicle) or excipient comprises one or more conventional non-toxic carriers (or vehicles) or excipients. Exemplary pharmaceutically acceptable carriers and excipients are known. Non-limiting examples include processing agents and drug delivery modifiers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, cyclodextrins such as hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low-melting waxes, ion exchange resins, and combinations of two or more of these. Pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences," 18th edition, A.R. Gennaro, Ed., Mack Pub. Co. New Jersey (1991), "Pharmaceutical Formulation Development of Peptides and Proteins," S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000), and "Handbook of Pharmaceutical Excipients," 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), all of which are incorporated herein by reference. IV. Use of BCMA variant molecules

[0254] Provided herein are methods of using any of the provided variant BCMA molecules, including those comprising a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, provided herein are methods of using a pharmaceutical composition comprising a variant BCMA molecule comprising a mutated CRD compared to the huBCMA CRD (SEQ ID NO: 1). In some embodiments, provided herein are methods of using a variant BCMA polypeptide and / or a BCMA-Ig-Fc fusion polypeptide. In some embodiments, provided herein are methods of using a BCMA-Ig-Fc fusion polypeptide dimer, and / or a BCMA-Ig-Fc fusion polypeptide conjugate. In some embodiments, provided herein are methods of using any of the provided variant BCMA molecules and / or any combination thereof.

[0255] In some embodiments, the provided variant BCMA molecules, and compositions comprising the same, are used to inhibit the interaction of one or more of the receptors BCMA, TACI, and BR3 with the ligands BAFF and APRIL. In some embodiments, the provided variant BCMA molecules, and compositions comprising the same, are used to inhibit the interaction of one or more of the endogenous receptors BCMA, TACI, and BR3 with the endogenous ligands BAFF and APRIL. In some embodiments, the provided variant BCMA molecules, and compositions comprising the same, are used to inhibit the interaction of one or more of the endogenous human receptors BCMA, TACI, and BR3 with the endogenous human ligands BAFF and APRIL. In some embodiments, the activity of BAFF and APRIL is suppressed by inhibiting the interaction of one or more of the described receptors with the ligands BAFF and APRIL.

[0256] In some embodiments, the provided variant BCMA molecules and compositions thereof are used to bind, e.g., specifically bind, to BAFF and APRIL. In some embodiments, the provided variant BCMA molecules and compositions thereof are used to bind, e.g., specifically bind, to endogenous BAFF and APRIL. In some embodiments, the provided variant BCMA molecules and compositions thereof are used to bind, e.g., specifically bind, to endogenous human BAFF and APRIL. In some embodiments, binding, e.g., specifically binding, to BAFF and APRIL inhibits the activity of BAFF and APRIL. A. Methods of Treatment and Therapeutic Uses

[0257] In some aspects, provided herein are methods of administration and use, e.g., therapeutic and prophylactic uses, of the variant BCMA-binding molecules provided herein, including fusion proteins, dimers, and conjugates thereof, and / or compositions comprising same, or any of the polynucleotides, vectors, or cells provided herein. In some embodiments, the provided methods of treatment and therapeutic uses include administering the molecule or a composition containing same to a subject having, e.g., a BCMA-associated disease, condition, or disorder, e.g., a disease, condition, or disorder associated with BCMA expression and / or in which BCMA is expressed, e.g., specifically expressed, in cells or tissues. In some embodiments, the variant BCMA molecules, compositions, polynucleotides, vectors, or cells provided herein are administered in an amount effective to provide treatment for the disease or disorder.

[0258] Also provided herein are uses of BCMA variant molecules, including fusion proteins, dimers, and conjugates thereof, or polynucleotides, vectors, or cells provided herein in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods are carried out by administering a variant BCMA molecule provided herein, or a composition comprising the same, or a polynucleotide, vector, or cell, to a subject who has, has had, or is suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. Also provided herein are uses of any of the compositions, e.g., pharmaceutical compositions, provided herein, for treating a BCMA-associated disease or disorder, e.g., for use in a treatment regimen.

[0259] In some embodiments, the provided variant BCMA molecules are used for therapeutic treatment of a subject. In some embodiments, the subject receiving therapeutic treatment exhibits symptoms or signs of a pathological deviation, disease, or disorder, and the treatment is administered to the subject with the intent to reduce or eliminate those signs or symptoms. In some embodiments, the provided variant BCMA molecules are used for prophylactic treatment of a subject. In some embodiments, the subject receiving prophylactic treatment does not exhibit signs or symptoms of a disease, pathological deviation, or disorder, or exhibits only early signs or symptoms of a disease, pathological deviation, or disorder. In some embodiments, prophylactic treatment is administered with the intent to prevent a disease, pathological deviation, or disorder, or to reduce the risk of developing a disease, pathological deviation, or disorder. In some embodiments, the provided variant BCMA molecules are administered in a therapeutically effective amount. In some embodiments, a therapeutically effective amount is a dosage or amount of a substance sufficient to produce a desired result. In some embodiments, the desired result may include an objective or subjective improvement in the recipient of the dosage or amount. In some embodiments, the desired result may include the induction, promotion, enhancement, or modulation of a measurable, detectable, or testable immune response in a subject.

[0260] Inhibition of BAFF and APRIL affects B cell development, survival, and function. Provided herein are methods for preventing or treating B cell or antibody-mediated disorders, comprising administering to a subject any of the provided variant BCMA molecules, e.g., polypeptides, fusion polypeptides, dimers, conjugates, polynucleotides, vectors, cells, or pharmaceutical compositions. In some embodiments, the variant BCMA molecule is or comprises a variant BCMA polypeptide, BCMA-Ig-Fc fusion protein or dimer, or a conjugate thereof. In some embodiments, methods are provided for administering a composition, e.g., a pharmaceutical composition, comprising any of the described variant BCMA molecules and / or any combination thereof. In some embodiments, the variant BCMA molecule is administered in a therapeutically effective amount. In some embodiments, a therapeutically effective amount is a dosage or amount of a substance sufficient to produce a desired result. In some embodiments, the desired result may include an objective or subjective improvement in the recipient of the dosage or amount. In some embodiments, the desired result may include inducing, promoting, enhancing, or modulating a measurable, detectable, or testable immune response in a subject. In some embodiments, the subject is a non-human mammal or a human. In some embodiments, the subject has a B cell or antibody-mediated disorder. In some embodiments, the B cell or antibody-mediated disorder is an autoimmune disease, transplant rejection, or B cell malignancy.

[0261] In some embodiments, provided herein are methods of treating a subject having a B cell or antibody-mediated disease or disorder, the methods comprising administering a therapeutically effective amount of a provided variant BCMA molecule, polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector, cell, or pharmaceutical composition. In some embodiments, provided herein are uses of provided variant BCMA molecules in the manufacture of a medicament for treating a subject having a B cell or antibody-mediated disease or disorder. In some embodiments, provided herein are uses of provided variant BCMA molecules for treating a subject having a B cell or antibody-mediated disease or disorder. In some embodiments, the variant BCMA molecule is or comprises a variant BCMA polypeptide, BCMA-Ig-Fc fusion protein or dimer, or a conjugate thereof. In some embodiments, provided are methods of administering a composition, e.g., a pharmaceutical composition, comprising any of the described variant BCMA molecules and / or any combination thereof.

[0262] In some embodiments, the B cell or antibody-mediated disease or disorder is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is an immune-mediated disease or disorder of a subject's tissues, bones, joints, blood vessels, thyroid, kidney, nervous system, brain, lung, and / or skin. In some embodiments, the autoimmune disease or disorder is a kidney disease or disorder, lupus, arthritis, spinal joint disorder, vasculitis disorder, hemolytic anemia disorder, thrombocytopenic disorder, thyroiditis disorder, demyelinating disease of the central nervous system and / or peripheral nervous system, inflammatory and / or fibrotic lung disorder, skin disorder, or allergic disorder.

[0263] In some embodiments, the autoimmune disease or disorder is systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berge's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), renal sarcoidosis, rheumatoid arthritis, juvenile chronic arthritis, arthritis associated with inflammatory bowel disease, ankylosing spondylitis, spondylitis associated with psoriasis, juvenile spondyloarthropathy, unclassifiable spondyloarthropathy, Reiter's syndrome, scleroderma, Sjögren's syndrome, systemic necrotizing vasculitis, polyarteritis nodosa, allergic vasculitis and granulomatosis, polyangiitis, Wegener's granulomatosis, lymphomatoid granulomatosis, mucocutaneous lymph node syndrome (MLNS), or vasculitis of the genital tract. or Kawasaki disease), isolated central nervous system vasculitis, Behçet's disease, thromboangiitis obliterans (Buerger's disease), cutaneous necrotizing venulitis, sarcoidosis, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, thrombocytopenic purpura, immune-mediated thrombocytopenia, Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis, type 1 diabetes mellitus, glomerulonephritis and tubulointerstitial nephritis, multiple sclerosis (MS), idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, bullous dermatosis, erythema multiforme, contact dermatitis, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, or urticaria.

[0264] In some embodiments, the renal disease or disorder is selected from systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berger's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), or renal sarcoidosis. In some embodiments, the renal disease or disorder is selected from systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), or IgA nephropathy (Berger's disease).

[0265] In some embodiments, the B cell or antibody-mediated disease or disorder is tissue or organ transplant rejection, hi some embodiments, the tissue or organ transplant rejection is acute or chronic B cell or antibody-mediated rejection of tissue allografts consisting of bone marrow, stem cells, skin, and solid organs, acute or chronic graft-versus-host disease (GVHD), antibody-mediated rejection (AMR) of solid organs, hyperacute organ transplant rejection, acute organ transplant rejection, and / or chronic organ transplant rejection.

[0266] In some embodiments, the B cell or antibody-mediated disease or disorder is a B cell malignancy, hi some embodiments, the B cell malignancy is non-Hodgkin's lymphoma, multiple myeloma (MM), B cell chronic lymphocytic leukemia, plasmacytoma, macroglobulinemia, or Waldenstrom's macroglobulinemia (WM). B. Medication

[0267] In some embodiments, the provided variant BCMA molecule and / or composition thereof can be administered as the only active agent. In some aspects, the specific dose level for any particular subject or patient may depend on various factors, including the activity of the specific compound used, age, body weight, overall health, route of administration, severity of the disorder, and excretion rate. The therapeutically effective amount in a given situation can be readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. The terms "therapeutically effective amount" and "therapeutically effective dose" are used interchangeably herein and refer to an amount of a compound that results in the prevention or improvement of a systemic or desired biological outcome in a patient. The terms "subject" and "patient" are used interchangeably herein and refer to a mammal, for example, a human, a non-human primate (e.g., baboon, orangutan, monkey, gorilla), or a non-primate mammal (e.g., mouse, rat, dog, pig).

[0268] In some embodiments, the therapeutically effective dose of the provided variant BCMA molecule ranges from 0.1 μg or about 0.1 μg / kg / day to 20 mg or about 20 mg / kg / day, from about 10 μg / kg / day to 1 mg or about 1 mg / kg / day, or from 100 μg or about 100 μg / kg / day to 1 mg or about 1 mg / kg / day. In some embodiments, the variant BCMA molecule is a variant BCMA polypeptide, a fusion protein or dimer thereof, or a conjugate thereof, or a composition, e.g., a pharmaceutical composition. In some embodiments, any combination of the above molecules is administered in the provided doses. In some embodiments, the provided variant BCMA molecule is delivered to the subject in one dose. In some embodiments, the provided variant BCMA molecule is delivered to the subject in more than one dose.

[0269] In some embodiments, the provided variant BCMA molecules can be administered at the maximum recommended clinical dosage and / or at a lower dosage. In some embodiments, the dosage level of any of the provided variant BCMA molecules can be varied to achieve a desired therapeutic response, depending, for example, on the route of administration, the severity of the disease, and the patient's response. In some embodiments, the BCMA molecule is a variant BCMA polypeptide, a fusion protein or dimer thereof, or a conjugate thereof, or a composition, e.g., a pharmaceutical composition. In some embodiments, any combination of the above-mentioned molecules is administered at the provided dosage.

[0270] In some embodiments, provided variant BCMA molecules and / or compositions thereof can be administered in combination with one or more other agents, see, e.g., US2008 / 0260737, incorporated herein by reference. In some embodiments, suitable agents include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, prednisone, disease-modifying antirheumatic drugs (DMARDs) (e.g., hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab, rituximab, azathioprine, D-penicillamine, gold (oral or intramuscular), minocycline, cyclosporine, retinoids, staphylococcal protein A immunoadsorbents, topical treatments (e.g., steroids, anthralin, calcipotriene, clobetasol, tazarotene, etc.). In some embodiments, combinations comprising variant BCMA molecules comprising a mutated CRD relative to the huBCMA CRD (SEQ ID NO: 1) may be administered as separate compositions. In some embodiments, the separate compositions may be administered at the same time or at different times. In some embodiments, the huBCMA Combinations comprising variant BCMA molecules that comprise a mutated CRD relative to the CRD (SEQ ID NO: 1) may be administered as a single dosage form containing two or more agents. V. Definition

[0271] Unless otherwise defined, all terms of art, notations, and other scientific and technical terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0272] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that embodiments and variations described herein include embodiments and variations that "consist of" and / or "consisting essentially of."

[0273] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to represent all possible subranges specifically disclosed as well as individual numerical values ​​falling within that range. For example, when a range of values ​​is provided, it is understood that each value falling between the upper and lower limits of that range, and any other stated value or values ​​falling between that stated range, is encompassed within the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the scope of the claimed subject matter. This is true regardless of the broadness of the range.

[0274] The term "about," as used herein, refers to a normal error range for each value, which is readily apparent to one of ordinary skill in the art. Reference herein to "about" a value or parameter encompasses (and describes) embodiments directed to the value or parameter itself. For example, a statement referring to "about X" encompasses the statement "X." The term "about" can encompass variations that can be up to ±5%, but can also be ±4%, ±3%, ±2%, ±1%, etc. Whether modified by the term "about," the claims encompass equivalents to quantities.

[0275] As used herein, a "domain" (typically a sequence of three or more amino acids, typically five or seven or more amino acids, e.g., 10-200 amino acid residues) refers to a portion of a molecule, such as a protein or an encoding nucleic acid, that is structurally and / or functionally distinct and identifiable from the rest of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independently folded structure composed of one or more structural motifs within a protein and / or that is recognized by a functional activity, such as binding activity. A protein can have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by primary sequence or structural homology with related family members, e.g., homology with a motif such as the cysteine-rich motif of a TNF receptor. In another example, a domain can be distinguished by its function, such as its ability to interact with a biomolecule. A domain can exhibit an independent biological function or activity, and thus, a domain can exert an activity, e.g., binding, either independently or fused to another molecule. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Although definitions are provided herein for illustrative purposes, in some embodiments, specific domains can be identified by name. If necessary, appropriate software can be used to identify domains.

[0276] The terms "extracellular domain" or "ectodomain" may be used interchangeably and, as used herein, refer to the region of a membrane protein, such as a transmembrane protein, that is outside the vesicle membrane. The extracellular domain often includes a binding domain that specifically binds to a ligand or cell surface receptor, e.g., via a binding domain that specifically binds to a ligand or cell surface receptor.

[0277] The terms "endodomain" or "intracellular domain" may be used interchangeably and, as used herein, refer to the region found in some membrane proteins, such as transmembrane proteins, that extends into the internal space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. In cells, the endodomain may interact with intracellular components and play a role in signal transduction, and thus, in some cases, may be an intracellular signaling domain. Alternatively, the endodomain of a cellular transmembrane protein may be referred to as a cytoplasmic domain, and in some cases, may be an intracytoplasmic signaling domain.

[0278] The term "transmembrane domain," as used herein, refers to a domain found in a membrane protein that substantially or completely spans a lipid bilayer, such as a lipid bilayer found in a biological membrane, such as a mammalian cell, or in an artificial construct, such as a liposome. Transmembrane domains can generally be predicted from the amino acid sequence by any number of commercially available bioinformatics software applications based on the increased hydrophobicity of the transmembrane domain compared to regions of the protein that interact with the aqueous environment (e.g., cytosol, extracellular fluid). Transmembrane domains are often hydrophobic alpha helices that span the membrane. Transmembrane proteins may pass through both layers of the lipid bilayer once or multiple times.

[0279] The term "specifically binds" as used herein refers to the ability of a protein to bind to a target protein under specific binding conditions, and thus its affinity or avidity, which is at least 5 times, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or avidity of the same protein for a population of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not exclusively bind to a single target molecule, but may also specifically bind to non-target molecules (e.g., paralogs or orthologs) due to similarities in the structural conformation between the target and non-target molecules. Those skilled in the art will understand that specific binding to molecules with the same function in different animal species (i.e., orthologs) or non-target molecules (e.g., paralogs) with epitopes substantially similar to the target molecule may occur without compromising the specificity of binding as determined by comparison with a statistically verified population of unique non-targets (e.g., random polypeptides). Thus, a polypeptide may specifically bind to target molecules of more than one distinct species due to cross-reactivity. Specific binding between two proteins can be determined using solid-phase ELISA immunoassays or Biacore measurements.

[0280] As used herein, a "variant BCMA polypeptide" or "protein" refers to a polypeptide comprising a BCMA cysteine-rich domain (CRD) amino acid sequence which sequence differs at one or more amino acid positions from the corresponding CRD amino acid sequence of the wild-type human BCMA extracellular domain (SEQ ID NO: 1), e.g., by a substitution, deletion, or insertion. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acids, which may be naturally occurring amino acids or artificial amino acid analogs.

[0281] As used herein, the term "variant BCMA molecule" refers to a molecule comprising a provided BCMA polypeptide variant, and encompasses variant BCMA polypeptides, variant BCMA fusion polypeptides (e.g., including bispecific fusion polypeptides, variant BCMA-Ig fusion polypeptides) and dimers thereof (e.g., dimers in which variant BCMA-Ig fusion polypeptides are covalently linked by disulfide bonds through the Ig component of the fusion polypeptide), as well as conjugates thereof. In various embodiments, the term "variant BCMA molecule" refers to a polypeptide comprising a provided variant BCMA CRD or variant BCMA ECD.

[0282] As used herein, the terms "mutant" and "variant," with respect to a polypeptide, both refer to a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide (such as, for example, a wild-type (WT) polypeptide sequence) by one or more amino acid residues. In one aspect, a variant polypeptide comprises a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, or more of the total number of residues in the parent or reference polypeptide sequence. In another embodiment, a variant polypeptide comprises a polypeptide sequence having at least 50%, or at least about 50%, at least 60%, or at least about 60%, at least 70%, or at least about 70%, at least 80%, or at least about 80%, at least 85%, or at least about 85%, at least 86%, or at least about 86%, at least 87%, or at least about 87%, at least 88%, or at least about 88%, at least 89%, or at least about 89%, at least 90%, or at least about 90%, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, at least 96%, or at least about 96%, at least 97%, or at least about 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to the polypeptide sequence of a parent or reference polypeptide.In another embodiment, a variant polypeptide comprises a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide by 1 to 50 or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid residues). A variant polypeptide can comprise a polypeptide sequence that differs from the polypeptide sequence of a parent or reference polypeptide by, for example, deletions, additions, or substitutions of one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) of the parent or reference polypeptide, or any combination of such deletion(s), addition(s), and / or substitution(s). The reference or parent polypeptide may itself be a variant polypeptide.

[0283] With respect to the numbering of positions within a given amino acid polymer or nucleic acid polymer, the terms "corresponding to," "with reference to," or "compared to" the numbering of a selected amino acid polymer or nucleic acid polymer refer to the position of any given polymer component (e.g., amino acid, nucleotide, also commonly referred to as "residue") specified with reference to the same or equivalent position within the selected amino acid or nucleic acid polymer, rather than the actual numerical position of the component of the given polymer. Thus, for example, the numbering of a given amino acid position within a given polypeptide sequence corresponds to the same or equivalent amino acid position within a selected polypeptide sequence used as a reference sequence.

[0284] An "equivalent position" (e.g., "equivalent amino acid position" or "equivalent nucleic acid position" or "equivalent residue position") is defined herein as a position (e.g., amino acid position or nucleic acid position or residue position, etc.) in a test polypeptide (or test polynucleotide) sequence that matches the corresponding position in a reference polypeptide (or reference polynucleotide) sequence when optimally aligned using the alignment algorithms described herein. The equivalent amino acid position in a test polypeptide need not have the same numerical position number as the corresponding position in the reference polypeptide. Similarly, the equivalent nucleic acid position in a test polynucleotide need not have the same numerical position number as the corresponding position in the reference polynucleotide.

[0285] Two polypeptide sequences are "optimally aligned" or "optimally aligned" when they are aligned using defined parameters, i.e., a defined amino acid substitution matrix, a gap existence penalty (also called a gap open penalty), and a gap extension penalty, to achieve the highest possible similarity score for the sequence pair. The BLOSUM62 matrix (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA 1992; 89 (22): 10915-10919) is often used as the default scoring substitution matrix in polypeptide sequence alignment algorithms (e.g., BLASTP, etc.). A gap existence penalty is imposed for the introduction of a single amino acid gap into one of the aligned sequences, and a gap extension penalty is imposed for each residue position within the gap. Unless otherwise specified, the alignment parameters used herein are the BLOSUM62 scoring matrix, a gap existence penalty of 11, and a gap extension penalty of 1. The alignment score is defined by the amino acid position in each sequence where the alignment begins and ends (e.g., the alignment window), and, if necessary, by inserting a gap or gaps in one or both sequences to reach the highest possible similarity score.

[0286] When determining amino acid positions by optimal alignment with a reference sequence, the amino acid positions in the test amino acid sequence correspond to the positions in the reference sequence that the residues are paired with in the alignment. "Position" is indicated by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Due to deletions, insertions, truncations, fusions, and similar modifications that must be taken into account when determining optimal alignment, the amino acid residue number in the test sequence determined simply by counting from the N-terminus is generally not necessarily the same as the number of the corresponding position in the reference sequence. For example, if there is a deletion in the aligned test sequence, there is no amino acid corresponding to the position in the reference sequence that corresponds to the deletion site. If there is an insertion in the aligned reference sequence, the insertion does not correspond to any amino acid position in the reference sequence. In the case of truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0287] The term "corresponding control protein" or "reference polypeptide," as used herein, refers to a protein having an amino acid sequence that is the same as the variant BCMA polypeptide amino acid sequence, except that the BCMA ECD component comprises the [S40G]huBCMA CRD (SEQ ID NO:237) or wild-type huBCMA CRD (SEQ ID NO:1); or the [S40G]huBCMA ECD (SEQ ID NO:224) or wild-type huBCMA ECD (SEQ ID NO:152). The terms "[S40G]huBCMA," "[S40G]huBCMA ECD," "[BCMA[S40G]," and "[S40G]BCMA" are used interchangeably herein and refer to a polypeptide comprising SEQ ID NO:224. The term "[S40G]huBCMA CRD" is used interchangeably herein and refers to a polypeptide comprising SEQ ID NO:237. Thus, as noted above, the corresponding control protein may be a corresponding BCMA polypeptide, a corresponding BCMA fusion polypeptide, a corresponding BCMA fusion polypeptide dimer, or a corresponding BCMA conjugate in which the BCMA ECD component is either the human wild-type BCMA ECD (SEQ ID NO: 152) or the [S40G]huBCMA ECD amino acid sequence (SEQ ID NO: 224); or in which the BCMA CRD component is either the [S40G]huBCMA CRD (SEQ ID NO: 237) or the wild-type huBCMA CRD (SEQ ID NO: 1). Control proteins comprising an [S40G]huBCMA ECD or CRD are also referred to herein by the terms "human BCMA surrogate control protein" and "human BCMA surrogate control," which refer to a polypeptide comprising an amino acid sequence encoding the [S40G] substituted form of the human BCMA extracellular domain. A [S40G]huBCMA ECD or CRD contains a single mutation ([S40G]) within the BCMA ECD or CRD amino acid sequence compared to the wild-type huBCMA ECD or CRD. Further exemplary control proteins comprising the [S40G]huBCMA ECD or CRD include mature [S40G]BCMA ECD or CRD-IgG2 Fc fusion protein (PIg18).As described in more detail below, the [S40G] substitution eliminates an N-glycosylation site that may be recognized by glycosylating host cells and is believed to result in a more homogeneous protein due to the absence of alternative glycoforms. The [S40G] substitution is not believed to affect the huBAFF and huAPRIL binding properties described herein.

[0288] The term "non-polypeptide conjugation moiety," as used herein, refers to a non-polypeptide polymer, sugar moiety, or non-polymeric lipophilic moiety.

[0289] The term "non-polypeptide polymer," as used herein, refers to a water-soluble polymer that is not a peptide, polypeptide, or protein and can be a natural or synthetic polymer (eg, a homopolymer, copolymer, terpolymer).

[0290] The term "sugar moiety" as used herein refers to a carbohydrate molecule attached by an in vivo or in vitro glycosylation process, eg, an N- or O-glycosylation process.

[0291] The term "therapeutic treatment," as used herein, refers to treatment administered to a subject who exhibits symptoms or signs of a pathological deviation, disease, or disorder, the treatment being administered to the subject with the intent of reducing or eliminating those signs or symptoms.

[0292] The term "prophylactic treatment," as used herein, refers to treatment administered to a subject who does not exhibit signs or symptoms of a disease, pathological deviation, or disorder, or who exhibits only early signs or symptoms of a disease, pathological deviation, or disorder. Such treatment is administered with the intent to prevent a disease, pathological deviation, or disorder, or to reduce the risk of developing a disease, pathological deviation, or disorder.

[0293] The term "therapeutically effective amount," as used herein, refers to a dosage or amount of a substance sufficient to produce a desired result. The desired result may include an objective or subjective improvement in the recipient of the dosage or amount. For example, the desired result may include the induction, promotion, enhancement, or modulation of a measurable, detectable, or testable immune response in a subject. VI. Illustrative Embodiments

[0294] Provided embodiments include: 1. A variant B-cell maturation antigen (BCMA) polypeptide comprising a variant cysteine-rich domain (CRD) containing at least one amino acid substitution selected from the following, based on the amino acid positions in SEQ ID NO: 1: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A). 2. The variant BCMA polypeptide of embodiment 1, wherein said variant CRD comprises at least three amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), relative to the amino acid positions of SEQ ID NO: 1. 3. The variant BCMA polypeptide of embodiment 1 or 2, wherein the variant CRD comprises at least three amino acid substitutions selected from among: (1) histidine or arginine at position 12 (S12H or S12R); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine at position 15 (H15R); and (4) valine at position 22 (L22V), relative to the amino acid positions in SEQ ID NO: 1. 4. The variant BCMA polypeptide of any one of embodiments 1 to 3, wherein said variant CRD further comprises at least one modification selected from among: (1) a deletion of residue 38 (N38del) or a non-asparagine amino acid residue at position 38 (N38X, where X is any amino acid residue that is not asparagine) and (2) a non-serine or threonine amino acid residue at position 40 (S40X, where X is any amino acid residue that is not serine or threonine), relative to the amino acid positions of SEQ ID NO: 1. 5. The variant BCMA polypeptide of any one of embodiments 1 to 4, wherein the variant CRD further comprises a glycine at residue 40 (S40G), relative to the amino acid position in SEQ ID NO: 1. 6. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein said variant CRD comprises at least or at least about 85%, at least 86%, at least or at least about 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least about 98%, or at least 99% or at least about 99% sequence identity to SEQ ID NO: 1. 7. The variant BCMA polypeptide of any one of embodiments 1 to 6, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 1. 8. The variant BCMA polypeptide of any one of embodiments 1 to 7, wherein the variant CRD comprises at least 95%, or at least about 95%, sequence identity to SEQ ID NO: 1. 9. The variant BCMA polypeptide of any one of embodiments 1 to 8, wherein the variant CRD comprises at least 99%, or at least about 99%, sequence identity to SEQ ID NO: 1. 10. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to a sequence set forth in any one of SEQ ID NOs: 3-146. 11. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19. 12. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein the variant CRD comprises a sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19. 13. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein the variant CRD comprises at least three amino acid substitutions, relative to the amino acid positions of SEQ ID NO: 1, selected from the group consisting of: (1) a histidine at position 12 (S12H); (2) an isoleucine at position 14 (L14I); (3) an arginine at position 15 (H15R or H15N); and (4) a serine to glycine mutation at residue 40 (S40G), wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 1. 14. The variant BCMA polypeptide of any one of embodiments 1 to 5, wherein the variant CRD comprises each of the following amino acid substitutions relative to the amino acid positions in SEQ ID NO: 1: (1) a histidine at position 12 (S12H); (2) an isoleucine at position 14 (L14I); (3) an arginine at position 15 (H15R); and (4) a serine to glycine mutation at residue 40 (S40G), wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO: 1. 15. The variant BCMA polypeptide of any one of embodiments 1 to 14, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 3. 16. The variant BCMA polypeptide of any one of embodiments 1 to 15, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 3. 17. The variant BCMA polypeptide of any one of embodiments 1 to 12, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 4. 18. The variant BCMA polypeptide of any one of embodiments 1 to 12 and 17, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 4. 19. The variant BCMA polypeptide of any one of embodiments to 12, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 8. 20. The variant BCMA polypeptide of any one of embodiments 1 to 12 and 19, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 8. 21. The variant BCMA polypeptide of any one of embodiments 1 to 12, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 9. 22. The variant BCMA polypeptide of any one of embodiments 1 to 12 and 21, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 9. 23. The variant BCMA polypeptide of any one of embodiments 1 to 12, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 19. 24. The variant BCMA polypeptide of any one of embodiments 1 to 12 and 23, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 19. 25. A fusion polypeptide comprising a variant BCMA polypeptide according to any one of embodiments 1 to 24 and an additional polypeptide. 26. The fusion polypeptide of embodiment 25, wherein the additional polypeptide is an immunoglobulin (Ig) Fc polypeptide. 27. A fusion polypeptide comprising a variant BCMA polypeptide according to any one of embodiments 1 to 24 and an immunoglobulin (Ig) Fc polypeptide. 28. A variant B-cell maturation antigen (BCMA) polypeptide comprising a variant cysteine-rich domain (CRD) containing at least one amino acid substitution selected from the following, based on the amino acid positions of SEQ ID NO: 1: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A); Immunoglobulin (Ig) Fc polypeptide and A fusion polypeptide comprising: 29. The fusion polypeptide of embodiment 28, wherein the variant CRD comprises at least three amino acid substitutions selected from among: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A), based on the amino acid positions of SEQ ID NO: 1. 30. The fusion polypeptide of embodiment 28 or 29, wherein the variant CRD comprises at least three amino acid substitutions selected from among (1) histidine or arginine at position 12 (S12H or S12R); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine at position 15 (H15R); and (4) valine at position 22 (L22V), based on the amino acid positions of SEQ ID NO: 1. 31. The fusion polypeptide of any one of embodiments 28 to 30, wherein the variant CRD further comprises at least one modification selected from among (1) a deletion of residue 38 (N38del) or a non-asparagine amino acid residue at position 38 (N38X, where X is any amino acid residue that is not asparagine) and (2) a non-serine or threonine amino acid residue at position 40 (S40X, where X is any amino acid residue that is not serine or threonine), relative to the amino acid positions of SEQ ID NO: 1. 32. A fusion polypeptide described in any one of embodiments 28 to 31, wherein the variant CRD further comprises a glycine at residue 40 (S40G), relative to the amino acid position of SEQ ID NO: 1. 33. A fusion polypeptide described in any one of embodiments 28 to 32, wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 3 to 146. 34. A fusion polypeptide described in any one of embodiments 28 to 33, wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19. 35. A fusion polypeptide described in any one of embodiments 28 to 34, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 19. 36. A fusion polypeptide described in any one of embodiments 28 to 35, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 3. 37. A fusion polypeptide described in any one of embodiments 28 to 36, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 3. 38. A fusion polypeptide described in any one of embodiments 28 to 35, wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO:4. 39. A fusion polypeptide described in any one of embodiments 28 to 35 and 38, wherein the variant CRD comprises the sequence set forth in SEQ ID NO:4. 40. A fusion polypeptide described in any one of embodiments 28 to 35, wherein the variant CRD comprises at least 90% or at least about 90% sequence identity to SEQ ID NO:8. 41. A fusion polypeptide described in any one of embodiments 28 to 35 and 40, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 8. 42. A fusion polypeptide described in any one of embodiments 28 to 35, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 9. 43. A fusion polypeptide described in any one of embodiments 28 to 35 and 42, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 9. 44. A fusion polypeptide described in any one of embodiments 28 to 35, wherein the variant CRD comprises at least 90%, or at least about 90%, sequence identity to SEQ ID NO: 19. 45. A fusion polypeptide described in any one of embodiments 28 to 35 and 44, wherein the variant CRD comprises the sequence set forth in SEQ ID NO: 19. 46. ​​The fusion polypeptide of any one of embodiments 26 to 45, wherein the variant BCMA polypeptide is fused directly or indirectly to the N-terminus or C-terminus of the Ig Fc polypeptide. 47. A fusion polypeptide according to any one of embodiments 26 to 46, wherein the Ig Fc polypeptide is or is derived from an isotype G immunoglobulin (IgG) or a variant thereof. 48. The fusion polypeptide of any one of embodiments 26 to 47, wherein the Ig Fc polypeptide is or is derived from an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, or an IgG4 Fc. 49. A fusion polypeptide according to any one of embodiments 26 to 48, wherein the Ig Fc polypeptide is or is derived from human IgG Fc. 50. The fusion polypeptide of any one of embodiments 26 to 49, wherein the Ig Fc polypeptide is or is derived from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. 51. A fusion polypeptide described in any one of embodiments 26 to 50, wherein the Ig Fc polypeptide is or is derived from IgG1 Fc. 52. A fusion polypeptide described in any one of embodiments 26 to 51, wherein the Ig Fc polypeptide is or is derived from human IgG1 Fc. 53. The fusion polypeptide of any one of embodiments 26 to 52, wherein the Ig Fc polypeptide is a human IgG1 Fc and comprises a sequence set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, or SEQ ID NO: 230, or a sequence having at least 90% sequence identity thereto. 54. The fusion polypeptide of any one of embodiments 26 to 53, wherein the Ig Fc polypeptide is human IgG1 Fc and comprises the sequence set forth in SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, or SEQ ID NO: 230. 55. A fusion polypeptide described in any one of embodiments 26 to 50, wherein the Ig Fc polypeptide is or is derived from IgG2 Fc. 56. A fusion polypeptide according to any one of embodiments 26 to 50 and 55, wherein the Ig Fc polypeptide is or is derived from human IgG2 Fc. 57. The fusion polypeptide of any one of embodiments 26 to 50, 55 and 56, wherein the Ig Fc polypeptide is a human IgG2 Fc and comprises the sequence set forth in SEQ ID NO: 171, SEQ ID NO: 235, or SEQ ID NO: 236, or a sequence having at least 90% sequence identity thereto. 58. A fusion polypeptide described in any one of embodiments 26 to 50 and 55 to 57, wherein the Ig Fc polypeptide is a human IgG2 Fc and comprises the sequence set forth in SEQ ID NO: 171, SEQ ID NO: 235, or SEQ ID NO: 236. 59. A fusion polypeptide according to any one of embodiments 26 to 50, wherein the Ig Fc polypeptide is or is derived from an IgG4 Fc. 60. The fusion polypeptide of any one of embodiments 26 to 50 and 59, wherein the Ig Fc polypeptide is or is derived from human IgG4 Fc. 61. The fusion polypeptide of any one of embodiments 26 to 50, 59 and 60, wherein the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 172, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, or SEQ ID NO: 234, or a sequence having at least 90% sequence identity thereto. 62. The fusion polypeptide of any one of embodiments 26 to 50 and 59 to 61, wherein the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 172, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, or SEQ ID NO: 234. 63. The fusion polypeptide of any one of embodiments 26 to 50 and 59 to 62, wherein the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 161, or a sequence having at least 90% sequence identity thereto. 64. The fusion polypeptide of any one of embodiments 26 to 50 and 59 to 63, wherein the Ig Fc polypeptide comprises SEQ ID NO: 161. 65. The fusion polypeptide of any one of embodiments 26 to 50 and 59 to 62, wherein the Ig Fc polypeptide is a human IgG4 Fc and comprises the sequence set forth in SEQ ID NO: 163, or a sequence having at least 90% sequence identity thereto. 66. The fusion polypeptide of any one of embodiments 26 to 50, 59 to 62, and 65, wherein the Ig Fc polypeptide comprises SEQ ID NO: 163. 67. The fusion polypeptide of any one of embodiments 26 to 66, wherein the Ig Fc polypeptide comprises isoleucine or valine substituted for one, two, three, four, or more native methionine residues. 68. The fusion polypeptide of any one of embodiments 26 to 37, 46 to 50, and 59 to 64, wherein the variant BCMA polypeptide comprises SEQ ID NO: 3 and the Ig Fc polypeptide comprises SEQ ID NO: 161. 69. The fusion polypeptide of any one of embodiments 26 to 37, 46 to 50, 59 to 62, 65, and 66, wherein the variant BCMA polypeptide comprises SEQ ID NO: 3 and the Ig Fc polypeptide comprises SEQ ID NO: 163. 70. The fusion polypeptide of any one of embodiments 26 to 35, 38, 39, 46 to 50, and 59 to 64, wherein the variant BCMA polypeptide comprises SEQ ID NO: 4 and the Ig Fc polypeptide comprises SEQ ID NO: 161. 71. The fusion polypeptide of any one of embodiments 26 to 35, 38, 39, 46 to 50, 59 to 62, 65, and 66, wherein the variant BCMA polypeptide comprises SEQ ID NO: 4 and the Ig Fc polypeptide comprises SEQ ID NO: 163. 72. The fusion polypeptide of any one of embodiments 26 to 35, 40, 41, 46 to 50, and 59 to 64, wherein the variant BCMA polypeptide comprises SEQ ID NO: 8 and the Ig Fc polypeptide comprises SEQ ID NO: 161. 73. The fusion polypeptide of any one of embodiments 26 to 35, 40, 41, 46 to 50, 59 to 62, 65, and 66, wherein the variant BCMA polypeptide comprises SEQ ID NO: 8 and the Ig Fc polypeptide comprises SEQ ID NO: 163. 74. The fusion polypeptide of any one of embodiments 26 to 35, 42, 43, 46 to 50, and 59 to 64, wherein the variant BCMA polypeptide comprises SEQ ID NO: 9 and the Ig Fc polypeptide comprises SEQ ID NO: 161. 75. The fusion polypeptide of any one of embodiments 26 to 35, 42, 43, 46 to 50, 59 to 62, 65, and 66, wherein the variant BCMA polypeptide comprises SEQ ID NO: 9 and the Ig Fc polypeptide comprises SEQ ID NO: 163. 76. The fusion polypeptide of any one of embodiments 26 to 35, 44 to 50, and 59 to 64, wherein the variant BCMA polypeptide comprises SEQ ID NO: 19 and the Ig Fc polypeptide comprises SEQ ID NO: 161. 77. The fusion polypeptide of any one of embodiments 26 to 35, 44 to 50, 59 to 62, 65, and 66, wherein the variant BCMA polypeptide comprises SEQ ID NO: 19 and the Ig Fc polypeptide comprises SEQ ID NO: 163. 78. The fusion polypeptide of any one of embodiments 27 to 77, further comprising a peptide linker connecting the variant BCMA polypeptide and the Ig Fc polypeptide. 79. The fusion polypeptide of embodiment 78, wherein the peptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. 80. The fusion polypeptide of embodiment 78 or 79, wherein the peptide linker is 4, 5, 6, or 7 amino acids in length. 81. A fusion polypeptide described in any one of embodiments 78 to 80, wherein the peptide linker comprises a residue selected from the group consisting of glycine, serine, alanine, and threonine. 82. A fusion polypeptide described in any one of embodiments 78 to 81, wherein the peptide linker comprises a sequence set forth in any one of SEQ ID NOs: 156, 158, 175-186, 188-213, GS, GGS, and GSA. 83. A fusion polypeptide described in any one of embodiments 78 to 82, wherein the peptide linker comprises SEQ ID NO: 156. 84. A fusion polypeptide described in any one of embodiments 78 to 82, wherein the peptide linker comprises SEQ ID NO: 158. 85. A fusion polypeptide described in any one of embodiments 25 to 84, comprising, in order from N-terminus to C-terminus, the variant BCMA polypeptide, a peptide linker, and an Ig Fc polypeptide. 86. The fusion polypeptide of any one of embodiments 25 to 85, comprising a variant BCMA polypeptide set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:19, a peptide linker comprising the sequence set forth in any one of SEQ ID NOs:156, 158, 175-186, 188-213, GS, GGS, and GSA, and an Ig Fc polypeptide comprising the sequence set forth in SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:172, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234. 87. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 3, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. 88. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 3, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. 89. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 4, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. 90. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 4, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. 91. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 8, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. 92. The fusion polypeptide of any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 8, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. 93. The fusion polypeptide of any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. 94. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 9, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. 95. A fusion polypeptide described in any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 156, and an Ig Fc polypeptide comprising SEQ ID NO: 161. 96. The fusion polypeptide of any one of embodiments 25 to 86, comprising a variant BCMA polypeptide comprising SEQ ID NO: 19, a peptide linker comprising SEQ ID NO: 158, and an Ig Fc polypeptide comprising SEQ ID NO: 163. 97. A fusion polypeptide described in any one of embodiments 25 to 37, 46 to 50, 59 to 69, and 78 to 88, comprising the sequence set forth in SEQ ID NO: 167 or a sequence having at least 90% sequence identity thereto. 98. The fusion polypeptide of any one of embodiments 25 to 37, 46 to 50, 59 to 69, and 78 to 88, comprising the sequence set forth in SEQ ID NO: 167. 99. The fusion polypeptide of any one of embodiments 26 to 98, comprising a first monomer comprising a first variant BCMA polypeptide fused directly or indirectly to a first Ig Fc polypeptide, and one or more second monomers comprising a second variant BCMA polypeptide fused directly or indirectly to a second Ig Fc polypeptide, wherein the first monomer and the one or more second monomers are fused in tandem. 100. The fusion polypeptide of embodiment 99, wherein the first variant BCMA polypeptide and the second variant BCMA polypeptide are the same. 101. The fusion polypeptide of embodiment 99, wherein the first variant BCMA polypeptide and the second variant BCMA polypeptide are different. 102. The fusion polypeptide of embodiment 99, wherein the first monomer and the second monomer are the same. 103. The fusion polypeptide of embodiment 99, wherein the first monomer and the second monomer are different. 104. A dimer comprising a first monomer comprising a variant BCMA polypeptide of any one of embodiments 1 to 24 or a fusion polypeptide of any one of embodiments 25 to 98, and a second monomer comprising a variant BCMA polypeptide of any one of embodiments 1 to 24 or a fusion polypeptide of any one of embodiments 25 to 98. 105. The dimer of embodiment 104, wherein the first monomer comprises a first variant BCMA polypeptide fused directly or indirectly to a first Ig Fc polypeptide, and the second monomer comprises a second variant BCMA polypeptide fused directly or indirectly to a second Ig Fc polypeptide. 106. The dimer described in embodiment 104 or 105, wherein the first variant BCMA polypeptide and the second variant BCMA polypeptide are the same. 107. The dimer described in embodiment 104 or 105, wherein the first variant BCMA polypeptide and the second variant BCMA polypeptide are different. 108. The dimer of any one of embodiments 105 to 107, wherein the first Ig Fc polypeptide and the second Ig Fc polypeptide are the same. 109. The dimer of any one of embodiments 105 to 107, wherein the first Ig Fc polypeptide and the second Ig Fc polypeptide are different. 110. A dimer described in any one of embodiments 104 to 109, wherein the first monomer and the second monomer are the same. 111. The dimer of any one of embodiments 104 to 109, wherein the first monomer and the second monomer are different. 112. A dimer described in any one of embodiments 104 to 111, wherein the first monomer and the second monomer are linked by at least one disulfide bond between a cysteine ​​residue of the first monomer and a cysteine ​​residue of the second monomer. 113. The dimer described in embodiment 112, wherein the disulfide bond is between a cysteine ​​residue of the Ig Fc polypeptide of the first monomer and a cysteine ​​residue of the Ig Fc polypeptide of the second monomer. 114. A conjugate comprising a variant BCMA polypeptide according to any one of embodiments 1 to 24, a fusion polypeptide according to any one of embodiments 25 to 103, or a dimer according to any one of embodiments 104 to 113, and an additional moiety covalently linked to said variant BCMA polypeptide, said fusion polypeptide, or said dimer. 115. The conjugate of embodiment 114, wherein the additional moiety is selected from a therapeutic moiety, a polymer moiety, a sugar moiety, and a lipophilic moiety. 116. The conjugate of embodiment 114 or 115, wherein the additional moiety is selected from one or more of polyalkylene oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), branched polyethylene glycol in which two or more polyethylene glycol chains are linked by a linker group, polyvinyl alcohol (PVA), polycarboxylate, poly(vinylpyrrolidone), polyethylene-co-maleic anhydride, and dextran. 117. A variant BCMA polypeptide according to any one of embodiments 1 to 24, a fusion polypeptide according to any one of embodiments 25 to 103, a dimer according to any one of embodiments 104 to 113, or a conjugate according to any one of embodiments 113 to 116, which binds to B-cell activating factor of the TNF family (BAFF) and / or proliferation-inducing ligand (APRIL) or a variant thereof. 118. exhibits a higher binding affinity for BAFF and / or APRIL compared to the binding affinity of a reference BCMA polypeptide or reference binding molecule; and / or exhibit greater inhibition of BAFF and / or APRIL activity or function compared to inhibition of BAFF and / or APRIL activity or function by a reference BCMA polypeptide or reference binding molecule. A variant BCMA polypeptide according to any one of embodiments 1 to 24, a fusion polypeptide according to any one of embodiments 25 to 103, a dimer according to any one of embodiments 104 to 113, or a conjugate according to any one of embodiments 113 to 116, or a variant BCMA polypeptide, fusion polypeptide, dimer or conjugate according to embodiment 117. 119. A variant BCMA polypeptide according to any one of embodiments 1 to 24, a fusion polypeptide according to any one of embodiments 25 to 103, a dimer according to any one of embodiments 104 to 113, or a conjugate according to any one of embodiments 113 to 116, or a variant BCMA polypeptide, fusion polypeptide, dimer or conjugate according to embodiment 117 or 118, which reduces B cell proliferation or reduces B cell proliferation mediated by BAFF and / or APRIL. 120. A variant BCMA polypeptide according to any one of embodiments 1 to 24, a fusion polypeptide according to any one of embodiments 25 to 103, a dimer according to any one of embodiments 104 to 113, or a conjugate according to any one of embodiments 113 to 116, or a variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate according to any one of embodiments 117 to 119, which reduces the production of pro-inflammatory cytokines. 121. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of embodiment 120, wherein the inflammatory cytokine is one or more of IFNγ or IL-17A. 122. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 117 to 121, wherein BAFF is human BAFF and APRIL is human APRIL. 123. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 117 to 121, wherein BAFF is mouse BAFF and APRIL is mouse APRIL. 124. A variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate according to any one of embodiments 118 to 122, wherein the reference BCMA polypeptide is the wild-type human BCMA CRD set forth in SEQ ID NO: 1. 125. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 118 to 122 and 124, wherein said reference BCMA polypeptide is a human BCMA CRD comprising a serine to glycine substitution at position 40 (S40G) comprising the sequence set forth in SEQ ID NO: 237. 126. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 118 to 125, wherein the reference binding molecule is selected from among atacicept, telitacicept, belimumab, or BION-1301. 127. the binding affinity of said variant BCMA polypeptide, said fusion polypeptide, said dimer, or said conjugate to human BAFF is at least 1-fold or at least about 1-fold, at least 2-fold or at least about 2-fold, at least 3-fold or at least about 3-fold, at least 4-fold or at least about 4-fold, at least 5-fold or at least about 5-fold, at least 10-fold or at least about 10-fold, at least 20-fold or at least about 20-fold, at least 25-fold or at least about 25-fold, at least 50-fold or at least about 50-fold, at least 100-fold or at least about 100-fold, at least 200-fold or at least about 200-fold, at least 250-fold or at least about 250-fold, or at least 500-fold or at least about 500-fold greater than the binding affinity of said reference BCMA polypeptide or said reference binding molecule to human BAFF; and / or Inhibition of human BAFF activity or function by the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate is at least 5-fold, or at least about 5-fold, at least 10-fold, or at least about 10-fold, at least 20-fold, or at least about 20-fold, at least 25-fold, or at least about 25-fold, at least 50-fold, or at least about 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least about 250-fold, or at least 500-fold, greater than inhibition of human BAFF activity or function by the reference BCMA polypeptide or reference binding molecule. 127. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 117 to 126. 128. the binding affinity of said variant BCMA polypeptide, said fusion polypeptide, said dimer, or said conjugate to human APRIL is at least 1-fold or at least about 1-fold, at least 2-fold or at least about 2-fold, at least 3-fold or at least about 3-fold, at least 4-fold or at least about 4-fold, at least 5-fold or at least about 5-fold, at least 10-fold or at least about 10-fold, at least 20-fold or at least about 20-fold, at least 25-fold or at least about 25-fold, at least 50-fold or at least about 50-fold, at least 100-fold or at least about 100-fold, at least 200-fold or at least about 200-fold, at least 250-fold or at least about 250-fold, or at least 500-fold or at least about 500-fold greater than the binding affinity of said reference BCMA polypeptide or said reference binding molecule to human APRIL; and / or Inhibition of human APRIL activity or function by the variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate is at least 5-fold, or at least about 5-fold, at least 10-fold, or at least about 10-fold, at least 20-fold, or at least about 20-fold, at least 25-fold, or at least about 25-fold, at least 50-fold, or at least about 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, or at least about 250-fold, or at least 500-fold, greater than inhibition of human APRIL activity or function by the reference BCMA polypeptide or reference binding molecule. 128. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 117 to 127. 129. Ratio of binding selectivity of human BAFF to that of human APRIL (huBAFF K D / huAPRIL K D 131. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 130, wherein: 130. Equilibrium dissociation constant (K D 130. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 129, wherein the BCMA IgE concentration is less than 600 pM, less than 550 pM, less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, less than 200 pM, or less than 150 pM. 131. Equilibrium dissociation constant (K D 131. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1-130, wherein the BCMA activity is in the picomolar (pM) range. 132. The equilibrium dissociation constant (K D131. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1-130, wherein the BCMA activity is in the sub-picomolar (pM) range. 133. The equilibrium dissociation constant (K D 133. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 129 and 132, wherein the BCMA IgE concentration is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. 134. The equilibrium dissociation constant (K D 134. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 133, wherein the BCMA IgE concentration is less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM, less than 50 pM, less than 40 pM, or less than 30 pM. 135. The equilibrium dissociation constant (K D 135. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 134, wherein the BCMA activity is in the picomolar (pM) range. 136. The equilibrium dissociation constant (K D 135. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 134, wherein the BCMA activity is in the sub-picomolar (pM) range. 137. The equilibrium dissociation constant (K D 137. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 133 and 136, wherein the BCMA IgE concentration is less than 1.0 pM, less than 0.9 pM, less than 0.8 pM, less than 0.7 pM, less than 0.6 pM, less than 0.5 pM, less than 0.4 pM, less than 0.3 pM, less than 0.2 pM, or less than 0.1 pM. 138. The equilibrium dissociation constants (K D 138. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 137, wherein the BCMA activity is less than 120 pM. 139. The equilibrium dissociation constants (K D 139. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 138, wherein the BCMA activity is less than 0.3 pM. 140. The equilibrium dissociation constant (K D 140. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 139, wherein the BCMA activity is measured by equilibrium exclusion binding or surface plasmon resonance (SPR). 141. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 140, which does not substantially bind to heparan sulfate proteoglycans (HSPGs). 142. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of any one of embodiments 1 to 141, wherein the HSPG is selected from one or more of syndecan-1 and syndecan-2. 143. A variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate according to any one of embodiments 1 to 142, which inhibits the activity or function of BAFF and / or APRIL or variants thereof. 144. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of embodiment 143, wherein the activity or function of BAFF and / or APRIL is selected from B cell survival, B cell proliferation, and / or immunoglobulin production. 145. A polynucleotide comprising a nucleotide sequence encoding a variant BCMA polypeptide of any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide of any one of embodiments 25 to 103 and 117 to 144, or the first monomer and / or the second monomer of a dimer of any one of embodiments 104 to 113 and 117 to 144. 146. A vector comprising a polynucleotide according to embodiment 136, or a variant BCMA polypeptide according to any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide according to any one of embodiments 25 to 103 and 117 to 144, or a polynucleotide comprising a nucleotide sequence encoding the first monomer and / or the second monomer of a dimer according to any one of embodiments 104 to 113 and 117 to 144. 147. A cell comprising the polynucleotide described in embodiment 145 or the vector described in embodiment 146. 148. A method for producing a variant BCMA polypeptide, fusion polypeptide, or dimer, comprising: 1) introducing the polynucleotide of embodiment 145 or the vector of embodiment 146 into a cell; 2) culturing the host cell under conditions suitable for expression of the polypeptide; 3) recovering or isolating said polypeptide; and optionally 4) purifying the polypeptide A method comprising: 149. A pharmaceutical composition comprising a variant BCMA polypeptide of any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide of any one of embodiments 25 to 103 and 117 to 144, a dimer of any one of embodiments 104 to 113 and 117 to 144, a conjugate of any one of embodiments 114 to 144, a polynucleotide of embodiment 145, a vector of embodiment 146, or a cell of embodiment 147. 150. The pharmaceutical composition of embodiment 149, further comprising one or more pharmaceutically acceptable excipients. 151. A pharmaceutical composition according to embodiment 149 or 150, wherein the one or more excipients comprise a pharmaceutically acceptable liquid carrier. 152. A pharmaceutical composition according to any one of embodiments 149 to 151, wherein the one or more excipients comprise a pharmaceutically acceptable processing agent. 153. A pharmaceutical composition according to any one of embodiments 149 to 152, which is a liquid formulation, an intravenous formulation, a solid dosage form, or an inhalable preparation. 154. A pharmaceutical composition according to any one of embodiments 149 to 153 for treating a disease or disorder. 155. A pharmaceutical composition for use according to embodiment 154, to be administered to a subject having said disease or disorder. 156. A variant BCMA polypeptide according to any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide according to any one of embodiments 25 to 103 and 117 to 144, a dimer according to any one of embodiments 104 to 113 and 117 to 144, a conjugate according to any one of embodiments 114 to 144, a polynucleotide according to embodiment 145, a vector according to embodiment 146, or a cell according to embodiment 147, for treating a disease or disorder. 157. A variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell for use according to embodiment 156, administered to a subject having said disease or disorder. 158. A method for treating a disease or disorder, comprising administering to a subject having said disease or disorder a variant BCMA polypeptide according to any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide according to any one of embodiments 25 to 103 and 117 to 144, a dimer according to any one of embodiments 104 to 113 and 117 to 144, a conjugate according to any one of embodiments 114 to 144, a polynucleotide according to embodiment 145, a vector according to embodiment 146, a cell according to embodiment 147, or a pharmaceutical composition according to any one of embodiments 149 to 155. 159. Use of a variant BCMA polypeptide according to any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide according to any one of embodiments 25 to 103 and 117 to 144, a dimer according to any one of embodiments 104 to 113 and 117 to 144, a conjugate according to any one of embodiments 114 to 144, a polynucleotide according to embodiment 145, a vector according to embodiment 146, a cell according to embodiment 147, or a pharmaceutical composition according to any one of embodiments 149 to 155 in the manufacture of a medicament for treating a disease or disorder. 160. Use of a variant BCMA polypeptide according to any one of embodiments 1 to 24 and 117 to 144, a fusion polypeptide according to any one of embodiments 25 to 103 and 117 to 144, a dimer according to any one of embodiments 104 to 113 and 117 to 144, a conjugate according to any one of embodiments 114 to 144, a polynucleotide according to embodiment 145, a vector according to embodiment 146, a cell according to embodiment 147, or a pharmaceutical composition according to any one of embodiments 149 to 155 for treating a disease or disorder. 161. The use described in embodiment 159 or 160, wherein the variant BCMA polypeptide, the fusion polypeptide, the dimer, the conjugate or the pharmaceutical composition is administered to a subject having the disease or disorder. 162. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 161, wherein said disease or disorder is a B-cell or antibody-mediated disease or disorder. 163. A pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 162, wherein the disease or disorder is an autoimmune disease or disorder. 164. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 163, wherein said autoimmune disease or disorder is an immune-mediated disease or disorder of the tissues, bones, joints, blood vessels, thyroid, kidneys, nervous system, brain, lungs, and / or skin of said subject. 165. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 163 or 164, wherein said autoimmune disease or disorder is selected from among a kidney disease or disorder, lupus, arthritis, a spondyloarthritic disorder, a vasculitis disorder, a hemolytic anemia disorder, a thrombocytopenic disorder, a thyroiditis disorder, a demyelinating disease of the central and / or peripheral nervous system, an inflammatory and / or fibrotic lung disorder, a skin disorder, or an allergic disorder. 166. The disease or disorder is systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berge's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), renal sarcoidosis, rheumatoid arthritis, juvenile chronic arthritis, arthritis associated with inflammatory bowel disease, ankylosing spondylitis, spondylitis associated with psoriasis, juvenile spondylitis, spondylitis associated with psoriasis, spondylitis associated with inflammatory bowel disease, spondylitis associated with psoriasis, spondylitis associated with inflammatory bowel disease, spondylitis associated with inflammatory bowel disease, spondylitis associated with spondylitis ... Spondyloarthropathy, unclassifiable spondyloarthropathy, Reiter's syndrome, scleroderma, Sjögren's syndrome, systemic necrotizing vasculitis, polyarteritis nodosa, allergic vasculitis and granulomatosis, polyangiitis, Wegener's granulomatosis, lymphomatoid granulomatosis, mucocutaneous lymph node syndrome (MLNS or Kawasaki disease), isolated central nervous system vasculitis, Behçet's disease, thromboangiitis obliterans (Buerger's disease), cutaneous necrotizing venulitis, sarcoidosis 166. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use according to any one of embodiments 158 to 165, wherein the therapeutic target is selected from among inflammatory bowel disease, autoimmune hemolytic anemia, immune pancytopenia, paroxysmal nocturnal hemoglobinuria, thrombocytopenic purpura, immune-mediated thrombocytopenia, Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis, type 1 diabetes, glomerulonephritis and tubulointerstitial nephritis, multiple sclerosis (MS), idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, bullous dermatitis, erythema multiforme, contact dermatitis, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, or urticaria. 167. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 166, wherein said disease or disorder is selected from among systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), IgA nephropathy (Berger's disease), Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Henoch-Schönlein purpura, polyarteritis nodosa (PAN), or renal sarcoidosis. 168. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 167, wherein said disease or disorder is selected from among systemic lupus erythematosus (SLE) and / or lupus nephritis (LN), or IgA nephropathy (Berge's disease). 169. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 161, wherein the disease or disorder is tissue or organ transplant rejection. 170. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 169, wherein said tissue or organ transplant rejection is selected from among acute or chronic B-cell or antibody-mediated rejection of tissue allografts consisting of bone marrow, stem cells, skin, and solid organs, acute or chronic graft-versus-host disease (GVHD), antibody-mediated rejection (AMR) of solid organs, hyperacute organ transplant rejection, acute organ transplant rejection, chronic organ transplant rejection. 171. A pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 161, wherein the disease or disorder is a B-cell malignancy. 172. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 171, wherein said B-cell malignancy is selected from among non-Hodgkin's lymphoma, multiple myeloma (MM), B-cell chronic lymphocytic leukemia, plasmacytoma, macroglobulinemia, or Waldenström's macroglobulinemia (WM). 173. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 172, wherein a therapeutically effective amount of the variant BCMA polypeptide, the fusion polypeptide, the dimer, the conjugate, the polynucleotide, the vector, the cell or the pharmaceutical composition is administered to the subject. 174. A pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 to 173, wherein said method or said use is therapeutic use or prophylactic use. 175. A pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 174, wherein said therapeutic use is for induction therapy. 176. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 175, wherein said induction therapy lasts for up to or up to about 1 week, up to or up to about 2 weeks, up to or up to about 3 weeks, or up to or up to about 4 weeks. 177. A pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 174, wherein said therapeutic use is for maintenance therapy. 178. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 177, wherein said maintenance therapy lasts for up to or up to about 1 week, up to or up to about 2 weeks, up to or up to about 3 weeks, or up to or up to about 4 weeks. 179. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to any one of embodiments 158 and 161 to 178, wherein said administration is selected from intravenous, oral, parenteral, sublingual, by inhalation, rectal or topical. 180. The pharmaceutical composition, variant BCMA polypeptide, fusion polypeptide, dimer, conjugate, polynucleotide, vector or cell, method or use for use according to embodiment 179, wherein said administration is intravenous administration. [Example]

[0295] VII. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 Construction of plasmid vectors encoding Z-TACI-Ig and BCMA ECD-Ig fusion proteins

[0296] This example provides a description of the generation of a [S40G]huBCMA ECD-Ig fusion protein (i.e., a huBCMA surrogate control protein) and a TACI ECD-Ig fusion protein ("Z-TACI-Ig"). These fusion proteins were used as controls and for comparison in BIAcore™ and KinExA™ binding assays, cell-based activity assays, and in vivo experiments. Both the huBCMA ECD and the TACI ECD have been reported to exhibit binding activity for both BAFF and APRIL.

[0297] As described in Example 18, removal of the N-glycosylation site at positions 38-40 (i.e., by a S40G substitution relative to amino acid positions in the wild-type huBCMA ECD CRD, SEQ ID NO: 1) was believed to result in a protein preparation with a more uniform molecular weight distribution compared to protein without this substitution. This substitution is not believed to affect binding activity to huBAFF and huAPRIL. This molecule was used as a control and benchmark for quantifying the binding activity of the variant BCMA polypeptides described herein. A. Construction of a DNA plasmid vector encoding the [S40G]huBCMA ECD-Ig fusion protein

[0298] This example describes the generation of a DNA plasmid vector encoding the [S40G]huBCMA ECD-Ig fusion protein shown in Figure 6. In this fusion protein, the [S40G]huBCMA extracellular domain (ECD) is covalently linked at its C-terminus to the N-terminus of a mutant human IgG2 Fc domain in which three of the four hinge cysteines have been mutated to serines (referred to herein as "PIg18").

[0299] Unless otherwise noted, the methods described for generating and purifying the [S40G]huBCMA ECD-Ig fusion protein were similarly used for preparing and purifying other variants of huBCMA ECD / huBCMA ECD-Ig described herein. A polypeptide comprising [S40G]huBCMA ECD (SEQ ID NO: 224), e.g., DNA encoding the native human BCMA ECD amino acid sequence shown in Figure 2B, with the substitution S40G included to remove a glycosylation site, was created by PCR assembly using overlapping oligonucleotides designed based on the sequence of the BCMA gene (NCBI AB052772.1 (CDS sequence); BAB60895.1 (polypeptide sequence)). The oligonucleotides were designed, generated, and assembled using standard procedures and included stop and start codons, as well as restriction sites as needed. See, for example, Sambrook, Joseph. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001; Verma and Eckstein, Annu. Rev. Biochem. 1998; 67:99-134; Tang et al., "Gene Assembly," Synthetic Biology, Academic Press, 2013: 3-21; Ulyanov and James "Chemical Synthesis of Oligonucleotides," Comprehensive Natural Products II, American Chemical Society, 2010; 10:247-278. Specifically, oligonucleotides were assembled in a 100 μl PCR reaction with 1 μM oligonucleotide, 1×Taq buffer (Qiagen; #201225) and 200 μM dNTPs, with 30 amplification cycles (94°C, 30 seconds; 60°C, 30 seconds; 72°C, 60 seconds).A nucleic acid sequence encoding the CTLA-4 signal peptide was added to the 5' end of the BCMA-ECD coding fragment using, for example, the splicing by overlap extension (SOE) method described in Horton et al., Gene 1989;77:61-68, incorporated herein by reference. Similarly, a nucleic acid sequence encoding a variant form of the human IgG2-Fc peptide, "PIg18," was fused to the 3' end of the CTLA-4 signal-BCMA-ECD coding fragment, and the resulting DNA fragment was subcloned into the vector CET1019AS (EMD Millipore, a division of Merck KGaA) to generate the vector CET1019-BCMA-PIg18. PIg18 differs from the corresponding native human IgG2-Fc in that it contains serine at positions 4, 5, and 11. In contrast, the corresponding native IgG2-Fc contains cysteines at each of these positions. The resulting plasmid was sequenced to confirm the DNA sequence encoding the entire fusion polypeptide.

[0300] A schematic diagram of CET1019-BCMA-PIg18 is provided in Figure 4. This vector contains promoter A from Caviid herpesvirus 2 directing expression of a transcription cassette containing the 5'UTR / intron from pCI-Neo (Promega) and a nucleic acid sequence encoding the CTLA-4 signal peptide fused in-frame to BCMA-PIg18, the SV40 early poly(A) tail signal sequence, the mouse PGK promoter driving expression of the puromycin resistance gene, the Bla promoter driving expression of the ampicillin resistance gene, a ColE1 origin of replication, and a UCOE (ubiquitous chromatin opening element) sequence (Rps3) that confers high expression of polypeptides encoded on the plasmid.

[0301] Polynucleotide and amino acid sequence encoding the predicted preform of the [S40G]BCMA ECD-Ig fusion protein containing the CTLA-4 signal peptide coding sequence. The predicted amino acid sequence includes the CTLA-4 signal peptide, [S40G] human BCMA ECD (SEQ ID NO: 224), and a mutant human IgG2 heavy chain Fc domain polypeptide (PIg18).

[0302] Signal peptides are generally cleaved during processing, and thus the secreted protein (i.e., the mature form) of a [S40G]BCMA ECD-Ig fusion protein usually does not contain a signal peptide sequence. In some cases, the expressed [S40G]BCMA ECD-Ig fusion protein may not include a C-terminal lysine (K) residue because the C-terminal lysine may be cleaved during processing or secretion. In some cases, the penultimate glycine (G) residue is missing.

[0303] In some embodiments, BCMA ECD-Ig and variants thereof generally exist in solution as dimeric fusion proteins composed of two identical monomeric polypeptides. In some embodiments, the two monomeric BCMA ECD-Ig polypeptides are covalently linked by disulfide bonds formed between cysteine ​​residues in each monomer, thereby forming a BCMA ECD-Ig fusion protein dimer. BCMA ECD-Ig dimers and variants thereof are the form of the fusion protein molecules used in the assays described herein, unless otherwise specified. Creation of a DNA plasmid vector encoding the BZ-TACI-Ig fusion protein

[0304] This example describes the generation of a DNA plasmid vector encoding the Z-TACI-Ig fusion protein shown in Figure 6. Z-TACI-Ig comprises a portion of the human TACI extracellular domain (ECD) containing both CRD1 and CRD2 (SEQ ID NO: 147) covalently linked at the N- and C-termini of a mutant human IgG1 Fc polypeptide. ZIg differs from the corresponding native human IgG1-Fc in having serine at position 5, alanine at position 19, glutamic acid at position 20, alanine at position 22, serine at position 115, and serine at position 116. These correspond to the following substitutions compared to the corresponding native IgG1 Fc: C5S+L19A+L20E+G22A+A115S+P116S. The predicted amino acid sequence of the preform of Z-TACI-Ig includes the TPA signal peptide, part of the human TACI ECD domain (this sequence has a polymorphism at position 3 of CRD1 relative to the reference, a K instead of an E), and ZIg.

[0305] To produce this fusion protein, the plasmid vector CET1019AS-Z-TACI-Ig was constructed as follows. A DNA sequence encoding a fusion of the human tissue plasminogen activator (TPA) signal peptide, Z-TACI-ECD, and mutant IgG1 Fc was synthesized (based on the polypeptide sequence in the publication: International Nonproprietary Names for Pharmaceutical Substances (INN) Recommended INN: List 57, WHO Drug Information, Vol. 21, No. 1, 2007). The synthetic gene contained appropriate start and stop codons and introduced flanking AgeI and SalI restriction sites that were used for subcloning into the expression vector. The DNA was digested with AgeI and SalI. The resulting fragment was separated by agarose gel electrophoresis, purified using a Qiaquick™ Gel Extraction Kit (Qiagen, Catalog No. 28704) as recommended by the manufacturer, and ligated into similarly digested plasmid CET1019AS (EMD Millipore, a division of Merck KGAa). The ligation was transformed into TOP10™ E. coli cells (Invitrogen Catalog No. C404010) as recommended by the manufacturer. The resulting cells were incubated overnight in LB medium containing 50 μg / ml carbenicillin at 37°C with shaking at 250 rpm, and then subjected to Maxiprep™ (Qiagen; Catalog No. 12362) purification to prepare a plasmid DNA stock. A schematic diagram of this vector is provided in Figure 5.

[0306] A schematic diagram of CET1019-BCMA-PIg18 is provided in Figure 4. This vector contains promoter A from Caviid herpesvirus 2 directing expression of a transcription cassette containing the 5'UTR / intron from pCI-Neo (Promega) and a nucleic acid sequence encoding the TPA signal peptide fused in-frame with Z-TACI-Ig, the SV40 early poly(A) tail signal sequence, the mouse PGK promoter driving expression of the puromycin resistance gene, the Bla promoter driving expression of the ampicillin resistance gene, a ColE1 origin of replication, and a UCOE (ubiquitous chromatin opening element) sequence (Rps3) that confers high expression of polypeptides encoded on the plasmid.

[0307] Signal peptides are generally cleaved during processing, and thus secreted fusion proteins (i.e., mature forms) of Z-TACI-Ig usually do not contain a signal peptide sequence. In some cases, the expressed Z-TACI-Ig polypeptide sequence may not include a C-terminal lysine (K) residue because the C-terminal lysine may be cleaved during processing or secretion. In some cases, the penultimate glycine (G) residue is missing.

[0308] In some embodiments, Z-TACI-Ig exists in solution as a dimeric fusion protein composed of two identical monomeric polypeptides. In some embodiments, the two monomeric Z-TACI-Ig polypeptides are covalently linked by a disulfide bond formed between the cysteine ​​residues of each monomer, thereby forming a Z-TACI-Ig fusion protein dimer. Unless otherwise specified, the Z-TACI-Ig dimer is the form of the fusion protein molecule used in the assays described in these examples. Example 2 Creation of stable cell lines expressing Z-TACI-Ig and BCMA-PIg18 fusion proteins and protein expression and purification

[0309] This example describes the creation of stable cell lines for the production of the Z-TACI-Ig and BCMA-PIg18 fusion proteins discussed above in multi-milligram quantities. A. Transfection of CHO-K1 cells

[0310] To produce multimilligram quantities of [S40G]huBCMA ECD-Ig and Z-TACI-Ig fusion proteins, stable CHO-K1 cell lines adapted for suspension growth were created by transfecting CHO-K1 cells (ATCC CCL-61) with the above-described plasmid vectors (see "Animal Cell Technology: Products from Cells, Cells as Products," Proceedings of the 16th ESACT Meeting, April 25-29, 1999, Lugano, Switzerland, edited by A. Bernard, et al. (2002)). Naive Opti-CHO-K1 cells were maintained in 500 ml shake flasks (SF500 flasks; VWR#29445-058) containing 100 ml of growth medium (OptiCHO medium, Invitrogen Catalog No. 12681) supplemented with 4 mM L-glutamine (Invitrogen Catalog No. 25031). For each electroporation, 2 × 10 cells were cultured. 6The cells were suspended in 400 μL of growth medium in a 0.4 cm cuvette (Bio-Rad Cat. No. 165-2088), and 20 μg of maxiprep plasmid DNA (e.g., the Z-TACI-Ig-encoding plasmid vector and the [S40G]huBCMA ECD-Ig-encoding plasmid vector) was added. Electroporation was performed in duplicate using a Gene Xcell Pulser (Bio-Rad Cat. No. 165-2661) at 320 V and a square-wave pulse width of 15 ms. The cells were pooled and transferred to a T25 flask containing 5 mL of growth medium and incubated at 37°C, 5% CO2, for 48 hours without selection. The cells were collected by centrifugation, resuspended in selection medium (growth medium containing 9% puromycin), and incubated for 8–10 days to obtain a pool of stably transfected cells ("stable pool"). Protein expression was determined by Protein A HPLC on culture supernatants from stable pools. For Z-TACI-Ig production, stable pools were further subcloned to identify suitable clonal cell lines. Meanwhile, good quality [S40G]huBCMA ECD-Ig protein was obtained from stable pools, and no further subcloning was required. Isolation of unique clones expressing BZ-TACI-Ig

[0311] Stable pools of Z-TACI-Ig-expressing cells were subcloned by limiting dilution. Stably transfected cells were resuspended in conditioned medium (harvested from the parent CHO-K1 cell culture) at a final density of 3.8 cells per ml and seeded into flat-bottom 96-well plates at 200 μL per well (75 cells per plate). Plates were incubated for 10–12 days, and 52 wells that showed colonies were expanded to 12-well plates. After 5 days of incubation, 40 clones were selected for further expansion to 125 ml shake flasks. Culture supernatants were assayed for protein expression using Protein A HPLC and cell density to measure their productivity in pg / cell / day. Expressed proteins from 18 clones that showed good productivity were further analyzed by Western blot analysis. Equal volumes (6 μl) of medium from each cell culture were separated by SDS-PAGE on a 4-12% Bis-Tris NuPAGE gel (Invitrogen Cat. No. NP0322BOX) in MOPS running buffer (Invitrogen) according to the manufacturer's recommendations. Proteins were transferred from the gel to a nitrocellulose membrane (Invitrogen Cat. No. LC2001) by electrophoretic transfer according to the manufacturer's recommendations. Z-TACI-Ig fusion protein was visualized by probing the nitrocellulose membrane with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human Ig antibody (Bethyl Cat. No. A80-104P) and detecting the signal using ECL Western blot detection reagents (Amersham Cat. No. RPN2132) according to the manufacturer's recommendations. Clones that showed good productivity and a high percentage of full-length Z-TACI-Ig protein were used for protein production. C. Protein A HPLC to measure protein expression

[0312] Protein A HPLC was used to quantify the amount of Fc fusion protein in the culture supernatant. The culture supernatant was loaded onto a Poros Protein AA / 20 column using 50 mM phosphate, 150 mM potassium chloride, pH 7.5 and 50 mM phosphate, 150 mM potassium chloride, pH 2.5 at a ratio of 42% / 58%, respectively. The Fc fusion protein was separated from process impurities by decreasing the pH of the mobile phase using a gradient elution to 50 mM phosphate, 150 mM potassium chloride, pH 7.5 and 50 mM phosphate, 150 mM potassium chloride, pH 2.5 at a ratio of 12% / 88%, respectively. The elution profile was monitored for 6 minutes by detecting absorbance (A) at 220 nanometers (nm). For a given peak area, the amount of Fc fusion protein was determined from a calibration curve of peak area versus amount, generated by injecting a set volume of a reference control of known concentration. D. Protein Purification

[0313] Supernatants from pooled or clonal cultures (e.g., containing cells expressing Z-TACI-Ig or [S40G]huBCMA ECD-Ig) were collected by centrifugation at 1000 × g for 10 minutes at room temperature and filtered through a 0.2 μm membrane (Nalgene, VWR Catalog No. 73520-982). Proteins were purified by Protein A affinity chromatography using a Hitrap MAb Select Sure (GE Healthcare Catalog No. 11-0034-93 or 11-0034-95) column with an AKTA Explorer HPLC system (GE Healthcare). Briefly, the column was equilibrated with 1 × PBS / pH 7.4, and the clarified harvest was applied to the column, which was then washed with 1 × PBS / pH 7.4 (Invitrogen). The bound protein was eluted with elution buffer (80 mM citric acid / 160 mM NaCl / pH 3.5) and immediately neutralized by adding 1 / 10 volume of 2 M Tris base / pH 8.6. The eluted protein was dialyzed into PBS pH 7.4 buffer using a 20 kDa MWCO membrane (Pierce, Cat. No. 66003 for 0.5-3 mL, Cat. No. 66012 for 3-12 mL, or Cat. No. 66030 for 12-30 mL). The protein was placed in PBS pH 7.4 and stored at -80°C. Example 3 Protein characterization: Z-TACI-Ig, [S40G]BCMA ECD-Ig, and BCMA variant molecules

[0314] This example describes the assessment of protein quality. A.SDS / PAGE analysis

[0315] The apparent molecular weights (MW) of purified Z-TACI-Ig fusion protein and purified [S40G]huBCMA ECD-Ig fusion protein (described below) were determined by SDS / PAGE analysis under non-reducing conditions. Under non-reducing conditions, these fusion proteins generally exist in the form of a dimeric fusion protein composed of two (mature) monomeric fusion polypeptides. The two monomeric fusion polypeptides are linked by a disulfide bond formed between the cysteine ​​residues of each monomer. The data presented in the following examples refer to the homodimeric form of the molecule, unless otherwise specified.

[0316] SDS / PAGE analysis was performed as follows. For each fusion protein, 2 μg of purified fusion protein was added to 20 μl of LDS Sample Buffer (Invitrogen Cat. No. NP0007) and run on a NuPAGE 4-12% Bis-Tris gel (Invitrogen Cat. No. NP0321BOX) in 1× Tris-Glycine sodium dodecyl sulfate (SDS) / PAGE running buffer (Invitrogen Cat. No. NP0002) according to the manufacturer's recommended conditions. The gel was stained by incubating in 50 ml of SimplyBlue SafeStain (Invitrogen Cat. No. LC6060) for 1 hour at room temperature with gentle agitation. The gel was destained by two 1-hour incubations with 200 ml of water at room temperature with gentle agitation and processed with drying buffer (Bio-Rad Cat. No. 161-0752) according to the manufacturer's recommended conditions.

[0317] Gel analysis indicated that the Z-TACI-Ig fusion protein dimer and the [S40G]huBCMA ECD-Ig fusion protein dimer were formed by two identical (in terms of size) Z-TACI-Ig or [S40G]huBCMA ECD-PIg18 protein monomers, respectively, linked by disulfide bonds formed between the cysteine ​​residues of each monomer. SDS / PAGE analysis was performed on all protein preparations to verify the protein quality in terms of apparent molecular weight, protein concentration, and purity. The results of SDS / PAGE analysis were similar for all protein preparations. Based on the gel results, the apparent MW of the purified Z-TACI-Ig or [S40G]huBCMA ECD-Ig fusion protein dimer was approximately 70 kDa or 62 kDa, respectively, which is consistent with the predicted MW of an exemplary homodimeric protein. B. Endotoxin Analysis

[0318] Measurement of endotoxin levels in fusion protein preparations was performed using a QCL-1000 Limulus Amoebocyte Lysate (LAL) assay kit at Nelson Laboratories, Inc. (Taylorsville, UT). The maximum endotoxin level for proteins used in cell-based assays was set at 10 endotoxin units (EU) per mg of protein. C. Size Exclusion Chromatography (SEC) Analysis

[0319] Protein aggregation levels (including the aggregation levels of Z-TACI-Ig fusion protein or [S40G]huBCMA ECD-Ig fusion protein) were measured by size exclusion chromatography using an HPLC system. Protein (20 μg) was loaded onto a TSK 3000 SWxL column (Tosoh Bioscience) equipped with a TSK Guard SWxL column using a mobile phase of 200 mM potassium phosphate, 150 mM potassium chloride, pH 6.8. The elution profile was monitored for 30 minutes by detecting absorbance (A) at 220 nanometers (nm). The maximum level of protein aggregation used in further assays was set at 10%.

[0320] SEC analysis was performed on all protein preparations to verify protein quality with respect to protein aggregation levels. Results showed that purified Z-TACI-Ig dimers were nearly uniform in size and did not contain high levels of aggregated species (data not shown). Example 4 Construction, production, and detection of phage displaying variant BCMA polypeptides

[0321] This example describes an exemplary method used to create a library of BCMA ECD variants and screen them by phage display for altered binding activity to human BAFF and / or human APRIL.

[0322] A portion of the BCMA ECD was displayed on the surface of filamentous phage using the monovalent phage display vector pSB0124. pSB0124 is a helper-dependent phagemid vector containing the bla gene for ampicillin resistance selection in E. coli, the filamentous phage M13 origin of replication, the STII secretion signal sequence, a 6-His Tag (SEQ ID NO: 253), a suppressible amber codon (TAG), and the C-terminal portion of M13 gene III. Unique SfiI and NotI sites engineered between the STII secretion signal sequence and the suppressible amber codon allow for cloning of a DNA sequence encoding a polypeptide to be displayed to generate an in-frame fusion protein, along with a His tag, on a copy of the pIII coat protein expressed from the phagemid vector in an amber suppressor E. coli strain (e.g., TG1).

[0323] The double-stranded phagemid DNA was transformed into TG1 E. coli cells (Stratagene Cat. No. 200123), which harbor the F plasmid, thus exhibiting F pili and being permissive for phage infection. The resulting transformants were infected with M13 helper phage to produce phage bearing mature recombinant coat proteins consisting of full-length pIII derived from the helper phage plus a polypeptide for fusion display as the M13 protein pIII.

[0324] DNA encoding the human BCMA ECD (SEQ ID NO:152, corresponding to amino acid residues 5-54 of huBCMA set forth in SEQ ID NO:149) was prepared by PCR assembly using overlapping oligonucleotides. These oligonucleotides contained flanking SfiI and NotI restriction sites that were used to subclone the PCR product into pSB0124. The assembled BCMA PCR product was digested with restriction enzymes SfiI and NotI, the fragments separated by agarose gel electrophoresis, and the BCMA fragment was purified using a Qiaquick™ Gel Extraction Kit (Qiagen, #28704) and ligated into similarly digested vector pSB0124 to generate pSB0124-BCMA. DNA was transformed into E. coli TOP10 cells, DNA was prepared, transformed into TG1 cells, and transformants were used for phage production as described in Example 5 below.

[0325] The following phage-displayed variants of BCMA were tested for functionality using a phage ELISA. NUNC MaxiSorp™ plates were coated with BAFF (R&D Systems, Catalog Number 2149-BF / CF) or APRIL (R&D Systems Catalog Number 884-AP / CF) at concentrations of 0.5 μg / ml and 4 μg / ml (respectively) in 50 μl / well of PBS overnight at 4°C. After washing with PBS-T (PBS + 0.02% TWEEN®-20), plates were blocked with PBS containing 3% milk (nonfat dry milk; Sigma-Aldrich). After blocking and washing, phage dilutions (two-fold titrations in PBS-T + 1% milk) were added to the coated plates and incubated at room temperature. After 2 hours of incubation, the plates were washed with PBS-T and bound phage was detected by incubating with horseradish peroxidase (HRP)-anti-M13 antibody conjugate (GE Healthcare) for 30 minutes, washed with PBS-T, and immobilized HRP was detected using TMB-H2O2 reagent (Pierce). After color development, the reaction was stopped using 2M H2SO4, and the plates were read using a spectrophotometer and SoftMaxPro™ software (Molecular Devices, Sunnyvale, CA) for absorbance at 450 nM. The absorbance was plotted against phage dilutions to obtain binding curves and compare the binding of different variants (data not shown). Example 5 Generation and screening of BCMA variant polypeptides by phage display

[0326] This example describes the generation of libraries of variant BCMA polypeptides and the screening of these libraries for altered binding activity to human BAFF and / or human APRIL by phage display. A. DNA sequences encoding BCMA variant polypeptides

[0327] Directed evolution methods were used to generate libraries of non-naturally occurring recombinant polynucleotides encoding BCMA ECD variant polypeptides. Directed evolution procedures included in vitro recombination and mutagenesis procedures substantially as described, for example, in Stemmer, Proc. Natl. Acad. Sci. USA 1994; 91:10747-10751; Chang et al., Nature Biotech. 1999; 17:793-797; International Patent Application Publication No. WO98 / 27230; and U.S. Patent Nos. 6,117,679 and 6,537,776, all of which are incorporated herein by reference.

[0328] The DNA sequence encoding the recombinant variant BCMA polypeptide was then amplified by PCR assembly using forward and reverse primers designed based on sequence homology. Exemplary forward and reverse primers include the following: 1 μM forward and reverse primers, Taq buffer (Qiagen; Cat. No. 201225), and 200 μM dNTPs. Five μl of the assembly reaction was used as template in a 100 μl PCR reaction, which was subjected to 15 amplification cycles (94° C. for 30 seconds; 50° C. for 30 seconds; 72° C. for 40 seconds). Amplified DNA encoding variant BCMA polypeptides was digested with restriction enzymes (SfiI and NotI), fragments were separated by agarose gel electrophoresis, purified using a Qiaquick™ Gel Extraction Kit (Qiagen, Catalog No. 28704) as recommended by the manufacturer, and ligated into similarly digested phage display vector pSB0124 (Chang et al., Nature Biotech 1999; 17:793-797). The resulting library ligation was transformed into TOP10 E. coli cells (Invitrogen, Inc., Catalog No. C4040-50) by electroporation according to the manufacturer's recommended conditions. Transformed cells were incubated overnight at 37°C, 250 rpm in LB (Luria broth) containing 50 μg / ml carbenicillin and then used to generate maxiprep (Qiagen Cat. No. 12362) stocks of library DNA as recommended by the manufacturer. B. Generation of a Phage Display Library of BCMA Variant Polypeptides

[0329] Library DNA (e.g., a library of DNA sequences encoding BCMA ECD variant polypeptides as variant BCMA-pIII fusions) was transformed into TG-1 E. coli cells (Stratagene Cat. No. 200123) by electroporation according to the manufacturer's recommendations. Cultures were grown for 1-2 generations under phagemid selection conditions (LB medium containing 50 μg / ml carbenicillin), infected with helper phage M13KO7 (at a multiplicity of infection level of 5-10), and incubated overnight at 37°C with shaking at 250 rpm under double selection for phagemid (50 μg / ml carbenicillin) and helper phage (70 μg / ml kanamycin). The culture was clarified by centrifugation (Sorvall 600TC rotor, 6000 rpm, 15 min, 4°C), and 32 ml of the culture supernatant was incubated with 8 ml of PEG / NaCl solution (20% PEG-8000; 2.5 M NaCl) on ice for 30 min, followed by centrifugation (Sorvall 600TC rotor, 9500 rpm, 40 min, 4°C) to precipitate phage particles. The phage pellet was suspended in 1 ml of PBS containing 1% BSA (bovine serum albumin, Sigma Cat. No. A7906), transferred to a microfuge tube, and clarified by centrifugation (Eppendorf tabletop centrifuge, maximum speed, 5 min, room temperature). The resulting phage library consisted of phage displaying variants of the BCMA ECD fused to the N-terminus of the pIII minor phage coat protein. Therefore, these variant BCMA polypeptides were not constructed as Ig fusion proteins and did not exist in a dimeric form. C. Panning of BCMA variant polypeptide phage libraries

[0330] The phage library was panned using standard conditions in up to five alternating or sequential rounds against BAFF or APRIL proteins. See, e.g., Lowman, et al., Biochemistry 1991; 30 (45): 10832-10838; Smith, GP et al., Chem. Rev. 1997; 97: 391-410, each of which is incorporated herein by reference. Each round of panning involved the following steps: (a) binding phage displaying a BCMA ECD variant polypeptide to 0.5 μg / ml or 0.05 μg / ml of BAFF (recombinant human BAFF / BLyS / TNFSF13B, CF; R&D Systems Catalog No. 2149-BF / CF; Accession No. Q9Y275, SEQ ID NO: 214) or 4 μg / ml of APRIL (recombinant human APRIL / TNFSF13, CF; R&D Systems Catalog No. 884-AP; Accession No. Q8NFH7, SEQ ID NO: 217, amino acid residues 110-250); (b) removing unbound phage; (c) eluting bound phage; and (d) amplifying the eluted phage for the next round of panning. An aliquot of phage from each round was used to transduce E. coli cells to obtain individual transductant colonies.

[0331] In some cases, the above procedure selected for phage with overlapping variant BCMA ECD coding sequences, resulting in tandem variant BCMA polypeptides fused to the phage pIII protein. To prevent such phage from dominating the selection, the following series of steps were introduced after step (c) above: (i) variant BCMA ECD coding sequences were PCR amplified from eluted phage using the same primers used for the original library cloning; (ii) amplified fragments were digested with SfiI and NotI and separated by gel electrophoresis, and only fragments with the predicted size of a single copy of the variant BCMA ECD sequence were recloned into a phagemid vector; (iii) the resulting phagemid library was used to generate a phage library as described above, which was used in the next round of selection. D. Identification of BCMA variant polypeptides with improved binding to human BAFF and / or human APRIL by phage ELISA

[0332] Individual colonies from each round of panning were inoculated into 96-well culture plates (NUNC, Cat. No. 243656) containing 150 μl / well of 2xYT (yeast-tryptone) medium containing 50 μg / ml carbenicillin and incubated overnight at 37°C and 250 rpm. The overnight cultures were used to inoculate deep-well blocks (Scienceware, Cat. No. 378600001) containing 600 μl / well of the same medium. The cultures were incubated at 37°C and 250 rpm for 2 hours, infected with M13K07 helper phage (multiplicity of infection (moi) 5–10), and then incubated overnight at 37°C and 250 rpm under double selection for the phagemid and helper phage markers (50 μg / ml carbenicillin and 70 μg / ml kanamycin, respectively). Cultures were clarified by centrifugation at 4000 rpm for 20 minutes at 4°C in a Beckman GH 3.8 rotor. ELISA plates (NUNC, Cat. No. 449824) were coated by adding 50 μl / well of TBS containing BAFF (R&D Systems, Cat. No. 2149-BF / CF) or APRIL (R&D Systems, Cat. No. 884-AP-010 / CF) at concentrations of 0.5 μg / ml or 4 μg / ml, respectively, and incubating overnight at 4°C. The plates were washed three times with 200 μl / well of TBST and blocked by adding 200 μl / well of TBS containing 3% nonfat dry milk and incubating at room temperature for 1 hour (hr). 25 μl / well of phage supernatant from the deep-well block was transferred to an ELISA plate containing 25 μl / well of 6% nonfat dry milk, and the plate was incubated at room temperature for 1 hour. Plates were washed three times with 200 μl / well of TBST and incubated for 1 hour at room temperature with 50 μl / well of HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare, Cat. No. 27-9421-01) diluted 1:5000 in TBST containing 3% nonfat dry milk.Plates were washed three times with 200 μl / well of TBST and signals were detected using a TMB substrate kit (Pierce, Cat. No. 34021) according to the manufacturer's recommended conditions.

[0333] Among the phage-displayed variant BCMA polypeptides, those that showed increased binding to human BAFF and / or human APRIL compared to the binding of phage-displayed human BCMA (Example 4) to human BAFF and / or human APRIL were selected for further analysis. Example 6 Cloning of nucleotide sequences encoding BCMA variant polypeptides into pcDNA-PIg18 fusion vectors

[0334] This example describes the generation of variant BCMA fusion polypeptides. Plasmids and post-translational modifications of the encoded polypeptides are also described.

[0335] To produce variant BCMA polypeptides as soluble Fc fusion proteins, DNA sequences encoding variant BCMA-ECDs that, through phage library screening, demonstrated improved binding to human BAFF and / or human APRIL (compared to native human BCMA-ECD), were subcloned from the phagemid vector into a modified PIg18-Fc fusion vector. A schematic diagram of this fusion is provided in Figure 6. The DNA sequence encoding the entire CTLA-4 signal peptide-BCMA ECD variant-PIg18 fusion described above was subcloned into the pcDNA3.1 vector (Invitrogen). Plasmid pcDNA3.1 is a mammalian expression vector containing an expression cassette controlled by a CMV promoter and bGH (bovine growth hormone) terminator, as well as neomycin and ampicillin resistance selection markers for mammalian and bacterial selection, respectively. The DNA sequence encoding the fusion polypeptide contains two unique restriction sites, AgeI and KpnI, located within the CTLA-4 signal coding region and at the 3' end of the BCMA ECD variant-encoding sequence, respectively. Plasmid pcDNA-BCMA-PIg18 was digested with AgeI and KpnI to release fragments encoding the BCMA ECD variants and the pcDNA3.1-PIg18-Fc fusion vector fragment, which was used for cloning of fragments encoding the BCMA ECD variants below.

[0336] The sequences encoding the BCMA ECD variants were amplified by PCR from the selected phage clones using forward and reverse primers containing AgeI and KpnI sites at the 5' and 3' ends, respectively. The DNA fragments were digested with AgeI and KpnI, gel-purified, and ligated into the pCDNA3.1-PIg18 fusion vector generated above. The plasmids were sequenced to confirm that the DNA sequence of the entire coding sequence for the fusion polypeptide consisting of the CTLA-4 signal peptide, BCMA ECD variants, and PIg18-Fc (mutant IgG2-Fc) was correct.

[0337] The resulting plasmid expression vector contained a nucleic acid fragment encoding a fusion polypeptide consisting of a CTLA-4 signal sequence, a BCMA ECD variant, and a PIg18-Fc region, expression of which was driven by a CMV promoter. A bovine growth hormone (bGH) poly(A) tail signal sequence was located 3' to the DNA encoding the fusion polypeptide. This vector also contained a Bla promoter; an ampicillin resistance gene; a pUC origin of replication; an SV40 polyadenylation (poly(A)) signal sequence; an f1 origin of replication; an SV40 promoter; and a neomycin resistance gene. The signal peptide is typically cleaved during processing; therefore, the secreted BCMA ECD variant-PIg18 fusion protein (i.e., the mature form) typically does not contain the signal peptide sequence. BCMA ECD variant-Ig fusion proteins typically exist in solution as dimeric fusion proteins. In this case, the mature BCMA ECD variant-Ig fusion protein contains a BCMA ECD variant fused at the N- and C-termini of human PIg18-Fc, forming a BCMA ECD variant-Ig fusion protein dimer. The C-terminal lysine of the fusion protein may also be lost as a result of post-translational processing in the host cell. The BCMA ECD variant-Ig fusion protein dimer is the form of the fusion protein molecule used in the assays described in these examples, unless otherwise specified. Example 7 Transient expression of BCMA ECD variant-PIg18 polypeptides in CHO-S cells

[0338] To collect BCMA ECD variant-Ig polypeptides, plasmid expression vectors containing polyn...

Claims

1. A variant B-cell maturation antigen (BCMA) polypeptide comprising a variant cysteine-rich domain (CRD) containing at least one amino acid substitution selected from the following, based on the amino acid positions of SEQ ID NO: 1: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A).

2. a variant B-cell maturation antigen (BCMA) polypeptide comprising a variant cysteine-rich domain (CRD) containing at least one amino acid substitution selected from the following, based on the amino acid positions of SEQ ID NO: 1: (1) histidine, arginine, proline, or asparagine at position 12 (S12H, S12R, S12P, or S12N); (2) isoleucine or valine at position 14 (L14I or L14V); (3) arginine or asparagine at position 15 (H15R or H15N); (4) serine at position 16 (A16S); and (5) valine, isoleucine, or alanine at position 22 (L22V, L22I, or L22A); Immunoglobulin (Ig) Fc polypeptides and A fusion polypeptide comprising:

3. A fusion polypeptide comprising the variant BCMA polypeptide described in claim 1 and an additional polypeptide.

4. A first monomer comprising a variant BCMA polypeptide of claim 1, or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; a second monomer comprising the variant BCMA polypeptide or fusion polypeptide of claim 1; A dimer containing 5. A variant BCMA polypeptide according to claim 1; A fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or a first monomer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; a second monomer comprising the variant BCMA polypeptide or fusion polypeptide of claim 1; a dimer comprising:

10. The variant BCMA polypeptide of claim 1, the fusion polypeptide, or the dimer. , a conjugate comprising:

6. A variant BCMA polypeptide according to claim 1; A fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; a first monomer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; a second monomer comprising the variant BCMA polypeptide or fusion polypeptide of claim 1; or a dimer comprising A variant BCMA polypeptide of claim 1; a fusion polypeptide comprising the variant BCMA polypeptide and an additional polypeptide; or A variant BCMA polypeptide of claim 1; or a first monomer comprising a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; a second monomer comprising the variant BCMA polypeptide or fusion polypeptide of claim 1; a dimer comprising:

10. The variant BCMA polypeptide of claim 1, the fusion polypeptide, or the dimer. A conjugate comprising:

2. The variant BCMA polypeptide, fusion polypeptide, dimer, or conjugate of claim 1, wherein the variant BCMA polypeptide, the fusion polypeptide, the dimer, or the conjugate binds to at least one selected from the group consisting of B-cell activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL) or variants thereof.

7. A variant BCMA polypeptide according to claim 1. a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or At least one selected from the group consisting of a first monomer and a second monomer of a dimer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or the fusion polypeptide. A polynucleotide comprising a nucleotide sequence encoding 8. A variant BCMA polypeptide according to claim 1. a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or At least one selected from the group consisting of a first monomer and a second monomer of a dimer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or the fusion polypeptide. A vector comprising a polynucleotide comprising a nucleotide sequence encoding 9. A variant BCMA polypeptide according to claim 1. a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or At least one selected from the group consisting of a first monomer and a second monomer of a dimer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or the fusion polypeptide. A cell comprising a polynucleotide comprising a nucleotide sequence encoding 10. A variant BCMA polypeptide according to claim 1. a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or At least one selected from the group consisting of a first monomer and a second monomer of a dimer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or the fusion polypeptide. A cell comprising a vector comprising a polynucleotide comprising a nucleotide sequence encoding 11. A method for producing a variant BCMA polypeptide, fusion polypeptide, or dimer, comprising: 1) introducing the polynucleotide of claim 7 into a cell; 2) culturing the host cell under conditions suitable for expression of said polypeptide; 3) recovering or isolating said polypeptide; and optionally 4) purifying the polypeptide A method comprising:

12. A method for producing a variant BCMA polypeptide, fusion polypeptide, or dimer, comprising: 1) introducing the vector according to claim 8 into a cell; 2) culturing the host cell under conditions suitable for expression of said polypeptide; 3) recovering or isolating said polypeptide; and optionally 4) purifying the polypeptide A method comprising:

13. A variant BCMA polypeptide according to claim 1; A fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; A dimer comprising a first monomer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or said fusion polypeptide; A conjugate comprising the variant BCMA polypeptide of claim 1; the fusion polypeptide; or the dimer; and an additional moiety covalently linked to the variant BCMA polypeptide of claim 1, the fusion polypeptide, or the dimer. A variant BCMA polypeptide of claim 1; A fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; or At least one selected from the group consisting of a first monomer comprising the variant BCMA polypeptide of claim 1 or a fusion polypeptide comprising the variant BCMA polypeptide of claim 1 and an additional polypeptide; and a second monomer comprising the variant BCMA polypeptide of claim 1 or the fusion polypeptide. a polynucleotide comprising a nucleotide sequence encoding a vector comprising the polynucleotide; a cell comprising the polynucleotide; or A cell containing the vector A pharmaceutical composition comprising:

14. A pharmaceutical composition for the treatment of a disease or disorder, comprising a variant BCMA polypeptide according to claim 1.

15. The pharmaceutical composition of claim 14, wherein the disease or disorder is a B-cell or antibody-mediated disease or disorder.

16. Use of a variant BCMA polypeptide according to claim 1 in the manufacture of a medicament for the treatment of a disease or disorder.

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