Baff receptor-binding polypeptides, compositions, and methods of use thereof

Abstract

BAFF receptor-binding polypeptides, related compositions, and related methods of use.

Description

Attorney Docket No.: PRG-068WO BAFF RECEPTOR-BINDING POLYPEPTIDES, COMPOSITIONS, AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 697,387, filed on September 20, 2024, the entire contents of which are incorporated by reference herein for all purposes. BACKGROUND

[0002] The B-cell activating factor-receptor (BAFF-R) is a member of the tumor necrosis factor receptor superfamily and is encoded by the TNFRSF13C gene. BAFF-R is one of the main pro-survival receptors in B cells. A homozygous deletion within exon 2 of BAFF-R leads to an almost complete block of B cell development at the stage of immature / transitional B cells. BAFF-R specifically binds to the tumor necrosis family member B-cell activating factor (BAFF; also known as BlyS).

[0003] Overexpression of either BAFF or BAFF-R in mice leads to B-cell hyperplasia and development of systemic auto-immunity with classical features of systemic lupus erythematosus (SLE). BAFF-induced signaling also appears to be involved in survival of malignant B-cells. Therefore, blockade of the BAFF / BAFF-R interaction is being investigated for the treatment of both B-cell related malignancies (e.g., chronic lymphocytic leukemia (CLL)) and autoimmune disorders.

[0004] Currently available treatments for autoimmune diseases and B-cell malignancies are limited by, for example, severe side effects and / or limited therapeutic effects.

[0005] There remains a need for improved therapeutics which target BAFF-R. SUMMARY

[0006] The present disclosure addresses this need with BAFF-R binding proteins, as well as related compositions and methods. In some embodiments, provided binding proteins and compositions exhibit improved therapeutic efficacy and / or half-lives relative to existing therapeutics which target BAFF-R. IPTS / 200129533.1 1Attorney Docket No.: PRG-068WO

[0007] In one aspect, provided are B-cell-activating factor receptor (BAFF-R) binding protein comprising: (a) a heavy chain variable region (VH) comprising (i) a CDR-H1 having an amino acid sequence according to SEQ ID NO: 3, (ii) a CDR-H2 having an amino acid sequence according to SEQ ID NO: 4, and (iii) a CDR-H3 having an amino acid sequence according to SEQ ID NO: 5; (b) a light chain variable region (VL) comprising (i) a CDR-L1 having an amino acid sequence according to SEQ ID NO: 6, (ii) a CDR-L2 having an amino acid sequence according to SEQ ID NO: 7, and (iii) a CDR-L3 having an amino acid sequence according to SEQ ID NO: 8; and (c) an afucosylated Fc region comprising an Fc modification that extends the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the Fc modification.

[0008] In some embodiments, the VH comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 1 and the VL comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 2.

[0009] In one aspect, provided are B-cell-activating factor receptor (BAFF-R) binding proteins which (a) specifically bind to an epitope of BAFF-R, which epitope comprise the sequence of SEQ ID NO: 20, and (b) comprises an afucosylated Fc region comprising Fc modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF- R binding protein that does not comprise the Fc modifications.

[0010] In some embodiments, the BAFF-R binding protein is an antibody or antigen- binding fragment thereof, for example, a human antibody or antigen-binding fragment thereof. In some embodiments, the antigen binding fragment is a Fab, a F(ab′)2, a Fab′, a single-chain Fv (scFv), an Fv fragment, a Fd fragment, or a diabody.

[0011] In some embodiments, the Fc region is an IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region, e.g., a human IgG1 Fc region. In some embodiments, the modifications comprise a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE).

[0012] In one aspect, provided are BAFF-R binding proteins comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) an afucosylated human IgG1 Fc polypeptide (i) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 65, 140 or 251 and (ii) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E IPTS / 200129533.1 2Attorney Docket No.: PRG-068WO (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE).

[0013] In one aspect, provided are BAFF-R binding proteins comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) an afucosylated human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275.

[0014] In some embodiments, the BAFF-R binding protein comprises (i) a first polypeptide comprising the immunoglobulin heavy chain variable domain and the afucosylated human IgG1 Fc polypeptide; and (ii) a second polypeptide comprising the immunoglobulin light chain variable domain.

[0015] In one aspect, provided are BAFF-R antibodies comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) an afucosylated human IgG1 Fc polypeptide (1) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 65, 140 or 251 and (2) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); and (b) two immunoglobulin light chains, each immunoglobulin light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2.

[0016] In one aspect, provided are BAFF-R antibodies comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) an afucosylated human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; and (b) two immunoglobulin light chains, each immunoglobulin light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2.

[0017] In some embodiments, the BAFF-R binding protein or antibody of any one of claims 1-15, further comprising an amino acid mutation of K214R. In some embodiments, the BAFF-R binding protein or antibody of any one of claims 1-16, further comprising an amino acid mutation of D356E. In some embodiments, the BAFF-R binding protein or antibody of any one of claims 1-17, further comprising an amino acid mutation of L358M. In some embodiments, the BAFF-R binding protein or antibody of any one of claims 1-15, further comprising an amino acid mutation of K214R / D356E / L358M. IPTS / 200129533.1 3Attorney Docket No.: PRG-068WO

[0018] In one aspect, provided are BAFF-R binding proteins comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 264-271, and the light chain comprises the amino acid sequence of SEQ ID NO: 272.

[0019] In one aspect, provided are isolated nucleic acids encoding a BAFF-R binding protein or antibody as disclosed herein.

[0020] In one aspect, provided are sets of nucleic acids collectively encoding a BAFF-R binding protein or antibody as disclosed herein.

[0021] In one aspect, provided are expression vectors comprising a nucleic acid encoding a BAFF-R binding protein or antibody as disclosed herein.

[0022] In one aspect, provided are sets of expression vectors which collectively encode a BAFF-R binding protein or antibody as disclosed herein.

[0023] In one aspect, provided are host cells comprising an isolated nucleic acid, a set of nucleic acids, an expression vector, or set of expression vectors as disclosed herein.

[0024] In some embodiments, host cells further comprise a fucosylation-disrupting modification.

[0025] In one aspect, provided are host cells comprising (a) a fucosylation-disrupting modification and (b) an expression vector which encodes, or a set of expression vectors which collectively encode: (1) a BAFF-R binding protein comprising: (a) an immunoglobulin heavy chain variable region (VH) comprising (i) a CDR-H1 having an amino acid sequence according to SEQ ID NO: 3, (ii) a CDR-H2 having an amino acid sequence according to SEQ ID NO: 4, and (iii) a CDR-H3 having an amino acid sequence according to SEQ ID NO: 5; (b) an immunoglobulin light chain variable region (VL) comprising (i) a CDR-L1 having an amino acid sequence according to SEQ ID NO: 6, (ii) a CDR-L2 having an amino acid sequence according to SEQ ID NO: 7, and (iii) a CDR-L3 having an amino acid sequence according to SEQ ID NO: 8; and (c) a Fc region comprising modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the modifications; (2) a BAFF-R binding protein which (a) specifically binds to an epitope of BAFF-R, which epitope comprise the sequence of SEQ ID NO: 20, and (b) comprises an Fc region comprising modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the modifications; (3) a BAFF- R binding protein comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) a human IgG1 Fc polypeptide (i) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 65, 140 or 251 IPTS / 200129533.1 4Attorney Docket No.: PRG-068WO and (ii) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); (4) a BAFF-R binding protein comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) a human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; (5) a BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) a human IgG1 Fc polypeptide (1) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 65, 140 or 251 and (2) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); or (6) a BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) a human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; and (b) two immunoglobulin light chains, each light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2.

[0026] In some embodiments, the fucosylation-disrupting modification is characterized by a deficiency in a fucosyltransferase, e.g.., α-1,6-fucosyltransferase (FUT8).

[0027] In some embodiments, the host cell is a mammalian host cell.

[0028] In one aspect, provided are pharmaceutical compositions comprising a BAFF-R binding protein or antibody as disclosed herein and a pharmaceutically acceptable carrier.

[0029] In one aspect, provided are methods comprising a step of administering to a subject in need thereof an effective amount of a BAFF-R binding protein or antibody or a pharmaceutical composition as disclosed herein. In some embodiments, the subject has an autoimmune or inflammatory disease, for example, a disorder selected from the group consisting of IgA nephropathy, myasthenia gravis, systemic lupus erythematosus, membranous glomerulonephritis, Sjögren syndrome, lupus nephritis, immune thrombocytopenia, acquired (autoimmune) hemolytic anemia, cold agglutinin disease, autoimmune hepatitis, multiple sclerosis, pemphigus vulgaris, and rheumatoid arthritis.

[0030] In some embodiments, the subject has cancer, for example, chronic lymphocytic leukemia. IPTS / 200129533.1 5Attorney Docket No.: PRG-068WO

[0031] In some embodiments, the step of administering comprises systemic administration of the BAFF-R binding protein or antibody, for example, systemic administration comprising intravenous or subcutaneous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGs.1A-1B show the gating strategy to identify CD19+CD20+ B cells (FIG.1A) and the gating strategy used to identify Live, Apoptotic and Dead cells within a given gate (FIG.1B).

[0033] FIGs. 2A-2C show the cytotoxicity results when human peripheral blood mononuclear cells were incubated in the absence (vehicle, 0.1% PBS) or presence of BAFF-R binding proteins. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0034] In various embodiments, provided are BAFF-R binding proteins, compositions thereof, and methods of use thereof. Definitions

[0035] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0036] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

[0037] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0038] As used herein, the terms “about,” “approximately,” and “comparable to,” when used herein in reference to a value, refer to a value that is similar to the referenced value in the context of that referenced value. In general, those skilled in the art, familiar with the IPTS / 200129533.1 6Attorney Docket No.: PRG-068WO context, will appreciate the relevant degree of variance encompassed by “about,” “approximately,” and “comparable to” in that context. For example, in some embodiments, the terms "about," “approximately,” and “comparable to” may encompass a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] As used herein, the term “afucosylated,” when used in reference to an Fc polypeptide or a binding protein which comprises an Fc polypeptide, refers to the absence of fucosylation on the core glycan of the N297 amino acid residue of the Fc polypeptide (wherein position numbering is according to the EU index of Kabat). As used herein, the term “hypofucosylated,” when used in reference to a plurality of proteins (e.g., BAFF-R binding proteins) comprising of Fc polypeptides, refers to a lower percentage of molecules in the plurality comprising extent of fucosylation on the core glycan of the N297 amino acid residue of the Fc polypeptide (wherein position numbering is according to the EU index of Kabat).

[0040] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen- binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (V) and at least three constant regions (CH1, CH2 and CH3). The variable regions determine the binding specificity of the antibody. Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242; and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917). The three CDRs in each variable region (e.g., light chain variable region or heavy chain variable region, with six CDRs total in a typical IPTS / 200129533.1 7Attorney Docket No.: PRG-068WO antibody format), referred to as CDR1, CDR2, and CDR3, collectively contribute to antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0041] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No.5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0042] An “antigen-binding fragment” of an antibody, or “antibody fragment" comprises a portion of an intact antibody, which portion is still capable of antigen binding. In some embodiments, the antibody has a function in addition to that of antigen-binding, and an antigen-binding fragment retains that function. Typically, an antigen-binding fragment comprises the variable region of the antibody. Papain digestion of antibodies produce two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire light chain along with the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab IPTS / 200129533.1 8Attorney Docket No.: PRG-068WO fragments having different antigen-binding activity and that is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH designates a Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0043] As used herein, the term “bivalent,” when used in reference to a binding protein, such as an antibody or an antibody-based binding protein, means that the binding protein is capable of binding two molecules of the antigen to which it specifically binds.

[0044] As used herein, the term “chimeric antibody” refers to an antibody that has a portion of its heavy and / or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass.

[0045] A “complementarity determining region” (abbreviated “CDR”) is a region of hypervariability interspersed within regions that are more conserved, termed “framework regions” (abbreviated “FR”). In some embodiments, the sequences of the framework regions are identical to the framework regions in human germline sequences. In some embodiments, the sequences of the framework regions are modified with respect to the human germline sequence.

[0046] As used herein, the phrase “complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0047] As used herein, the terms “decrease,” “decreased,” “increase,” “increased,” or “reduction,” “reduced,” (e.g., in reference to therapeutic outcomes or effects) have meanings relative to a reference level, as further explained herein. IPTS / 200129533.1 9Attorney Docket No.: PRG-068WO

[0048] As used herein, antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and which typically vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0049] As used herein, the phrases “effective amount” and “therapeutically effective amount” of an agent (e.g., a binding protein or as described herein) are used interchangeably and refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof. An effective amount may vary according to factors such as the type of disease (e.g., disease state, age, sex, and / or weight of the individual, and the ability of a binding protein (or pharmaceutical composition thereof) to elicit a desired response in the individual. An effective amount may also be an amount for which any toxic or detrimental effects of the binding protein or pharmaceutical composition thereof are outweighed by therapeutically beneficial effects.

[0050] As used herein, the term “epitope” is an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule (or binding protein), known as the paratope, and which is comprised of the six complementary- determining regions of the antibody (or binding protein). A single antigen may have more than one epitope. Epitopes may be conformational or linear. A conformational epitope is comprised of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is comprised of adjacent amino acid residues in a polypeptide chain.

[0051] An “Fc chain” of a dimeric Fc as used herein refers to one of the two polypeptides forming the dimeric Fc region, i.e., a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable association with another similar polypeptide. For example, an Fc chain of a dimeric IgG Fc comprises an IgG CH2 and an IgG IPTS / 200129533.1 10Attorney Docket No.: PRG-068WO CH3 constant domain sequence. An Fc chain or a dimeric Fc (“Fc region”) can be of any of a variety of Ig classes, e.g., IgA, IgD, IgE, IgG, or IgM. These classes are also designated α, δ, ε, γ, and μ, respectively. Several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0052] The terms “Fc receptor” and “FcR” are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR can be a native sequence human FcR. Generally, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Immunoglobulins of other isotypes can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed in Daëron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976); and Kim et al., J. Immunol.24:249 (1994)).

[0053] An “functional fragment” of polypeptide comprises a portion of an intact polypeptide, which portion still retains the functions of the intact polypeptide. For example, a functional fragment of a BAFF-R binding protein would retain the functions of an intact BAFF-R binding proteins, e.g., ability to selectively bind BAFF. As another example, a functional fragment of an Fc polypeptide would retain the functions of the intact Fc polypeptide, such as binding to certain Fc receptors such as FcRn. For Fc polypeptides exhibiting altered binding to certain Fc receptors, a functional fragment of such Fc polypeptides would retain the same functions, including the same altered binding to certain Fc receptors. In some embodiments, a functional fragment of a polypeptide represents at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, or at least 97.5% of the sequence of the intact polypeptide. IPTS / 200129533.1 11Attorney Docket No.: PRG-068WO

[0054] As used herein, the term “humanized,” when used in reference to an antibody (or binding protein or binding protein comprising an antibody fragment), refers to a form of a non-human (e.g., murine) antibody that is chimeric. A “humanized antibody” contains minimal sequences derived from non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having a desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence although the framework regions may include one or more amino acid substitutions that improve binding affinity. In some embodiments, no more than six amino acid substitutions in the heavy chain and no more than three amino acid substitutions are used in the light chain in the framework region. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0055] As used herein, the term “linker” is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, those of ordinary skill in the art appreciate that a polypeptide (e.g., binding protein) whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, the linker is an “amino acid linker,” that is, it comprises amino acid residues, e.g., an amino acid linker may comprise at least 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acid residues. In some embodiments, a linker is IPTS / 200129533.1 12Attorney Docket No.: PRG-068WO characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

[0056] “Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0057] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0058] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0059] As used herein, “polypeptide,” which may be used interchangeably with “protein,” refers to a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides can include one or more “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may comprise two or more individual polypeptide chains, which may in some cases be linked to one another, for example by one or more disulfide bonds or other means. IPTS / 200129533.1 13Attorney Docket No.: PRG-068WO

[0060] In some instances, the present disclosure refers to a molecule that is used as a “reference,” such as a “reference binding protein.” Generally, such reference molecules are identical to the molecule against which it is being compared except for a key aspect, e.g., presence or absence of an Fc modification.

[0061] As used herein, the phrase “reference level” generally refers to a level considered “normal” for comparison purposes, e.g., a level of an appropriate control. For example, in the context of half-life (e.g., serum half-life) of a polypeptide (e.g., a binding protein), a reference level may refer to the half-life of a “reference binding protein” as described herein.

[0062] As used herein, the phrase “specifically binds” or “selectively binds” to a target (e.g., BAFF-R), when referring to a binding protein as described herein, refers to a binding reaction by which the binding protein binds to the target with higher affinity, higher avidity and / or or longer duration than it binds to a structurally different target. In typical embodiments, the binding protein has an affinity of at least 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater by a specific target compared to an unrelated target when tested under the same affinity assay conditions. The term “specific binding,” “binds specifically to,” or “is specific to” a particular target, as used herein, may be presented, for example, by a molecule that has an equilibrium dissociation constant Kdfor the target to which it binds, e.g., on the order of 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12M, or less. In some embodiments, a binding protein can specifically bind to an epitope on a target that is conserved between species (e.g., structurally conserved between species), e.g., conserved between human and non-human primate species (e.g., structurally conserved between human and non-human primate species). In some embodiments, a binding protein may bind exclusively to a given target or set of defined targets, e.g., exclusively to BAFF-R but not to other molecules.

[0063] The terms “subject,” “recipient”, “individual”, “host”, and “patient”, are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel. IPTS / 200129533.1 14Attorney Docket No.: PRG-068WO

[0064] As used herein, to “treat” a condition or “treatment” of the condition (e.g., the conditions described herein) is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

[0065] The terms “variable domain” and “variable region” are used interchangeably and refer to the portions of the antibody (or binding protein) or immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody. Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These sub-domains are called “hypervariable regions” or “complementarity determining regions” (CDRs). The more conserved (i.e., non- hypervariable) portions of the variable domains are called the “framework” regions (FRM or FR) and provide a scaffold for the six CDRs in three-dimensional space to form an antigen- binding surface.

[0066] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0067] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls. IPTS / 200129533.1 15Attorney Docket No.: PRG-068WO BAFF-R binding proteins

[0068] In one aspect, provided are binding proteins that are capable of binding to BAFF-R (B-cell activating factor receptor). In some embodiments, provided binding proteins are capable of binding to an epitope of human BAFF-R which comprises the sequence: PTPCVPAECFDLLVRHCVACGLLR (SEQ ID NO: 20).

[0069] In some embodiments, the binding proteins are antibodies or fragments thereof. In some embodiments, the antibodies or fragments thereof are monoclonal antibodies or fragments thereof. In some embodiments, the antibodies or fragments thereof are chimeric antibodies or fragments thereof. In some embodiments, the antibodies or fragments thereof are humanized antibodies or fragments thereof. In some embodiments, the antibodies or antigen-binding fragments are human antibodies.

[0070] Antigen-binding fragments may be, e.g., an scFv, an Fab, an scFab (single-chain Fab). As used herein, the term “scFv” is used in accordance with its common usage in the art to refer to a single chain in which the VH domain and the VL domain from an antibody are joined, typically via a linker. As used herein, the term “Fab fragment” is used in accordance with its common usage in the art. Fab fragments typically comprise an entire light chain (VL and CL1 domains), the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CH1).

[0071] In some embodiments, provided BAFF-R binding proteins comprise a heavy chain variable domain comprises complementarity determining regions CDR-H1, CDR-H2, and CDR-H3 with amino acid sequences as shown in Table 1A. In some embodiments, provided BAFF-R binding proteins further comprise a light chain variable domain comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3 with amino acid sequences as shown in Table 1A.

[0072] In some embodiments, provided BAFF-R binding proteins comprise a heavy chain variable domain with a heavy chain variable sequence as shown in Table 1A, e.g., SEQ ID NO: 1. In some embodiments, provided BAFF-R binding proteins comprise a heavy chain variable domain which is a variant of the heavy chain variable sequence shown in Table 1A, in that the heavy chain variable domain has (1) CDR-H1, CDR-H2, and CDR-H3 with sequences as shown in Table 1A and (2) an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at IPTS / 200129533.1 16Attorney Docket No.: PRG-068WO least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain variable domain sequence shown in Table 1A.

[0073] In some embodiments, provided BAFF-R binding proteins comprise a heavy chain variable domain as described herein and further comprise a light chain variable region which has (1) CDR-L1, CDR-L2, and CDR-L3 with sequences as shown in Table 1A, and (2) an amino acid sequence that is at least 85%, at least 87.5%, 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% identical to the amino acid sequence of the light chain variable domain sequence shown in Table 1A (e.g., SEQ ID NO: 2).

[0074] Without limitation, Table 1B provides some exemplary BAFF-R binding proteins comprising the heavy chain variable domain, light chain variable domain, and complementarity-determining region sequences that are shown in Table 1A. In some embodiments, provided BAFF-R binding proteins comprise a heavy chain with an amino acid sequence as shown in Table 1B. In some embodiments, provided BAFF-R binding proteins comprise a light chain with an amino acid sequence as shown in Table 1B. In some embodiments, a provided BAFF-R binding protein comprises a heavy chain with an amino acid sequence as shown in Table 1B and further comprises a light chain with an amino acid sequences for the same antibody (Ab) shown in Table 1B. Table 1A. Exemplary heavy chain variable domain, light chain variable domain, and complementarity-determining region sequences of BAFF-R binding proteins Heavy chain Light chainIPTS / 200129533.1 17Attorney Docket No.: PRG-068WO CDR-H2:RIYYRSKWYNSYAVSVKS CDR-L2: GSSSRATIPTS / 200129533.1 18Attorney Docket No.: PRG-068WO Table 1B. Exemplary heavy chain and light chain sequences of BAFF-R binding proteins Ab Full Heavy Chain Full Light Chain 1A V L SGPGLVKPS TLSLTCAISGD DIVLT SPATLSLSPGERATLSCRASQIPTS / 200129533.1 19Attorney Docket No.: PRG-068WO EYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGIPTS / 200129533.1 20Attorney Docket No.: PRG-068WO GPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKIPTS / 200129533.1 21Attorney Docket No.: PRG-068WO VSWNSGALTSGVHTFPAVLQSSGLYSL SVTEQDSKDSTYSLSSTLTLSKADYEK SSVVTVPSSSLGTQTYICNVNHKPSNT HKVYACEVTHQGLSSPVTKSFNRGECIPTS / 200129533.1 22Attorney Docket No.: PRG-068WO 1GQVQLQQSGPGLVKPSQTLSLTCAISGD DIVLTQSPATLSLSPGERATLSCRASQ SVSSNSAAWGWIRQSPGRGLEWLGRIY FISSSYLSWYQQKPGQAPRLLIYGSSSFc polypeptides

[0075] Binding proteins suitable for use in accordance with the present disclosure typically comprise an immunoglobulin Fc region. Fc regions typically comprise one or more Fc chains (e.g., Fc polypeptides, such as a first Fc polypeptide and a second Fc polypeptide). An IgG Fc polypeptide typically contains two constant heavy domains (CH2 and CH3) and a hinge region connected to the CH2 domain. Typical Fc regions comprise two Fc polypeptides which dimerize with one another; however, an Fc region may have a single Fc polypeptide or more than two Fc polypeptides, e.g., as may be present in some antibody formats.

[0076] In some embodiments, the binding proteins described herein comprise an IgG1 Fc region (e.g., human IgG1 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG1 Fc. In some embodiments, the wild type IgG1 Fc is a human IgG1 Fc, in which each Fc chain has an amino acid sequence of SEQ ID NO: 21, 65, 140 or 251. In some embodiments, the binding proteins described herein IPTS / 200129533.1 23Attorney Docket No.: PRG-068WO comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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% identical to that of an Fc chain within a wild-type IgG1 Fc, e.g., a polypeptide having an amino acid sequence of SEQ ID NO: 21, 65, 140 or 251. In certain embodiments, human IgG1 Fc regions include an SRDEL (SEQ ID NO: 260) allotype or an SREEM (SEQ ID NO: 261) allotype.

[0077] In some embodiments, the binding proteins described herein comprise an IgG2 Fc region (e.g., human IgG2 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG2 Fc. In some embodiments, the wild type IgG2 Fc is a human IgG2 Fc, in which each Fc chain has an amino acid sequence of SEQ ID NO: 23. In some embodiments, the binding proteins described herein comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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% identical to that of an Fc chain within a wild-type IgG2 Fc, e.g., a polypeptide having an amino acid sequence of SEQ ID NO: 23.

[0078] In some embodiments, the binding proteins described herein comprise an IgG4 Fc region (e.g., human IgG4 Fc region), that is, except for having particular residue(s) at certain positions as noted herein, the Fc region has an amino acid sequence that is substantially similar to that of the Fc region within a wild type IgG4 Fc. In some embodiments, the wild type IgG4 Fc is a human IgG4 Fc, in which each Fc chain has an amino acid sequence of SEQ ID NO: 22. In some embodiments, the binding proteins described herein comprise an Fc region, each Fc chain of which has an amino acid sequence that is at least 85%, at least 87.5%, 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% identical to that of an Fc chain within a wild-type IgG4 Fc, e.g., a polypeptide having an amino acid sequence of SEQ ID NO: 22.

[0079] In some embodiments, the Fc region comprises a means for extending the half-life of the binding protein. Examples of such means include Fc modifications (such as amino acid mutations or sets of amino acid mutations), including certain Fc modifications discussed herein. IPTS / 200129533.1 24Attorney Docket No.: PRG-068WO Fc modifications

[0080] In certain embodiments, Fc regions are modified (e.g., substituted) at one or more amino acid residues.In some embodiments, the Fc region comprises one or more modifications which modify binding to Fc-gamma receptors, e.g., by promoting selective binding, reducing binding, or enhancing binding thereto.

[0081] In certain embodiments, modifications to Fc regions alter the half-life of a molecule (e.g., binding protein) which comprises the Fc region by altering (e.g., enhancing) binding to an Fc receptor such as the neonatal Fc receptor (FcRn.) For example, in some embodiments, the Fc region is modified to enhance the half-life of the molecule (e.g., binding protein) which comprises the Fc region. Non-limiting examples of half-life-enhancing mutations or sets of mutations include, e.g., M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE).

[0082] In some embodiments, modifications to Fc regions prevent Fab arm (e.g., IgG4 Fab arm) exchange. An example of such a modification in the context of an IgG4 Fc region is the S228P mutation.

[0083] In some embodiments, modifications to Fc regions reduce or abrogate effector functions, e.g., Fcγ receptor-mediated effector functions. Non-limiting examples of effector- reducing mutations or sets of mutations include, e.g., aglycosylation mutations (e.g., N297A or N297Q or N297G), L234A / L235A (for IgG1 Fc regions), H268Q / V309L / A330S / P331S (for IgG2 Fc regions), and V234A / G237A / P238S / H268A / V309L / A330S / P331S (for IgG2 Fc regions). In some embodiments, effector function is reduced by modifying the attached sugar structures. Non-limiting examples of effector-reducing sugar modifications include increasing sialylation or galactosylation of the sugar chains.

[0084] In some embodiments, modifications to Fc regions enhance Fcγ receptor-mediated effector functions. Non-limiting examples of FcγRIIIa effector-enhancing mutations or sets of mutations include, e.g., F243L / R292P / Y300L / V305I / P396L, S298A / E333A / K334A, S239D / I332E, S239D / I332E / A330L, and L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one Fc chain and D270E / K326D / A330M / K334E in another Fc chain. In some embodiments, modification of the sugar chains (e.g., reduction or removal of fucosylation) can increase the affinity to FcγRIIIa resulting in enhanced ADCC activity. IPTS / 200129533.1 25Attorney Docket No.: PRG-068WO

[0085] In some embodiments, modifications to Fc regions enhance antibody-dependent cellular phagocytosis (ADCP). A non-limiting example of an ADCP-enhancing set of mutations is G236A / S239D / I332E (ADE). In some embodiments, modification of the structures of the sugar chains (e.g., reduction or removal of fucosylation) can result in increased ADCP activity.

[0086] In some embodiments, modifications to Fc regions enhance complement- dependent cytotoxicity (CDC) and / or binding to complement protein C1q. Non-limiting examples of an CDC-enhancing and / or C1q-binding-enhancing sets of mutations include, e.g., K326W / E333S, S267E / H268F / S324T, and E345R / E430G / S440Y. In some embodiments, modification of the structures of the sugar chains (e.g., reduction or removal of fucosylation) can result in increased CDC activity.

[0087] In some embodiments, modifications to Fc regions enhance co-engagement with antigens and Fcγ receptors. Non-limiting examples of co-engagement-enhancing sets of mutations include, e.g., S267E / L328F and N325S / L328F.

[0088] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0089] Amino acid sequences of exemplary Fc polypeptide sequences are provided in Tables 2A and 2B. Functional fragments (e.g., fragments which contain at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, or at least 97.5% and retain the function of) of the sequences shown in Tables 2A and 2B may also be used in accordance with the polypeptides (e.g., BAFF-R binding proteins), compositions, and methods disclosed herein.

[0090] In some embodiments, provided BAFF-R binding proteins comprise an Fc polypeptide having an amino acid sequence of SEQ ID NO: 252, 253, 254, 255, 256, 259 or 275. Table 2A. Exemplary Fc Sequences Name SEQ Fc chain sequence T VIPTS / 200129533.1 26Attorney Docket No.: PRG-068WO DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV P D G T V C V Q G T V V V E S T V C V Q G T V C V Q G T V E V P D G T V E VIPTS / 200129533.1Attorney Docket No.: PRG-068WO LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V EIPTS / 200129533.1 8Attorney Docket No.: PRG-068WO VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1Attorney Docket No.: PRG-068WO N434S)PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 30Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 31Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG T V E V P D G T V C V Q G T V C V Q G T V C V Q G T V C V Q G T V C V QIPTS / 200129533.1 32Attorney Docket No.: PRG-068WO EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG T V C V Q G T V C V Q G T V C V Q G T V C V Q G T V V V E S T V V V EIPTS / 200129533.1 33Attorney Docket No.: PRG-068WO EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG T V V D E S T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 34Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 35Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 36Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHWHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E VIPTS / 200129533.1 37Attorney Docket No.: PRG-068WO LQVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 38Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 39Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 40Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E VIPTS / 200129533.1 41Attorney Docket No.: PRG-068WO LQVLHVDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLD G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 42Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V C V Q G T V C V QIPTS / 200129533.1 43Attorney Docket No.: PRG-068WO EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLG T V C V Q G T V V V E S T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 44Attorney Docket No.: PRG-068WO PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 45Attorney Docket No.: PRG-068WO PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPG T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V P D G T V E V PIPTS / 200129533.1 46Attorney Docket No.: PRG-068WO 9D / I332E) PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD ADE SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGTable 2B. Further Exemplary Fc sequences Name SEQ Fc chain sequence ID A S M N Q P L A S T R P Y Y A S R Y E N H A S R Y E NIPTS / 200129533.1 47Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID H A S R Y E N H A S M A Q P L A S M N Q P L A S M N Q P L A S M N QIPTS / 200129533.1 8Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID P L A S M N Q P L A S M N Q P L A S Y N Q P L A S Y A QIPTS / 200129533.1 49Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID P L A S Y N Q P L A S Y N Q P L A S Y N Q P L A S Y N Q P L A S Y NIPTS / 200129533.1 50Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID Q P L A S M N Q P L A S M A Q P L A S M N Q P L A S M N Q P L A S MIPTS / 200129533.1 51Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M A Q PIPTS / 200129533.1 52Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N QIPTS / 200129533.1 53Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID P L A S R Y E N H A S R Y E N H A S R Y E N H A S R Y E N H A S R YIPTS / 200129533.1 54Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID E N H A S R Y E N H A S R Y E N H A S R Y E N H A S R Y E N H A S EIPTS / 200129533.1 55Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID R P Y Y A S T R P Y Y A S T R P Y Y A S M N Q P L A S M A Q P L A SIPTS / 200129533.1 56Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID M N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q P LIPTS / 200129533.1 57Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID A S M N Q P L A S M A Q P L A S M N Q P L A S M N Q P L A S M N Q PIPTS / 200129533.1 58Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID L A S M N Q P L A S M N Q P L A S M N Q P L A S M A Q P L A S M N QIPTS / 200129533.1 59Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M NIPTS / 200129533.1 60Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID Q P L A S M A Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S MIPTS / 200129533.1 61Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID N Q P L A S M N Q P L A S M N Q P L A S M A Q P L A S M N Q P L A SIPTS / 200129533.1 62Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID M N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S Y N Q P LIPTS / 200129533.1 63Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID A S Y A Q P L A S Y N Q P L A S Y N Q P L A S Y N Q P L A S Y N Q PIPTS / 200129533.1 64Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID L A S Y N Q P L A S M N Q P L A S M A Q P L A S M N Q P L A S M N QIPTS / 200129533.1 65Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q P LIPTS / 200129533.1 66Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID A S M A Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q PIPTS / 200129533.1 67Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID L A S M N Q P L A S M N Q P L A S R Y E N H A S R Y E N H A S R Y EIPTS / 200129533.1 68Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID N H A S T R P Y Y A S M A Q P L A S M N Q P L A S M N Q P L A S M NIPTS / 200129533.1 69Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S YIPTS / 200129533.1 70Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID N Q P L A S M N Q P L A S M N Q P L A S M N Q P L A S M A Q P LIPTS / 200129533.1 71Attorney Docket No.: PRG-068WO Name SEQ Fc chain sequence ID A S Y N Q P L A S M N Q P L A S M N Q P L

[0091] In some embodiments, the binding protein comprises an Fc region comprising one or more modifications in SEQ ID NO: 21 (hIgG1). In some embodiments, the binding protein comprises an Fc region comprising one or more modifications in SEQ ID NO: 23 (hIgG2). In some embodiments, the binding protein comprises an Fc region comprising one or more modifications in SEQ ID NO: 22 (hIgG4). In some embodiments, the Fc region comprises an Fc polypeptide whose amino acid sequence has 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to any one of SEQ IPTS / 200129533.1 72Attorney Docket No.: PRG-068WO ID NOs: 21-23. In some embodiments, the Fc region comprises an amino acid sequence according to any one of SEQ ID NOs: 21-23.

[0092] In certain embodiments, the binding protein comprises an Fc region (“modified Fc region”) with at least one amino acid modification relative to a wild type Fc region. Binding proteins with a modified Fc region may comprise mutations in one Fc polypeptide, or in multiple Fc polypeptide (e.g., when two or more Fc polypeptide are present). Within a binding protein with modifications in multiple Fc polypeptide, the modifications on each Fc polypeptide may be the same or different. In some embodiments the modified Fc region comprises a half-life extending mutation or set of mutations, e.g., M252Y, S254T, and T256E (YTE) and / or M428L and N434S (LS).

[0093] In some embodiments, the modified Fc region comprises a modification selected from the group consisting of: S298A, E333A, K334A, K326A, F243L, R292P, Y300L, V305I, P396L, F243L, R292P, Y300L, L235V, P396L, F243L, S239D, I332E, A330L, S267E, L328F, D265S, S239E, K326A, A327H, G237F, K326E, G236A, D270L, H268D, S324T, L234F, N325L, V266L, S267D, K214R, D356E, L358M, and combinations thereof. In some embodiments, the modified Fc region comprises a modification selected from the group consisting of S228P, M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, N434W, and combinations thereof.

[0094] In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of: L234A / L235A; V234A / G237A; L235A / G237A / E318A; S228P / L236E; H268Q / V309L / A330S / A331S; C220S / C226S / C229S / P238S; C226S / C229S / E3233P / L235V / L235A; L234F / L235E / P331S; C226S / P230S; L234A / G237A; L234A / L235A / G237A; Q311R / M428L; L234A / L235A / P329G; K214R / D356E / L358M; and combinations thereof.

[0095] In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of M428L / N434S (LS); M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV); T256D / H285D / T307R / Q311V / A378V (DDRVV); L309D / Q311H / N434S (DHS); S228P / L235E (SPLE); L234A / L235A (LALA); M428L / N434A (LA); L234A / G237A (LAGA); L234A / L235A / G237A (LALAGA); L234A / L235A / P329G (LALAPG); IPTS / 200129533.1 73Attorney Docket No.: PRG-068WO H433K / N434F (KF); N297A / YTE; D265A / YTE; LALA / YTE; LAGA / YTE; LALAGA / YTE; LALAPG / YTE; N297A / LS; D265A / LS; LALA / LS; LAGA / LS; LALAGA / LS; LALAPG / LS; N297A / DHS; D265A / DHS; LALA / DHS; LAGA / DHS; LALAGA / DHS; LALAPG / DHS; SP / YTE; SPLE / YTE; SP / LS; SPLE / LS; SP / DHS; SPLE / DHS; N297A / LA; D265A / LA; LALA / LA; LAGA / LA; LALAGA / LA; LALAPG / LA; N297A / N434A; D265A / N434A; LALA / N434A; LAGA / N434A; LALAGA / N434A; LALAPG / N434A; N297A / N434W; D265A / N434W; LALA / N434W; LAGA / N434W; LALAGA / N434W; LALAPG / N434W; N297A / DQ; D265A / DQ; LALA / DQ; LAGA / DQ; LALAGA / DQ; LALAPG / DQ; N297A / DW; D265A / DW; LALA / DW; LAGA / DW; LALAGA / DW; LALAPG / DW; N297A / YD; D265A / YD; LALA / YD; LAGA / YD; LALAGA / YD; LALAPG / YD; N297A / QVV; D265A / QVV; LALA / QVV; LAGA / QVV, LALAGA / QVV; LALAPG / QVV; N297A / DDRVV; D265A / DDRVV; LALA / DDRVV; LAGA / DDRVV; LALAGA / DDRVV; LALAPG / DDRVV; SP / Q311R / M428L; SPLE / Q311R / M428L; N297A / Q311R / M428L; D265A / Q311R / M428L; LALA / Q311R / M428L; LAGA / Q311R / M428L; LALAGA / Q311R / M428L; LALAPG / Q311R / M428L; and combinations thereof. In some embodiments, the modified Fc region comprises a specific combination of amino acid substitutions selected from the group consisting of M428L / N434S (LS), M252Y / S254T / T256E (YTE), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE). In some embodiments, the modified Fc region comprises M428L / N434S (LS) (e.g., SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 56, SEQ ID NO: 63, SEQ ID NO: 133, SEQ ID NO: 175, or SEQ ID NO: 182) modifications. In some embodiments, the modified Fc region comprises M252Y / S254T / T256E (YTE) (e.g., SEQ ID NO: 32, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 134, SEQ ID NO: 151, SEQ ID NO: 172, SEQ ID NO: 181, or SEQ ID NO: 253) modifications.

[0096] In some embodiments, provided binding proteins include modifications to improve their ability to mediate effector function. Such modifications are known in the art and include afucosylation, or engineering of the affinity of the Fc region towards an activating receptor, mainly FcγRIIIa for antibody-dependent cellular cytotoxicity (ADCC), and towards C1q for complement-dependent cytotoxicity (CDC).

[0097] In some aspects, provided binding proteins comprise an Fc region (e.g., an IgG1 Fc region) with reduced fucose content at position Asn 297 (EU numbering) compared to a naturally occurring Fc region. Such Fc regions are known to confer improved ADCC activity IPTS / 200129533.1 74Attorney Docket No.: PRG-068WO to the binding proteins which comprise them. In some aspects, such binding proteins do not comprise any fucose at position Asn 297.

[0098] In some embodiments, provided binding proteins include modifications to decrease their ability to mediate effector function. Such modifications are known in the art and include increased sialylation, or decreasing the affinity of the Fc region towards an activating receptor, mainly FCGR3a for antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, an antibody provided herein comprises an Fc region (e.g., an IgG1 Fc region) with increased sialyl content at position Asn 297 (EU numbering) compared to a naturally occurring Fc region.

[0099] In some embodiments, the binding protein comprises an Fc region with one or more amino acid substitutions which improve ADCC, such as a substitution at one or more of positions 298, 333, and 334 of an Fc polypeptide. In some embodiments, a binding protein provided herein comprises an Fc region with one or more amino acid substitutions at positions 239, 332, and 330.

[0100] In some embodiments, the binding protein further comprises an Fc region comprising a human IgG sequence selected from the group consisting of any one of SEQ ID NOs: 21-259 and 273-275. In some embodiments, the Fc region comprises an Fc polypeptide whose amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 21-259 and 273-275.

[0101] In some embodiments, the binding protein further comprises an Fc region comprising a human IgG sequence which lacks the C-terminal G446 (according to Kabat) of any of SEQ ID NO: 21-139, but is otherwise identical to one of the aforesaid sequences.

[0102] Although a C-terminal lysine may be present in the corresponding coding sequence of the constant heavy chain region (e.g., in a sequence such as SEQ ID NO: 158), it may be cleaved off during manufacture or after administration (resulting in, e.g., a constant heavy chain sequence such as SEQ ID NO: 39). Accordingly, any of the binding proteins described above may comprise a human IgG sequence containing a C-terminal lysine, a human IgG sequence lacking a C-terminal lysine, or a mixture thereof (e.g., a mixture of the same heavy chain constant sequence with and without a C-terminal lysine). IPTS / 200129533.1 75Attorney Docket No.: PRG-068WO

[0103] In some embodiments, the binding protein comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function.

[0104] In some embodiments, the binding protein comprises one or more alterations that improve or diminish C1q binding and / or CDC.

[0105] In certain embodiments, the binding protein comprises an Fc region with one or more amino acid substitutions, wherein the one or more substitutions result in an increase in one or more of antibody half-life, ADCC activity, ADCP activity, or CDC activity compared with a comparable binding protein whose Fc region lacks the one or more substitutions. In certain embodiments, the one or more amino acid substitutions results in increased binding protein half-life at pH 6.0 compared to a binding protein comprising a wild-type Fc region. In certain embodiments, the binding protein has an increased half-life that is about 10,000-fold, 1,000-fold, 500-fold, 100-fold, 50-fold, 20-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4.5-fold, 4-fold, 3.5-fold, 3-fold, 2.5-fold, 2-fold, 1.95-fold, 1.9-fold, 1.85-fold, 1.8-fold, 1.75-fold, 1.7-fold, 1.65-fold, 1.6-fold, 1.55-fold, 1.50-fold, 1.45-fold, 1.4-fold, 1.35-fold, 1.3-fold, 1.25-fold, 1.2-fold, 1.15-fold, 1.1-fold, or 1.05-fold longer compared to a binding protein comprising a wild-type Fc region.

[0106] In certain embodiments, the binding protein comprises an Fc region which comprise one or more amino acid substitutions, wherein the one or more substitutions result in a decrease in one or more of ADCC activity, ADCP activity, or CDC activity compared to a comparable binding protein whose Fc region lacks the one or more substitutions.

[0107] In certain embodiments, the Fc region binds an Fcγ Receptor selected from the group consisting of: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In certain embodiments, the Fc region binds an Fcγ Receptor with higher affinity at pH 6.0 compared to an antibody comprising a wild-type Fc region.

[0108] In some embodiments, the binding protein has an extended half-life (i.e., serum half-life) relative to the half-life of a reference binding protein. In some embodiments, the binding protein has a half-life of at least about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks. In some embodiments, the binding protein has a half-life of more than 52 weeks. In some embodiments, the binding protein has a half-life of at least about 14, 28, 42, 56, 70, 84, 96, or more than 96 days. In some embodiments, the binding protein has a half-life in a range of about 14 days to about 96 days, about 14 days to about 84 days, about 14 days to IPTS / 200129533.1 76Attorney Docket No.: PRG-068WO about 70 days, about 14 days to about 56 days, about 14 days to about 42 days, about 14 days to about 28 days, of about 28 days to about 96 days, about 28 days to about 84 days, about 28 days to about 70 days, about 28 days to about 56 days, about 28 days to about 42 days, of about 42 days to about 96 days, about 42 days to about 84 days, about 42 days to about 70 days, or about 42 days to about 56 days. In some embodiments, the binding protein has a half-life of at least about 50 days, at least about 55 days, at least about 60 days, at least about 65 days, at least about 70 days, at least about 75 days, at least about 80 days, at least about 85 days, or at least about 90 days. In some embodiments, the binding protein has a half-life of about 50 days, about 55 days, about 60 days, about 65 days, about 70 days, about 75 days, about 80 days, about 85 days, or about 90 days. Methods of measuring half-life are known in the art. In some embodiments, the half-life is measured in a non-human primate. In some embodiments, the half-life is measured in a human. In some embodiments, the half-life is measured following intravenous administration. In some embodiments, the half-life is measured following subcutaneous administration.

[0109] In some embodiments, provided binding proteins have a half-life that is at least 20% longer than a comparator antibody. In some embodiments, the comparator antibody comprises the same complementarity determining regions and variable regions but different Fc regions, or a comparator antibody which is bivalent and monospecific but has the same complementarity determining regions and variable regions. In some embodiments, the half- life of provided binding proteins is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than the half-life of the comparator antibody. In some embodiments, the half-life of provided binding proteins is longer than the half-life of the comparator antibody by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold. Afucosylation

[0110] Afucosylation of an Fc polypeptide (e.g., human IgG1 Fc polypeptide) may be achieved by any of a variety of means. For example, engineered host cells with fucosylation- disrupting modifications as described herein may be used to produce binding proteins having afucosylated Fc polypeptides. Alternatively or additionally, the Fc polypeptide may comprise a mutation at a residue which would otherwise typically comprise fucosylation, e.g., N297 (e.g., N297A or N297Q or N297G) mutations for IgG1 Fc polypeptides. Yet other methods of obtaining binding proteins having afucosylated Fc polypeptides include culturing host cells IPTS / 200129533.1 77Attorney Docket No.: PRG-068WO under conditions which prevent or reduce fucosylation (e.g., using biochemical inhibitors which disrupt fucosylation), post-translational removal of fucose (e.g., with a fucosidase enzyme), post-translational addition of a desired carbohydrate which lacks fucosylation (e.g., after recombinant expression of a non-glycosylated glycoprotein), purification of the glycoprotein so as to select for product which is not fucosylated, and any combination of these methods and other methods described herein. Linkers

[0111] In some embodiments, a linker may be present between various portions of a polypeptide, e.g., between the BAFF-R binding portion of a BAFF-R binding protein and the Fc polypeptide and / or, e.g., for polypeptides comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) in a single chain (e.g., an scFv), between the VHand the VL. In some embodiments, the linker comprises an amino acid linker.

[0112] For example, a linker as employed herein may comprise from about 1 to about 100 amino acid residues, e.g., about 1 to about 70, about 2 to about 70, about 1 to about 30, or about 2 to about 30 amino acid residues. In some embodiments, the linker comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues.

[0113] In certain embodiments, the linker comprises a glycine-serine sequence, e.g., a (GnS)msequence (e.g., GGS, GGGS (SEQ ID NO: 262), and / or GGGGS (SEQ ID NO: 263) sequence) that is present in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 copies within the linker. Dimerization

[0114] In certain embodiments, provided are BAFF-R binding proteins comprising more than one polypeptide chain (e.g., at least a first polypeptide and a second polypeptide). Thus, some provided polypeptides or polypeptide complexes comprise more than one Fc polypeptide (e.g., at least a first Fc polypeptide and a second Fc polypeptide).

[0115] In some such embodiments, the first Fc polypeptide and the second Fc polypeptide have identical amino acid sequences. IPTS / 200129533.1 78Attorney Docket No.: PRG-068WO

[0116] In some such embodiments, the first Fc polypeptide and the second Fc polypeptide do not have identical amino acid sequences. In some embodiments, the first and second Fc polypeptides comprise one or more features, e.g., in their respective CH3 domains, that promote formation of a heterodimer (e.g., of the first and second Fc polypeptide) over a homodimer (e.g., a homodimer of two first Fc polypeptides or a homodimer of two second Fc polypeptides). Nucleic acids

[0117] Also provide are nucleic acids including expression vectors which encode a BAFF- R binding protein (as disclosed herein) or a component thereof. For example, relevant to BAFF-R binding proteins which comprise more than one polypeptide chain, a set of nucleic acids or set of expression vectors is provided in which each nucleic acid or expression vector encodes one of the polypeptide chains of the BAFF-R binding protein, so that the set of nucleic acids or set of expression vectors collectively encode the BAFF-R binding protein.

[0118] Provided nucleic acids or expression vectors may comprise additional components to facilitate replication and expression in relevant host cells, e.g., promoters, enhancers, etc. See, e.g., the Methods of Preparation section of the present disclosure. Host Cells

[0119] A wide variety of types of host cells may be used in accordance with the present disclosure. Exemplary types of host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein.

[0120] In some embodiments, provided BAFF-R binding proteins are produced using engineered host cells with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells to produce BAFF-R binding proteins having altered glycosylation (e.g., altered fucosylation such as afucosylation).

[0121] In one aspect, also provided are host cells which comprise nucleic acids or sets of nucleic acids of the present disclosure which encode BAFF-R binding proteins of the present disclosure. IPTS / 200129533.1 79Attorney Docket No.: PRG-068WO

[0122] In some embodiments, the host cell comprises a fucosylation-disrupting modification, e.g., a deficiency in a fucosyltransferase. Such host cells can be used to produce binding proteins which are hypofucosylated or afucosylated. Examples of fucosyltransferases include, e.g., α-l,6-fucosyltransferase (encoded by the FUT8 gene), FucTI, FucTII, FucTIII, FucTIV, FucTV, FucTVI and FucTVII. In some embodiments, the host cell comprises a deficiency in α-l,6-fucosyltransferase, e.g., by having a functionally disrupted FUT8 gene. (See, e.g., US Pat. No.7,214,775.)

[0123] In some embodiments, the host cell comprises a deficiency in the GDP-mannose- dependent de novo pathway which produces fucosyltransferases. For example, a deficiency in the enzyme GDP-Mannose 4, 6-Dehydratase (a part of this pathway) also results in a disruption in fucosylation. (See, e.g., International Patent Publication WO03 / 035835 (describing Lecl3 cells (a variant CHO cell line with reduced ability to attach fucose to Asn(297)-linked carbohydrates); Shields, R. L. et al., “Lack of fucose on human IgG1 N- linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity,” 2002 J. Biol. Chem.277:26733-26740; and Ohyama C et al. “Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells,” J Biol Chem.1998 Jun 5;273(23):14582-7) Alternatively or additionally, expression of the bacterial GDP-6-deoxy-D-lyxo-4-hexulose reductase in the cytosol of mammalian cells (e.g., Chinese Hamster Ovary cells) can be used to disrupt this pathway.

[0124] In some embodiments, the host cell comprises a modification that results in increased complex N-linked oligosaccharides carrying bisecting GlcNac structures in binding proteins produced from the host cell. See, e.g., International Patent Publication WO 99 / 54342 by Umana et al., which describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)). Proteins expressed from these engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies.

[0125] In some embodiments, the host cells comprise a deficiency in a transporter gene involved in fucosylation, e.g., the SLC35C1 gene.

[0126] In some embodiments, the host cells are yeast cells or filamentous fungi engineered for mammalian-like glycosylation pattern and capable of producing antibodies IPTS / 200129533.1 80Attorney Docket No.: PRG-068WO lacking fucose as glycosylation pattern. (See, e.g., International Patent Publication WO2003056914A1.) Pharmaceutical Compositions

[0127] The present disclosure also includes pharmaceutical compositions that contain therapeutically effective amounts of the BAFF-R binding proteins disclosed herein. Compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527- 1533, 1990).

[0128] In some embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); IPTS / 200129533.1 81Attorney Docket No.: PRG-068WO delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0129] In some embodiments, provided pharmaceutical compositions are citrate-free.

[0130] In some embodiments, provided pharmaceutical composition contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles.

[0131] In some embodiments, provided pharmaceutical compositions contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(−)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0132] Pharmaceutical compositions containing a binding protein disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration, as discussed further herein in the “Methods of Treatment” section.

[0133] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some embodiments, the formulation for parenteral administration is citrate-free.

[0134] For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. IPTS / 200129533.1 82Attorney Docket No.: PRG-068WO

[0135] An intravenous or subcutaneous drug delivery formulation may be contained in a syringe, pen, or bag. In some embodiments, the bag is connected to a channel comprising a tube and / or a needle. In some embodiments, the formulation is a lyophilized formulation or a liquid formulation.

[0136] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.

[0137] A polyol, which acts as a tonicifier and may stabilize the binding protein, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In some embodiments, the aqueous formulation is isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) is added, compared to a disaccharide (such as trehalose). In some embodiments, the polyol which is used in the formulation as a tonicity agent is mannitol.

[0138] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In some embodiments, the formulation may include a surfactant which is a polysorbate. In some embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996).

[0139] In some embodiments, the binding protein is formulated as a liquid formulation. In some embodiments, the liquid formulation is prepared in combination with a sugar at stabilizing levels. In some embodiments, the liquid formulation is prepared in an aqueous carrier. In some embodiments, a stabilizer is added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugar is disaccharides, e.g., sucrose. In some embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0140] In some embodiments, the pH of the liquid formulation is set by addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the base is sodium hydroxide. IPTS / 200129533.1 83Attorney Docket No.: PRG-068WO

[0141] The aqueous carrier of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0142] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0143] The binding protein may be lyophilized to produce a lyophilized formulation including the proteins and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In some embodiments, the lyoprotectant is sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative.

[0144] The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In some embodiments, the protein to sucrose or maltose weight ratio is of from 1:2 to 1:5. In some embodiments, the pH of the formulation, prior to lyophilization, is set by addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the pharmaceutically acceptable base is sodium hydroxide. Methods of Preparation

[0145] BAFF-R binding proteins described herein can be made using recombinant DNA technology well known to a skilled person in the art. For example, one or more isolated polynucleotides (such as nucleic acids described herein) encoding the BAFF-R binding protein can be ligated to other appropriate nucleotide sequences, including, for example, constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired BAFF-R binding proteins. Production of defined gene constructs is within routine skill in the art.

[0146] Nucleic acids encoding desired BAFF-R binding proteins can be incorporated (ligated) into expression vectors, which can be introduced into host cells (such as those described herein) through conventional transfection or transformation techniques. IPTS / 200129533.1 84Attorney Docket No.: PRG-068WO Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode BAFF-R binding proteins.

[0147] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is typically first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. The expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication. The refractile bodies can then be solubilized, and the protein may be refolded and / or cleaved by methods known in the art.

[0148] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is typically first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers and introns. In embodiments involving polypeptides comprising a BAFF-R binding protein or portion thereof, the expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0149] In some embodiments, in order to express a BAFF-R binding protein, an N- terminal signal sequence is included in the protein construct. Exemplary N-terminal signal sequences include signal sequences from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0150] After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix. Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the BAFF-R binding proteins.

[0151] The BAFF-R binding proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze- thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. Methods of Treatment IPTS / 200129533.1 85Attorney Docket No.: PRG-068WO

[0152] Described herein, in certain embodiments, are methods of treating a subject in need thereof, the method comprising a step of administering to the subject an effective amount of a BAFF-R binding protein or a pharmaceutical composition as disclosed herein. Subjects

[0153] In certain embodiments, the subject is a mammal, such as a primate. In some embodiments, the subject is human.

[0154] In some embodiments, the subject suffers from, exhibits at least one symptom of, is diagnosed with, and / or is identified as at risk of an autoimmune or inflammatory disease. Examples of such autoimmune or inflammatory diseases include, but are not limited to, IgA nephropathy, myasthenia gravis, systemic lupus erythematosus, membranous glomerulonephritis, Sjögren syndrome, lupus nephritis, immune thrombocytopenia, acquired (autoimmune) hemolytic anemia, cold agglutinin disease, autoimmune hepatitis, multiple sclerosis, pemphigus vulgaris, and rheumatoid arthritis.

[0155] In some embodiments, the subject suffers from, exhibits at least one symptom of, is diagnosed with, and / or is identified as at risk of cancer, for example, a B-cell cancer, such as chronic lymphocytic leukemia (CLL). Routes of administration

[0156] In certain embodiments, the step of administering comprises systemic administration. In certain embodiments, systemic administration comprises parenteral administration, e.g., intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, or intradermal administration. In some embodiments, systemic administration comprises enteric administration, e.g., trans- gastroenteric administration or oral administration.

[0157] In some embodiments, the step of administering comprises intravenous administration. In some embodiments, the step of administering comprises subcutaneous administration. Outcomes

[0158] In many embodiments, methods disclosed herein result in a measurable improvement in the subject, e.g., in amelioration or resolution of symptoms. For example, such improvement may include an improvement in a clinical score or a score from a survey or questionnaire associated with, or suitable for assessing the autoimmune or inflammatory disease being treated. IPTS / 200129533.1 86Attorney Docket No.: PRG-068WO EXAMPLES Example 1. Generation, production, and purification of BAFF-R binding proteins

[0159] This Example describes the generation, production, and purification of BAFF-R binding proteins Antibody 1A, Antibody 1B, Antibody 1C, Antibody 1D, Antibody 1E, Antibody 1F, Antibody 1G, Antibody 1I, and Antibody 1J. Tables 1A and 1B describe characteristic sequences for these clones.

[0160] The coding sequences for immunoglobulin heavy and light chains of BAFF-R binding proteins were generated by DNA synthesis and PCR and subsequently subcloned into plasmids for mammalian cell expression. Gene sequences in the expression vectors were confirmed by DNA sequencing. Transient expression of binding proteins was performed by co-transfection of paired immunoglobulin heavy chain and light chain constructs into Chinese Hamster Ovary (CHO) cells using a polyethylenimine (PEI)-based method. Briefly, a fucosyltransferase 8 (Fut8) knockout CHO cell line was used to generate afucosylated antibodies (Antibodies 1B-1G) or a CHO-K1 cell line was used to generate fucosylated antibodies (Antibodies 1A, 1I, and 1J). The respective CHO cell line was added to a shake flask at approximately 5.5x106cells / mL. Transfection was initiated by adding a mixture of 1 mg / L DNA and 7 mg / L PEI in OptiMEMTM medium (Invitrogen) to the cells followed by gentle mixing. Cells were then cultured in an incubator shaker at 120 rpm, 37 °C, and 8% CO2 for about 9 days. Feeding with peptone and glucose was carried out 24 h later and every 2-3 days thereafter depending on the cell density and viability. The cell culture was terminated on or around day 9 when cell viability reduced to <80%. Conditioned medium was harvested for protein purification. Affinity chromatography and size exclusion chromatography were performed using an AKTA instrument (Cytiva). Conditioned medium expressing binding proteins was harvested by centrifugation at 4000 rpm for 50 min and filtered with a 0.22 µm filter. Harvested supernatants were loaded onto a column of MabSelectTM SuReTM (Cytiva) resin. After washing the column with Buffer A (PBS, PH 7.4), protein was eluted with Buffer B (1 M Glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of Buffer C (1 M sodium citrate, pH 6.0). Affinity purified antibodies were further purified and buffer exchanged into 20 mM His-HAc, 150 mM NaCl, pH 5.5 by size exclusion chromatography using a Superdex® 200 Increase 5 / 150GL column. IPTS / 200129533.1 87Attorney Docket No.: PRG-068WO Example 2. Determination of binding affinities to Fc receptors

[0161] Binding affinities (KD) of provided BAFF-R binding proteins to various Fc receptors was determined through surface plasmon resonance (SPR) using a BIACORE™ 8K SPR system (Cytiva). Tested BAFF-R binding proteins include isotype control, Antibody 1A, Antibody 1B, Antibody 1C, Antibody 1D, Antibody 1E, Antibody 1F, and Antibody 1G.

[0162] Antibody was immobilized onto a CM5 chip (Cytiva) using an anti-Fab capture antibody. Chips were prepared to achieve a loading level of 800-1500 RUs. 6-point dilution series of each protein was prepared and run over captured antibody to determine the affinity of each interaction.

[0163] Cyno neonatal Fc receptor FcRn (pH 5.8 & pH 7.4), human FcγRIIa 167H, human FcγRIIa 167R, human FcγRIIb, human FcγRIIIa 176V, human FcγRIIIa 176F, and cyno FcγRIIIb were analyzed using steady state analysis of each sample replicate where a report point was taken at the plateau of each injection response curve. After reference and blank subtractions, an assessment of affinity was made using a non-linear curve fit model. Human FcγRI and human FcRn (pH 5.8 & pH 7.4) were analyzed in the equilibrium state using a Langmuir 1:1 model calculating the association (ka) and dissociation (kd) constants of the interactions by a global fitting analysis to generate dissociation constant (KD). The calculated KD values of each antibody is shown in Table 4A and Table 4B below. Table 4A. KD values calculated for huFcγRI, huFcγRIIa 167H, huFcγRIIa 167R, huFcγRIIb, huFcγRIIIa 167V, and huFcγRIIIa 167F. huFc huFc RIIa huFc RIIa huFc RI huFc RIIIa huFc RIIIaIPTS / 200129533.1 88Attorney Docket No.: PRG-068WO 8.53E 1C -11 1.03E-06 7.41E-07 2.14E-06 2.36E-08 6.07E-08cynoFcRn pH5.8, huFcRn pH7.4, and cynoFcRn pH7.4. Antibo huFcγRI cynoFcγR huFcRn cynoFcRn huFcRn cynoFcRnIPTS / 200129533.1 89Attorney Docket No.: PRG-068WO Example 3. BAFF-R binding protein-induced cell death in in vitro cell assays

[0164] To assess whether disclosed BAFF-R binding proteins are capable of inducing cell death, e.g., by a mechanism such as antibody-dependent cellular cytotoxicity (ADCC), cancer cells are contacted with effector cells in the presence or absence of BAFF-R binding proteins in in vitro assays. Peripheral Blood Mononuclear Cells (PBMCs) assay

[0165] Unfractionated peripheral blood mononuclear cells (PBMCs) (containing both NK cells (effectors) and B cells (targets)) are isolated from healthy donors (Donor 1, Donor 2, or Donor 3) through SepMate™ (StemCell Technologies) density gradient centrifugation with Ficoll-Paque™ PLUS density gradient media (Cytiva Life Sciences) and incubated in the absence (vehicle, 0.1% PBS) or presence of BAFF-R binding proteins (Antibody 1B or, as a control, Antibody 1A) in X-VIVO™15 Serum-free Hematopoietic Cell Medium (Lonza Bioscience) for 72 hours. Cells are then stained with Apotracker™ Green (which detects apoptotic cells by binding to exposed phosphatidylserine residues) (Biolegend); one or more of the following antibodies: α-CD2 PE-Cy7, α-CD4 BV510, α-CD8 AF700, α-CD19 PE- Dazzle 594, α-CD20 PE, α-CD22 APC, α-CD25 BV650, α-CD56 BV605, α-CD69 BV421, α- CD71 PerCP-Cy5.5; and Zombie NIR™ viability dye (Biolegend) and analyzed by flow cytometry (FIGs.1A and 1B). Binding of all clones to BAFF-R was found to mediate potent antibody-dependent cytotoxicity, resulting in depletion of B cells from in vitro human PBMC cultures. The depletion was shown across donors for Antibodies 1A and 1B. Data is shown in FIGs.2A-2C. Natural killer cells assay

[0166] On the day of the experiment, fresh blood was drawn from three donors into a heparinized vacutainer tube (Donor 120, Donor 122, and Donor 129) and stored at room temperature until use. Natural Killer cells were isolated and stored at ambient temperature for a time period between 25-180 minutes. RosetteSep NK (Miltenyi) was used to isolate NK cells. Raji B cells were harvested from culture and grown in RPMI 1640 with 10% MP-26 (ATCC). Freshly isolated NK cells were added to a 96 well plate at 1:1 ratio and incubated for 21hr (+ / - 1hr). A Cytotox-glo assay was performed on the assay wells according the manufacturers protocol. Assay plates were read on a SpectraMax I3x plate reader, and analyzed on SoftMax IPTS / 200129533.1 90Attorney Docket No.: PRG-068WO Pro. Cell death was quantified using a CytoTox-Glo™ Assay (Promega) kit. Data is shown in Tables 5 and 6. Table 5: EC50values from Donor 122 in the NK Cell Assay Run 1 Run 2 Run 3 Run 4 Run 5 Antibody EC50 EC50 EC50 EC50 EC50Run 1 Run 2 Run 3 Run 4 Run 5IPTS / 200129533.1 91Attorney Docket No.: PRG-068WO Table 4: EC50 values from Donor 129 in the NK Cell Assay Run 1 Run 2 Run 3 Run 4 Run 5 Antibody EC50 EC50 EC50 EC50 EC50EQUIVALENTS

[0167] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0168] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein.

[0169] Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. IPTS / 200129533.1 92

Claims

Attorney Docket No.: PRG-068WO CLAIMS 1. A B-cell-activating factor receptor (BAFF-R) binding protein comprising: (a) a heavy chain variable region (VH) comprising (i) a CDR-H1 having an amino acid sequence according to SEQ ID NO: 3, (ii) a CDR-H2 having an amino acid sequence according to SEQ ID NO: 4, and (iii) a CDR-H3 having an amino acid sequence according to SEQ ID NO: 5; (b) a light chain variable region (VL) comprising (i) a CDR-L1 having an amino acid sequence according to SEQ ID NO: 6, (ii) a CDR-L2 having an amino acid sequence according to SEQ ID NO: 7, and (iii) a CDR-L3 having an amino acid sequence according to SEQ ID NO: 8; and (c) an afucosylated Fc region comprising an Fc modification that extends the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the Fc modification.

2. The BAFF-R binding protein of claim 1, wherein the VHcomprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 1 and the VL comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO:

2.

3. A B-cell-activating factor receptor (BAFF-R) binding protein which (a) specifically binds to an epitope of BAFF-R, wherein the epitope comprises the sequence of SEQ ID NO: 20, and (b) comprises an afucosylated Fc region comprising Fc modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the Fc modifications.

4. The BAFF-R binding protein of any one of claims 1-3, wherein the BAFF-R binding protein is an antibody or antigen-binding fragment thereof.

5. The BAFF-R binding protein of claim 4, wherein the BAFF-R binding protein is a human antibody or antigen-binding fragment thereof.

6. The BAFF-R binding protein of claim 4 or 5, wherein the antigen binding fragment is a Fab, a F(ab′)2, a Fab′, a single-chain Fv (scFv), an Fv fragment, a Fd fragment, or a diabody.

7. The BAFF-R binding protein of claim 6, wherein the Fc region is an IgG1, IgG2, or IgG4 Fc region. IPTS / 200129533.1 93Attorney Docket No.: PRG-068WO 8. The BAFF-R binding protein of claim 7, wherein the Fc region is an IgG1 Fc region.

9. The BAFF-R binding protein of claim 8, wherein the IgG1 Fc region is a human IgG1 Fc region.

10. The BAFF-R binding protein of any one of claims 1-3, wherein the modifications comprise a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE).

11. A BAFF-R binding protein comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) an afucosylated human IgG1 Fc polypeptide (i) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 140 or 251 and (ii) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE).

12. A BAFF-R binding protein comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) an afucosylated human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275.

13. The BAFF-R binding protein of claim 11 or 12, which comprises (i) a first polypeptide comprising the immunoglobulin heavy chain variable domain and the afucosylated human IgG1 Fc polypeptide; and IPTS / 200129533.1 94Attorney Docket No.: PRG-068WO (ii) a second polypeptide comprising the immunoglobulin light chain variable domain.

14. A BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) an afucosylated human IgG1 Fc polypeptide (1) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 140 or 251 and (2) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); and (b) two immunoglobulin light chains, each immunoglobulin light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO:

2.

15. A BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) an afucosylated human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; and (b) two immunoglobulin light chains, each immunoglobulin light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO:

2.

16. The BAFF-R binding protein or antibody of any one of claims 1-15, further comprising an amino acid mutation of K214R.

17. The BAFF-R binding protein or antibody of any one of claims 1-16, further comprising an amino acid mutation of D356E. IPTS / 200129533.1 95Attorney Docket No.: PRG-068WO 18. The BAFF-R binding protein or antibody of any one of claims 1-17, further comprising an amino acid mutation of L358M.

19. The BAFF-R binding protein or antibody of any one of claims 1-15, further comprising an amino acid mutation of K214R / D356E / L358M.

20. A BAFF-R binding protein comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 264-271, and the light chain comprises the amino acid sequence of SEQ ID NO:

272.

21. An isolated nucleic acid encoding the BAFF-R binding protein or antibody of any one of claims 1-20.

22. A set of nucleic acids collectively encoding the BAFF-R binding protein or antibody of any one of claims 1-20.

23. An expression vector comprising a nucleic acid encoding the BAFF-R binding protein or antibody of any one of claims 1-20.

24. A set of expression vectors which collectively encode the BAFF-R binding protein or antibody of any one of claims 1-20.

25. A host cell comprising the isolated nucleic acid of claim 21, the set of nucleic acids of claim 22, the expression vector of claim 23, or set of expression vectors of claim 24.

26. The host cell of claim 25, further comprising a fucosylation-disrupting modification.

27. A host cell comprising (a) a fucosylation-disrupting modification and (b) an expression vector which encodes, or a set of expression vectors which collectively encode: (1) a BAFF-R binding protein comprising: (a) an immunoglobulin heavy chain variable region (VH) comprising (i) a CDR-H1 having an amino acid sequence according to SEQ ID NO: 3, (ii) a CDR-H2 having an amino acid sequence according to SEQ ID NO: 4, and IPTS / 200129533.1 96Attorney Docket No.: PRG-068WO (iii) a CDR-H3 having an amino acid sequence according to SEQ ID NO: 5; (b) an immunoglobulin light chain variable region (VL) comprising (i) a CDR-L1 having an amino acid sequence according to SEQ ID NO: 6, (ii) a CDR-L2 having an amino acid sequence according to SEQ ID NO: 7, and (iii) a CDR-L3 having an amino acid sequence according to SEQ ID NO: 8; and (c) a Fc region comprising modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the modifications; (2) a BAFF-R binding protein which (a) specifically binds to an epitope of BAFF-R, wherein the epitope comprises the sequence of SEQ ID NO: 20, and (b) comprises an Fc region comprising modifications that extend the half-life of the BAFF-R binding protein as compared to a BAFF-R binding protein that does not comprise the modifications; (3) a BAFF-R binding protein comprising (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) a human IgG1 Fc polypeptide (i) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 140 or 251 and (ii) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); (4) a BAFF-R binding protein comprising IPTS / 200129533.1 97Attorney Docket No.: PRG-068WO (a) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (b) an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO: 2; and (c) a human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; (5) a BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) a human IgG1 Fc polypeptide (1) having an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 21, 140 or 251 and (2) comprising a set of amino acid mutations selected from the group consisting of M252Y / S254T / T256E (YTE), M428L / N434S (LS), M428L / N434A (LA), H433K / N434F (KF), L309D / Q311H / N434S (DHS), and G236A / S239D / I332E (ADE); or (6) a BAFF-R antibody comprising (a) two immunoglobulin heavy chains, each comprising (i) an immunoglobulin heavy chain variable domain (VH) having the sequence of SEQ ID NO: 1; (ii) a human IgG1 Fc polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 252, 253, 254, 255, 256, 259 and 275; and (b) two immunoglobulin light chains, each light chain comprising an immunoglobulin light chain variable domain (VL) having the sequence of SEQ ID NO:

2.

28. The host cell of claim 26 or 27, wherein the fucosylation-disrupting modification is characterized by a deficiency in a fucosyltransferase. IPTS / 200129533.1 98Attorney Docket No.: PRG-068WO 29. The host cell of claim 28, wherein the fucosyltransferase is α-1,6-fucosyltransferase (FUT8).

30. The host cell of any one of claims 25-29, wherein the host cell is a mammalian host cell.

31. A pharmaceutical composition comprising the BAFF-R binding protein or antibody of any one of claims 1-20 and a pharmaceutically acceptable carrier.

32. A method comprising a step of administering to a subject in need thereof an effective amount of the BAFF-R binding protein or antibody of any one of claims 1-20 or the pharmaceutical composition of claim 31.

33. The method of claim 32, wherein the subject has an autoimmune or inflammatory disease.

34. The method of claim 33, wherein the subject has a disorder selected from the group consisting of IgA nephropathy, myasthenia gravis, systemic lupus erythematosus, membranous glomerulonephritis, Sjögren syndrome, lupus nephritis, immune thrombocytopenia, acquired (autoimmune) hemolytic anemia, cold agglutinin disease, autoimmune hepatitis, multiple sclerosis, pemphigus vulgaris, and rheumatoid arthritis.

35. The method of claim 32, wherein the subject has cancer.

36. The method of claim 35, wherein the cancer is chronic lymphocytic leukemia.

37. The method of any one of claims 32-36, wherein the step of administering comprises systemic administration of the BAFF-R binding protein or antibody.

38. The method of claim 37, wherein the systemic administration comprises intravenous or subcutaneous administration. IPTS / 200129533.1 99

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