Antibodies targeting BAFF-R and uses thereof

Abstract

Disclosed are proteins having antibody heavy and light chain variable domains that can pair to form an antigen-binding site that targets BAFF-R on a cell, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, such as for the treatment of cancer or autoimmune disease.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 250,092, filed September 29, 2021, the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer-readable sequence listing in its entirety that has been submitted in XML file format via the Patent Center, the contents of which are incorporated herein by reference in their entirety. The XML file of the sequence listing submitted via the Patent Center is entitled "14247-699-228_seqlist.xml", was created on September 16, 2022, and is 114,456 bytes in size.

[0003] The present invention provides proteins having antibody heavy and light chain variable domains that can pair to form an antigen-binding site that targets BAFF-R on a cell, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, such as for the treatment of cancer or autoimmune disease. [Background technology]

[0004] Cancer remains a major health problem, despite considerable research efforts and scientific advances reported in the literature for the treatment of this disease. Some of the most frequently diagnosed cancers in adults include prostate cancer, breast cancer, and lung cancer. Hematological malignancies are less frequent than solid cancers, but have poorer survival rates. Current treatment options for these cancers are not effective for all patients and / or may have significant adverse side effects. Other types of cancer also remain difficult to treat using existing treatment options.

[0005] BAFF-R, also called BAFF receptor, TNF receptor superfamily member 13C (TNFRSF13C), CD268, or BR3, is a type III transmembrane protein of the TNF receptor superfamily. BAFF-R is expressed at the late transition (T2) B cell stage and on all mature B cells, downregulated on germinal center B cells, re-expressed on memory cells, and absent on plasma cells (Non-Patent Document 1). BAFF-R is the receptor for B cell activating factor (BAFF), a B cell survival factor. BAFF can associate with three receptors: BAFF-R, transmembrane activator and interactor of CAML (TACI), and B cell maturation antigen (BCMA). Among these three receptors, BAFF-R is the major receptor involved in the development of follicular and marginal zone splenic B cells (Non-Patent Document 2).

[0006] The BAFF / BAFF-R signaling axis may play a role in B cell hyperplasia. Increased expression of BAFF-R and elevated serum levels of BAFF have been observed in patients with non-Hodgkin's lymphoma (NHL) (Non-Patent Document 3). A specific single nucleotide polymorphism (SNP) in BAFF-R is associated with an increased risk of chronic lymphocytic leukemia (CLL) (Non-Patent Document 4). The BAFF / BAFF-R axis is also involved in autoimmune inflammatory diseases (Non-Patent Document 5). Some patients with systemic lupus erythematosus (SLE) have increased levels of BAFF in serum (Non-Patent Document 6), and BAFF-R is consistently occupied on blood B cells in SLE (Non-Patent Document 7). Given the observation that autoreactive B cells are more dependent on BAFF for their survival compared to protective B cells (Non-Patent Document 8), it has been proposed that abnormally high levels of BAFF may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Davidson(2012)Curr.Rheumatol.Rep.,14(4):295-302 [Non-Patent Document 2] Schiemann et al.(2001)Science,293:2111-14 [Non-Patent Document 3] Shen et al.(2016)Adv.Clin.Exp.Med.,25(5):837-44 [Non-Patent Document 4] Jesek et al. (2016) Tumor Biol.,37(10):13617-26 [Non-Patent Document 5] Mackay et al. (1999) J. Exp. Med., 190:1697-1710 [Non-Patent Document 6] Cheema et al.(2001)Arthritis Rheum.,44:1313-19 [Non-Patent Document 7] Carter et al.(2005)Arthritis Rheum.,52:3943-54 [Non-Patent Document 8] Lesley et al.(2004)Immunity,20:441-53 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need in the art for new and useful antibodies that bind to BAFF-R, particularly antibodies that bind to BAFF-R and inhibit its interaction with BAFF. [Means for solving the problem]

[0009] The present invention provides antigen-binding sites that bind to BAFF-R. Proteins and protein conjugates, such as antibodies, antibody-drug conjugates, bispecific T cell engagers (BiTEs), and immunocytokines, that contain such antigen-binding sites, as well as immune effector cells (e.g., T cells) expressing proteins that contain such antigen-binding sites (e.g., chimeric antigen receptors (CARs)), are useful for treating BAFF-R-associated diseases, such as cancer and autoimmune diseases.

[0010] Thus, in one aspect, the present invention provides an antigen-binding site that binds or is capable of binding to BAFF-R, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO:50, a complementarity determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO:51, and a complementarity determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO:52; and A light chain variable domain (VL) comprising a CDR1 sequence comprising the amino acid sequence of SEQ ID NO:4, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO:5, and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO:49. The present invention provides an antigen-binding site comprising:

[0011] In another aspect, provided herein is an antigen-binding site that binds to or is capable of binding to BAFF-R, comprising: (a) the VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 49, respectively; (b) the VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 1, 2, and 16, respectively; and the VL comprises sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (c) the VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 21, 2, and 22, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (d) the VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 20, 23, and 26, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or (e) VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively; and VL comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 49, respectively.

[0012] In one embodiment, the antigen-binding site comprises a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 49, respectively.

[0013] In one embodiment, the antigen-binding site comprises a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively.

[0014] In some embodiments, the antigen binding site comprises a VH comprising an amino acid sequence that is 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 SEQ ID NO: 40. In some embodiments, the antigen binding site comprises a VH that comprises a G44C substitution compared to SEQ ID NO: 40. In some embodiments, the antigen binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the antigen binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 42.

[0015] In some embodiments, the antigen binding site comprises a VL comprising an amino acid sequence 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 SEQ ID NO: 41. In some embodiments, the VL comprises a G100C substitution compared to SEQ ID NO: 41. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antigen binding site comprises a VL comprising the amino acid sequence of SEQ ID NO: 43.

[0016] In another aspect, provided herein is an antigen binding site comprising a VH comprising the amino acid sequence of SEQ ID NO: 40 and a VL comprising the amino acid sequence of SEQ ID NO: 41, or a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43. In one embodiment, the antigen binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 40 and a VL comprising the amino acid sequence of SEQ ID NO: 41. In one embodiment, the antigen binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 43.

[0017] In certain embodiments, the antigen binding site is present as a single chain fragment variable (scFv), a Fab fragment, or a monoclonal antibody.

[0018] In certain embodiments, the antigen binding site is present as a single chain fragment variable (scFv).

[0019] In some embodiments, the antigen binding site is present as an scFv comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 44.

[0020] In another aspect, provided herein is an antigen-binding site that competes for binding to BAFF-R with the antigen-binding site of any of the above embodiments.

[0021] In one embodiment, the antigen-binding site has a dissociation constant (K) of 5 nM or less as measured by surface plasmon resonance (SPR). D ) binds to human BAFF-R.

[0022] In one embodiment, the antigen-binding site inhibits (e.g., blocks) binding of BAFF-R to BAFF (e.g., by at least 50%, at least 75%, at least 90%, at least 95%, or at least 99%) as measured in a competitive binding assay.

[0023] In another aspect, provided herein is a protein comprising the antigen-binding site of any one of the above embodiments.

[0024] In some embodiments, the protein further comprises an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, each polypeptide chain of the antibody heavy chain constant region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a wild-type human IgG1 Fc region.

[0025] In certain embodiments, at least one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations relative to the amino acid sequence of a wild-type human IgG1 Fc region at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system.

[0026] In certain embodiments, at least one polypeptide chain of the antibody heavy chain constant region is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, numbered according to the EU numbering system. , T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0027] In one embodiment, one polypeptide chain of the antibody heavy chain constant region has a wild type human IgG1 one or more mutations to the amino acid sequence of the Fc region; and the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations to the amino acid sequence of a wild-type human IgG1 Fc region at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system.

[0028] In one embodiment, one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to the amino acid sequence of a wild-type human IgG1 Fc region; and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to the amino acid sequence of a wild-type human IgG1 Fc region, numbered according to the EU numbering system.

[0029] In one embodiment, one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to the amino acid sequence of a wild-type human IgG1 Fc region; and the other polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution relative to the amino acid sequence of a wild-type human IgG1 Fc region, numbered according to the EU numbering system.

[0030] In another aspect, provided herein is an antibody-drug conjugate comprising a protein of any one of the above embodiments and a drug moiety, in certain embodiments, the drug moiety is selected from the group consisting of an auristatin, an N-acetyl-gamma calicheamicin, a maytansinoid, a pyrrolobenzodiazepine, and SN-38.

[0031] In another aspect, provided herein is an immunocytokine comprising the antigen-binding site of any one of the above embodiments and a cytokine, in certain embodiments, the cytokine is selected from the group consisting of IL-2, IL-4, IL-10, IL-12, IL-15, TNF, and IFNα.

[0032] In another aspect, provided herein is a bispecific T cell engager comprising an antigen-binding site of any one of the above embodiments and an antigen-binding site that binds to CD3.

[0033] In another aspect, provided herein is a method for treating a pulmonary circulation disorder comprising: (a) an antigen-binding site of the present invention; (b) a transmembrane domain; and (c) Intracellular signaling domain It is a chimeric antigen receptor (CAR) comprising:

[0034] In certain embodiments, the transmembrane domain is selected from the transmembrane regions of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, BAFF-R, CD37, CD64, CD80, CD86, CD134, CD137, CD152, and CD154.

[0035] In some embodiments, the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain of CD3zeta, common FcRgamma (FCER1G), FcgammaRIIa, FcRbeta (FcεR1b), CD3gamma, CD3delta, CD3ε, CD79a, CD79b, DAP10, and DAP12. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain comprising a functional signaling domain of a costimulatory receptor. In some embodiments, the costimulatory receptor is selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), or any combination thereof.

[0036] In another aspect, provided herein is an isolated nucleic acid encoding a CAR of any one of the above embodiments.

[0037] In another aspect, provided herein is an expression vector comprising the isolated nucleic acid of the above aspect.

[0038] In another aspect, provided herein is an immune effector cell comprising a nucleic acid or expression vector of the above aspect.

[0039] In another aspect, provided herein is an immune effector cell expressing a CAR of any one of the above aspects. In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, a γδ T cell, or a NKT cell. In some embodiments, the immune effector cell is a NK cell.

[0040] In another aspect, provided herein is a pharmaceutical composition comprising a protein, antibody-drug conjugate, immunocytokine, bispecific T cell engager, or immune effector cell of any of the above aspects or embodiments; and a pharma- ceutically acceptable carrier.

[0041] In another aspect, provided herein is a method of treating cancer, comprising administering to a subject in need thereof an effective amount of a protein, antibody-drug conjugate, immunocytokine, bispecific T cell engager, immune effector cell, or pharmaceutical composition of any of the above aspects or embodiments. In some embodiments, the cancer is B-cell non-Hodgkin's lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, and acute lymphocytic leukemia (ALL). In some embodiments, the cancer expresses BAFF-R.

[0042] In another aspect, provided herein is a method of treating an autoimmune inflammatory disease, comprising administering to a subject in need thereof an effective amount of the protein, antibody-drug conjugate, immunocytokine, bispecific T cell engager, immune effector cell, or pharmaceutical composition of any of the above aspects or embodiments.

[0043] In certain embodiments, the protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager of any of the above aspects or embodiments is a purified antigen binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager.

[0044] In certain embodiments, the protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager of any of the above aspects or embodiments is purified by a method selected from the group consisting of centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0045] These and other aspects and advantages of the present invention are set forth in the following figures, detailed description and claims.

[0046] The present invention may be more fully understood with reference to the following drawings. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 is a graph showing the fluorescence output from a binding assay demonstrating blockade of binding of BAFF-biotin to human BAFF-R expressed on CHO cells by the indicated antibodies.

[0048] [Diagram 2] FIG. 2 is a graph depicting the fluorescence output from a blocking assay showing blockage by the indicated antibodies of binding of BAFF-biotin to human BAFF-R expressed on CHO cells.

[0049] [Figure 3-1] Figures 3A-3D are graphs of the fluorescence output from binding assays on CHO cells (Figures 3A, 3B) or from blocking assays of BAFF-biotin binding to BAFF-R with the indicated antibodies (Figures 3C, 3D). [Figure 3-2] Same as above.

[0050] [Figure 4-1]4A-4E are flow cytometry plots showing binding of AB0369scFv expressed in yeast to no antigen control (FIG. 4A), h-BAFF-R-hFc (FIG. 4B), irrelevant hFc (FIG. 4C), hBAFF-R-GST (FIG. 4D), or irrelevant GST (FIG. 4E). The vertical axis shows expression of the scFv as measured by detection of the Flag epitope tag; the horizontal axis shows binding of the biotinylated control of the BAFF-R construct to the scFv as measured by detection of streptavidin-PE. [Figure 4-2] Same as above.

[0051] [Diagram 5] 5A-5B are graphs depicting binding of AB0369 or the indicated controls to human (FIG. 5A) or cynomolgus monkey (FIG. 5B) BAFF-R.

[0052] [Figure 6-1] Figures 6A-6B detail a multispecificity assay of a multispecific binding protein with a BAFF-R binding site derived from AB0369. Figure 6A is a schematic of the assay. Figure 6B shows a graph of AB0369 (left panel), a graph of a trastuzumab negative control (middle panel), or a graph of an ixekizumab positive control (right panel) in the absence (top panel) or presence (bottom panel) of a multispecific reagent (PSR). [Figure 6-2] Same as above.

[0053] [Figure 7] FIG. 7 is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells as induced by a multispecific binding protein with a BAFF-R binding site derived from AB0369.

[0054] [Figure 8] FIG. 8 is a graph depicting the fluorescence output from a binding assay showing the prevention of binding of BAFF-biotin to human BAFF-R expressed on CHO cells by AB0369 or the indicated antibodies.

[0055] [Figure 9-1] Figures 9A-9D are flow cytometry plots showing binding of hBAFF-R-hFc-His to the parental AB0369scFv or to clones selected from the library, expressed in yeast and generated by affinity maturation after successive rounds of selection: Figure 9A shows binding to the parental AB0369scFv; Figure 9B shows binding to samples from the first round of clone selection; Figure 9C shows binding to samples from the second round of clone selection; and Figure 9D shows binding to output from the second round of clone selection. [Figure 9-2] Same as above.

[0056] [Figure 10-1] Figures 10A-10E are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity matured scFv clones expressed in yeast. Figure 10A shows binding to parent AB0369; Figure 10B shows binding to AB0605; Figure 10C shows binding to AB0622; Figure 10D shows binding to AB0622; Figure 10E shows binding to ianalumab-based scFv. [Figure 10-2] Same as above.

[0057] [Figure 11] Figures 11A-11C are graphs showing BAFF-R binding and cytotoxicity of multispecific binding proteins expressed from affinity maturation of AB0369. Figure 11A is a graph showing binding of multispecific binding proteins with BAFF-R binding sites derived from the indicated clones to human BAFF-R expressed on CHO cells. Figure 11B is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells as induced by multispecific binding proteins with BAFF-R binding sites derived from the indicated clones. Figure 11C is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells as induced by multispecific binding proteins with BAFF-R binding sites derived from AB0622.

[0058] [Figure 12] Figures 12A-12B detail a multispecificity assay of multispecific binding proteins with BAFF-R binding sites derived from AB00605 and AB0606. Figure 12A is a schematic of the assay. Figure 12B shows a graph of AB0605 (left panel) or AB0606 (right panel) in the absence (top panel) or presence (bottom panel) of the multispecific reagent (PSR).

[0059] [Figure 13] Figures 13A-13C are flow cytometry plots showing binding of hBAFF-R-hFc-His to the parental AB0369scFv or to clones selected from the library expressed in yeast and generated by affinity maturation after successive rounds of selection: Figure 13A shows binding to the parental AB0369scFv; Figure 13B shows binding to samples from the first round of clone selection; Figure 13C shows binding to samples from the second round of clone selection.

[0060] [Figure 14-1] Figures 14A-14E are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity matured scFv clones expressed in yeast. Figure 14A shows binding to parent AB0369; Figure 14B shows binding to AB0679; Figure 14C shows binding to AB0681; Figure 14D shows binding to AB0682; Figure 14E shows binding to ianalumab-based scFv. [Figure 14-2] Same as above.

[0061] [Figure 15]Figures 15A-15C are graphs showing the binding of BAFF-R to multispecific binding proteins expressed from affinity maturation of AB0369. Figure 15A is a graph showing the binding of multispecific binding proteins with BAFF-R binding sites from the indicated clones to human BAFF-R expressed on CHO cells. Figure 15B is a graph showing the binding of multispecific binding proteins with BAFF-R binding sites from the indicated clones to cynomolgus monkey BAFF-R expressed on CHO cells. Figure 15C is a graph showing the fluorescence output from a binding assay showing the interference of BAFF-biotin binding to BAFF-R expressed on CHO cells by the indicated antibodies.

[0062] [Figure 16] FIG. 16 is a graph showing KHYG-1-CD16aV cytotoxicity assay of BJAB cells when induced by multispecific binding proteins with BAFF-R binding sites from AB0679, AB0568, or the tool-F3 positive control.

[0063] [Figure 17] Figures 17A-17D are flow cytometry plots showing binding of hBAFF-R-hFc-His to parental AB0369scFv clones selected from a library generated by affinity maturation expressed in yeast after successive rounds of selection: Figure 17A shows binding to the parental AB0369scFv; Figure 17B shows binding to samples from the first round of clone selection; Figure 17C shows binding to samples from the second round of clone selection; and Figure 17D shows binding to samples from the third round of clone selection.

[0064] [Figure 18-1]Figures 18A-18F are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity matured scFv clones expressed in yeast. Figure 18A shows binding to parent AB0369; Figure 18B shows binding to AB0682; Figure 18C shows binding to AB0898; Figure 18D shows binding to AB0899; Figure 18E shows binding to AB0900; Figure 18F shows binding to ianalumab-based scFv. [Figure 18-2] Same as above. [Figure 18-3] Same as above.

[0065] [Figure 19] FIG. 19 is a graph showing KHYG-1-CD16aV cytotoxicity assay of BJAB cells when induced by multispecific binding proteins having a BAFF-R binding site derived from AB0898, AB0899, or AB0900.

[0066] [Figure 20] FIG. 20 shows graphs of the differential scanning calorimetry (DSC) profiles of AB0898 (top panel), AB0899 (middle panel), and AB0900 (bottom panel).

[0067] [Figure 21] FIG. 21 shows flow cytometry plots of binding of scFv clones expressed in yeast to biotinylated hBAFFR-Fc before (left) and after (right) loading by incubation with 1 mM non-biotinylated hBAFFR-Fc.

[0068] [Figure 22-1] Figure 22 shows flow cytometry plots of binding of yeast-expressed scFv clones to biotinylated hBAFFR-Fc before (top) and after (bottom) loading by incubation with 1 mM non-biotinylated hBAFFR-Fc. The clones tested are (from left to right): AB1080, AB1081, AB1084, AB1085, and ianalumab-based scFv. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 22-4] Same as above.

[0069] [Figure 23] Figures 23A-23B are graphs showing binding of the indicated antibody clones to human (Figure 23A) or cynomolgus monkey (Figure 23B) BAFF-R.

[0070] [Figure 24-1] Figures 24A-24B detail a multispecificity assay of multispecific binding proteins with BAFF-R binding sites derived from AB1080 or AB1081. Figure 24A is a schematic of the assay. Figure 24B shows graphs of AB1080 (left panel), AB1081 (middle left panel), trastuzumab negative control (middle right panel), or ixekizumab positive control (right panel) in the absence (top panel) or presence (bottom panel) of the multispecific reagent (PSR). [Figure 24-2] Same as above.

[0071] [Diagram 25] Figures 25A-25B show graphs of KHYG-1-CD16aV cytotoxicity assays of BJAB cells when induced by multispecific binding proteins with BAFF-R binding sites derived from AB1080 (Figure 25A) or AB1085 (Figure 25B) compared to tool positive controls.

[0072] [Figure 26] FIG. 26 is a graph depicting the fluorescence output from a binding assay showing the prevention of binding of BAFF-biotin to human BAFF-R expressed on CHO cells by the indicated antibody clones.

[0073] [Figure 27]FIG. 27 shows a graph of nano dual scanning fluorimetry (nanoDSF) analysis of multispecific binding proteins with BAFF-R binding sites derived from AB1080 (left panel), AB1081 (middle left panel), AB1084 (middle right panel), and AB1085 (right panel).

[0074] [Figure 28] FIG. 28 shows a graph of a hydrophobic interaction chromatography (HIC) analysis of multispecific binding proteins having BAFF-R binding sites derived from the indicated antibodies.

[0075] [Figure 29] FIG. 29 shows a graph of the HIC analysis of AB1612 compared to the indicated reference biologics.

[0076] [Diagram 30] Figures 30A-30B are graphs showing binding of the indicated antibody clones to human (Figure 30A) or cynomolgus monkey (Figure 30B) BAFF-R. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The present invention provides antigen-binding sites that bind to human BAFF-R. Proteins and protein conjugates, such as antibodies, antibody-drug conjugates, bispecific T cell engagers (BiTEs), and immunocytokines, that contain such antigen-binding sites, as well as immune effector cells (e.g., T cells) that express proteins that contain such antigen-binding sites (e.g., chimeric antigen receptors (CARs)), are useful for treating BAFF-R-associated diseases, such as cancer and autoimmune diseases. Various aspects of the invention are described in the following sections; however, aspects of the invention described in one particular section are not limited to any particular section.

[0078] To facilitate the understanding of this invention, several terms and phrases are defined below.

[0079] As used herein, the terms "a" and "an" mean "one or more" and include plurals unless the context requires otherwise.

[0080] As used herein, the term "antigen-binding site" refers to a portion of an immunoglobulin molecule that is involved in or capable of antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains are called "hypervariable regions", which are interposed between more conserved adjacent sections known as "framework regions", or "FR". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions", or "CDRs". In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single domain antibody". The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains an antigen-binding surface, or in a recombinant polypeptide such as an scFv, in which a peptide linker is used to link the heavy chain variable domain to the light chain variable domain in a single polypeptide. All amino acid positions in the heavy or light chain variable regions disclosed herein are numbered according to the Kabat numbering system.

[0081] The CDRs of an antigen-binding site can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). CDRs determined under these definitions typically contain overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term "CDR" refers to a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy and light chain CDRs of an antibody are defined using various conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to MacCallum (see above) and the light CDRs are defined according to Kabat (see above), with CDRH1, CDRH2 and CDRH3 representing the heavy chain CDRs and CDRL1, CDRL2 and CDRL3 representing the light chain CDRs.

[0082] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably, humans.

[0083] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, such as reducing, reducing, modulating, ameliorating, or eliminating, of a condition, disease, disorder, etc., which results in the amelioration or amelioration of the symptoms thereof.

[0084] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic uses in vivo or ex vivo.

[0085] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (such as oil-in-water or water-in-oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0086] As used herein, the term "pharmaceutical acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention, or its active metabolites or residues, that is capable of providing a compound of the present invention upon administration to a subject. As known to those skilled in the art, the "salts" of the compounds of the present invention can be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic acid, which are not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0087] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of the formula NW4 + wherein W is C 1~4 (alkyl) and the like.

[0088] Exemplary salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include Na + , NH4 + , and NW4 + (Wherein W is C 1~4 and the anion of a compound of the present invention combined with a suitable cation such as an alkyl group.

[0089] For therapeutic use, the salts of the compounds of the invention are considered to be pharma-ceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharma-ceutically acceptable compound.

[0090] As used herein, BAFF-R (also known as BAFF receptor, B-cell activating factor receptor, BR3, TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, TNF receptor superfamily member 13C, CD268, and BLyS receptor 3) refers to the protein with Uniprot accession number Q96RJ3 and related isoforms and orthologs.

[0091] Throughout this specification, where compositions are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of, or consist of, the components recited, and that there are processes and methods of the invention that consist essentially of, or consist of, the processing steps recited.

[0092] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition for the variable applies.

[0093] Various features and aspects of the invention are described in further detail below.

[0094] I. Antigen binding site In one aspect, the present invention provides an antigen-binding site that binds to human BAFF-R. Exemplary antigen-binding site VH, VL, CDR, and scFv sequences are listed in Table 1. CDR sequences are identified according to the Chothia numbering scheme. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

Table 8

Table 9

Table 10

Table 11

[0095] In certain embodiments, an antigen-binding site of the invention comprises an antibody heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of the same antibody disclosed in Table 1. In certain embodiments, an antigen binding site comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262:732-745), or any other CDR determination method known in the art of the VH and VL sequences of antibodies disclosed in Table 1. In certain embodiments, the antigen binding site comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of an antibody disclosed in Table 1.

[0096] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:49, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:50, 51, and 52, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:4, 5, and 49, respectively.

[0097] In certain embodiments, the antigen-binding site of the present invention is derived from AB0369. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 77, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0098] In certain embodiments, the antigen-binding site of the present invention is derived from 1203_A01. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 7, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 6, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 7, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 6, respectively.

[0099] In certain embodiments, the antigen-binding site of the present invention is derived from 1203_A02. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 10, 2, and 11, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 12, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 10, 2, and 11, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 12, respectively.

[0100] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:16. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:1, 2, and 16, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively.

[0101] In certain embodiments, the antigen-binding site of the present invention is derived from AB0605scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:64, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:1, 2, and 13, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 13, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0102] In certain embodiments, the antigen-binding site of the present invention is derived from AB0606scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:65, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:1, 2, and 14, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 14, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0103] In certain embodiments, the antigen-binding site of the present invention is derived from AB0622scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:66, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:1, 2, and 15, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 15, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0104] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:21, SEQ ID NO:2, and SEQ ID NO:22. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:21, 2, and 22, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively.

[0105] In certain embodiments, the antigen-binding site of the present invention is derived from AB0679scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:67, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:17, 2, and 14, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 17, 2, and 14, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0106] In certain embodiments, the antigen-binding site of the present invention is derived from AB0681scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:68, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:18, 2, and 19, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 18, 2, and 19, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0107] In certain embodiments, the antigen-binding site of the present invention is derived from AB0682scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:69, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:20, 2, and 14, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 2, and 14, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0108] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:20, 23, and 26, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively.

[0109] In certain embodiments, the antigen-binding site of the present invention is derived from AB0898. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 70, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 32, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 32, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0110] In certain embodiments, the antigen-binding site of the present invention is derived from AB0899. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 71, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 24, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 24, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 24, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0111] In certain embodiments, the antigen-binding site of the invention is derived from AB0900. For example, in certain embodiments, the antigen-binding site of the invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 72, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 25, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 23, and 25, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0112] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 49, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO: 35, 36, and 37, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively.

[0113] In certain embodiments, the antigen-binding site of the present invention is derived from AB1080scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 73, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 23, and 27, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 23, and 27, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively.

[0114] In certain embodiments, the antigen-binding site of the present invention is derived from AB1081scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:74, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:43. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:28, 29, and 30, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:4, 5, and 39, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively.

[0115] In certain embodiments, the antigen-binding site of the present invention is derived from AB1084scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 75, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 31, 23, and 32, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively. In certain embodiments, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 31, 23, and 32, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39.

[0116] In certain embodiments, the antigen-binding site of the present invention is derived from AB1085scFv. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 76, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 63. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 2, and 34, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 2, and 34, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0117] In certain embodiments, an antigen-binding site of the invention comprises a VH comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. In certain embodiments, an antigen-binding site of the invention comprises a VL comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 49, respectively. In certain embodiments, an antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO: 46, 47, and 48, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NO: 4, 5, and 49, respectively.

[0118] In certain embodiments, the antigen-binding site of the invention is derived from AB1424 or AB1612. For example, in certain embodiments, the antigen-binding site of the invention comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:40, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:41. In certain embodiments, an antigen-binding site of the invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 42, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 43. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively.In certain embodiments, the antigen binding site is present as an scFv, and the scFv comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 44 or 45.

[0119] In certain embodiments, the antigen-binding site of the present invention is derived from 3A1. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:54, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:55. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:80, 81, and 82, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:83, 84, and 85, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80, 81, and 82, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 83, 84, and 85, respectively.

[0120] In certain embodiments, the antigen-binding site of the present invention is derived from 7G4. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 56, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 57. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80, 86, and 87, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 83, 84, and 85, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80, 86, and 87, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 83, 84, and 85, respectively.

[0121] In certain embodiments, the antigen-binding site of the present invention is derived from 1B3-A7. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:58, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:59. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:88, 89, and 90, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:91, 92, and 93, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 88, 89, and 90, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 91, 92, and 93, respectively.

[0122] In certain embodiments, the antigen-binding site of the present invention is derived from 10H7-C5. For example, in certain embodiments, the antigen-binding site of the present invention comprises a VH comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:60, and a VL comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:79. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:94, 95, and 96, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:97, 92, and 53, respectively. In a specific embodiment, the antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 94, 95, and 96, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 97, 92, and 53, respectively.

[0123] In each of the above embodiments, it is contemplated herein that the VH and / or VL sequences that together bind BAFF-R may contain amino acid modifications (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) within the framework regions of the VH and / or VL without significantly affecting their ability to bind BAFF-R.

[0124] In certain embodiments, an antigen-binding site of the invention has a K of 1 nM or less, 5 nM or less, 10 nM or less, 15 nM or less, or 20 nM or less as measured by surface plasmon resonance (SPR) (e.g., using the methods described in Example 1 below) or by biolayer interferometry (BLI). DIn certain embodiments, an antigen-binding site of the invention binds to human BAFF-R with a dissociation constant (i.e., a dissociation constant) of 1×10 as measured by SPR (e.g., using the methods described in Example 1 below) or by BLI. -5 , 1×10 -4 , 1×10 -3 , 5×10 -3 , 0.01, 0.02, or 0.05 1 / s or less d (i.e., K off The off-rate is also called the off-rate.

[0125] In certain embodiments, an antigen-binding site of the invention has a K of 5 nM or less, 10 nM or less, 15 nM or less, 20 nM or less, or 30 nM or less as measured by surface plasmon resonance (SPR) (e.g., using the methods described in Example 1 below) or by biolayer interferometry (BLI). D In certain embodiments, an antigen-binding site of the invention binds to cynomolgus monkey BAFF-R and / or binds to BAFF-R derived from a subject's body fluids, tissues, and / or cells with a dissociation constant of 1×10 as measured by SPR (e.g., using the methods described in Example 1 below) or by BLI. -3 , 5×10 -3 , 0.01, 0.02, or 0.03 1 / s or less d (i.e., K off The off-rate is also called the off-rate.

[0126] In another aspect, the present invention provides an antigen-binding site that competes with the antigen-binding site described above for binding to BAFF-R (e.g., human BAFF-R). In a particular embodiment, the antigen-binding site of the present invention competes with the antigen-binding site derived from AB1424 disclosed above for binding to BAFF-R. In one embodiment, the antigen-binding site competes with AB1424 for binding to BAFF-R. In a particular embodiment, the antigen-binding site of the present invention competes with the antigen-binding site derived from humanized AB1423 disclosed above for binding to BAFF-R. In one embodiment, the antigen-binding site competes with humanized AB1424 for binding to BAFF-R. In a particular embodiment, the antigen-binding site of the present invention competes with the antigen-binding site derived from AB1612 disclosed above for binding to BAFF-R. In one embodiment, the antigen-binding site competes with AB1612 for binding to BAFF-R. In certain embodiments, the antigen-binding site of the present invention competes with an antigen-binding site derived from humanized AB1612 disclosed above for binding to BAFF-R. In one embodiment, the antigen-binding site competes with humanized AB1612 for binding to BAFF-R. In certain embodiments, the antigen-binding site of the present invention competes with an antigen-binding site derived from AB0369, 1203_A01, 1203_A02, AB0605, AB0606, AB0622, AB0679, AB0681, AB0682, AB0898, AB0899, AB0900, AB1080, AB1081, AB1084, AB1085, 3A1, 7G4, 1B3-A7, or 10H7-C5 disclosed above for binding to BAFF-R. In certain embodiments, the antigen binding site competes with AB0369, 1203_A01, 1203_A02, AB0605, AB0606, AB0622, AB0679, AB0681, AB0682, AB0898, AB0899, AB0900, AB1080, AB1081, AB1084, AB1085, 3A1, 7G4, 1B3-A7, or 10H7-C5 for binding to BAFF-R.

[0127] Proteins with antigen-binding sites The antigen-binding sites disclosed herein can be present in an antibody or an antigen-binding fragment thereof. The antibody can be a monoclonal antibody, a chimeric antibody, a diabody, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment, an Fv, a bispecific antibody, a bispecific Fab2, a bispecific (mab)2, a humanized antibody, an artificially generated human antibody, a bispecific T cell engager, a bispecific NK cell engager, a single chain antibody (e.g., a single chain Fv fragment or scFv), a triomab, a knobs-into-holes (kih) IgG with a common light chain, a crossmab, an ortho-FabIgG, a DVD-Ig, a 2-in-1-IgG, an IgG-scFv, a sdFv2-Fc, a bi-nanobody, a tandAb, a dual affinity retargeting antibody (DART), a DART-Fc, a scFv-HSA-scFv (where HSA = human serum albumin), or a dock-and-lock (DNL)-Fab3. In certain embodiments, the antibodies of the present disclosure are scFvs. In certain embodiments, the scFvs are in a VH-VL format.

[0128] In one embodiment, the single chain variable fragment (scFv) comprises a heavy chain variable domain and a light chain variable domain. In one embodiment, to enhance the stability of the scFv, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions being numbered under Kabat. In one embodiment, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker can be used, for example, the (G4S)4 linker ((GlyGlyGlyGlySer)4 (SEQ ID NO: 98). In one embodiment of the scFv, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In one embodiment of the scFv, the heavy chain variable domain is located at the C-terminus of the light chain variable domain.

[0129] It is contemplated that in an scFv, the VH and VL may be connected by a linker, such as (GlyGlyGlyGlySer)4, i.e., (G4S)4 linker (SEQ ID NO: 98). One of skill in the art will appreciate that any of the other disclosed linkers (see, e.g., Table 2) can be used in an scFv having the VH and VL sequences disclosed herein (e.g., Table 1).

[0130] The length of a linker (e.g., a flexible linker) can be "short," e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, or "long," e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues in length.

[0131] In certain embodiments, the linker is (GS)n (SEQ ID NO: 109), (GGS) n (SEQ ID NO: 110), (GGGS) n (SEQ ID NO: 111), (GGSG) n (SEQ ID NO: 112), (GGSGG) n (SEQ ID NO: 113), and (GGGGS) n (SEQ ID NO: 114), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker comprises or consists of an amino acid sequence selected from SEQ ID NOs: 98-108, as listed in Table 2. [Table 12]

[0132] In certain embodiments, the antigen-binding sites disclosed herein are linked to an antibody constant region, e.g., an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the heavy chain constant region of, for example, IgG1, IgG2, IgG3, and IgG4 (e.g., human), in particular; IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In another embodiment, the antigen-binding sites disclosed herein may be linked to a light chain constant region, e.g., a kappa or lambda (e.g., human) light chain constant region. The constant region may be modified, e.g., mutated, to alter the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In another embodiment, the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no ability to bind Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not aid in binding to Fc receptors, for example, it has a mutated or deleted Fc receptor binding region.

[0133] In certain embodiments, the antigen binding site is linked to an IgG constant region comprising the hinge, CH2 and CH3 domains, with or without the CH1 domain. In certain embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human antibody constant region, e.g., a human IgG1 constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In one embodiment, an antibody Fc domain or portion thereof sufficient to bind to CD16 has a wild-type human IgG1 Fc sequence [ka] In certain other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an antibody constant region from another mammal, e.g., rabbit, dog, cat, mouse, or horse. For example, one or more mutations may be incorporated into the constant region compared to the human IgG1 constant region at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S , L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0134] In certain embodiments, the antigen binding site is linked to a portion of the antibody Fc domain sufficient to bind to CD16. Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is primarily centered on amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 in the CH2 domain, and carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0135] In certain embodiments, mutations that may be incorporated into CH1 of the human IgG1 constant region may occur at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be incorporated into the Cκ of the human IgG1 constant region may occur at amino acids E123, F116, S176, V163, S174, and / or T164.

[0136] In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, such as a human IgG1 antibody. In some embodiments, mutations are introduced into the antibody constant domain to allow heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from a human IgG1 constant domain, the antibody constant domain may comprise an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to amino acids 234-332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0137] In order to promote the formation of asymmetric protein, Fc domain heterodimerization is assumed.Mutations (e.g., amino acid substitutions) in Fc domain that promote heterodimerization are described, for example, in International Application Publication No. WO2019157366, which is not incorporated herein by reference.

[0138] The above-mentioned proteins can be produced by recombinant DNA technology well known to those skilled in the art.For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding a first immunoglobulin light chain can be cloned into a third expression vector; a fourth nucleic acid sequence encoding a second immunoglobulin light chain can be cloned into a fourth expression vector; the first, second, third and fourth expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0139] To achieve the highest yield of protein, various ratios of the first, second, third and fourth expression vectors can be investigated to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell banking using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy or Clonepix.

[0140] Clones can be cultured under conditions suitable for bioreactor scale-up and maintained for expression of proteins comprising the antigen-binding sites disclosed herein. Proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0141] Thus, in another aspect, the invention provides one or more isolated nucleic acids comprising sequences encoding the immunoglobulin heavy chain and / or immunoglobulin light chain variable region of any one of the above antibodies. The invention provides one or more expression vectors expressing the immunoglobulin heavy chain and / or immunoglobulin light chain variable region of any one of the above antibodies. Similarly, the invention provides host cells comprising one or more of the above expression vectors and / or isolated nucleic acids.

[0142] In certain embodiments, the antibody has a K of 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM or less as measured using standard binding assays, such as surface plasmon resonance or biolayer interferometry. D In certain embodiments, the antibodies disclosed herein bind to BAFF-R with a K of less than 5 nM. D In certain embodiments, the antibodies bind to BAFF-R from bodily fluids, tissues and / or cells of a subject. In certain embodiments, the antibodies disclosed herein inhibit (e.g., block) the binding of BAFF-R to BAFF (e.g., by at least 50%, 75%, 90%, 95% or 99% as measured in a competitive binding assay).

[0143] Competitive assays for determining whether an antibody binds to the same epitope as the disclosed antibodies or competes for binding with the disclosed antibodies are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0144] Typically, a competitive assay involves the use of an antigen (e.g., human BAFF-R protein or fragment thereof) bound to a solid surface or expressed on a cell surface, a test BAFF-R-binding antibody, and a reference antibody. The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, or 100x). Antibodies identified by competitive assays (e.g., competing antibodies) include antibodies that bind to the same epitope as the reference antibody, or a similar (e.g., overlapping) epitope, and antibodies that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antibody such that steric hindrance occurs.

[0145] Competitive assays can be performed in both orientations to ensure that the presence of label does not interfere with or inhibit binding, for example, in one orientation, the reference antibody is labeled and the test antibody is unlabeled, and in the second orientation, the test antibody is labeled and the reference antibody is unlabeled.

[0146] A test antibody competes with a reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1x, 5x, 10x, 20x or 100x) inhibits binding of the other antibody by at least 50%, 75%, 90%, 95% or 99%, as measured, for example, in a competitive binding assay.

[0147] Two antibodies can be determined to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies can be determined to bind overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0148] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties, including affinity and / or specificity, improve biophysical properties, including aggregation, stability, precipitation and / or non-specific interactions, and / or reduce immunogenicity. Affinity-maturation procedures are within the skill of one of ordinary skill in the art. For example, diversity may be introduced into the immunoglobulin heavy chains and / or immunoglobulin light chains by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-directed mutagenesis.

[0149] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified (e.g., by a process called germlining) to human germline sequences to optimize the antibody.

[0150] Generally, an optimized antibody has at least the same, or substantially the same, affinity for an antigen as the non-optimized (or parent) antibody from which it is derived. Preferably, an optimized antibody has a higher affinity for an antigen when compared to the parent antibody.

[0151] If the antibody is to be used as a therapeutic agent, it may be conjugated to an effector agent, such as a small molecule toxin or radionuclide, using standard in vitro conjugation chemistry. If the effector agent is a polypeptide, the antibody may be chemically conjugated to the effector or attached to the effector as a fusion protein. Construction of fusion proteins is within the skill of one of ordinary skill in the art.

[0152] Antibodies can be conjugated to effector moieties, such as small molecule toxins or radionuclides, using standard in vitro conjugation chemistries. If the effector moiety is a polypeptide, the antibody can be chemically conjugated to the effector or attached to the effector as a fusion protein. Construction of fusion proteins is within the skill of one of ordinary skill in the art.

[0153] CAR T cells, BAFF-R / CD3-directed bispecific T cell engagers, immunocytokines, antibody-drug conjugates, and immunotoxins Another aspect of the invention provides a molecule or complex comprising an antigen binding site that binds to BAFF-R as disclosed herein. Exemplary molecules or complexes include, but are not limited to, chimeric antigen receptors (CARs), T cell engagers (e.g., BAFF-R / CD3-directed bispecific T cell engagers), immunocytokines, antibody-drug conjugates, and immunotoxins.

[0154] Any antigen-binding site that binds to BAFF-R disclosed herein may be used. In certain embodiments, the VH, VL, and / or CDR sequences of the antigen-binding site that binds to BAFF-R are shown in Table 1. In certain embodiments, the antigen-binding site that binds to BAFF-R is an scFv. In certain embodiments, the scFv comprises an amino acid sequence that is at least 90% (e.g., 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 an amino acid sequence selected from SEQ ID NOs: 44 and 45. In certain embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NOs: 44 and 45.

[0155] In certain embodiments, the antigen-binding site that binds to BAFF-R in a molecule or complex (e.g., a CAR, a T cell engager, an immunocytokine, an antibody-drug conjugate, or an immunotoxin) comprises a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively; and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively. In certain embodiments, the antigen-binding site comprises a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 40 or 42; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 41 or 43. In certain embodiments, the antigen binding site comprises an scFv comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 44 or SEQ ID NO: 45. In certain embodiments, the antigen binding site comprises an scFv comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 44.

[0156] Chimeric antigen receptors (CARs) In certain embodiments, the present invention provides a BAFF-R-targeted CAR that comprises an antigen-binding site that binds to BAFF-R as disclosed herein (see, e.g., Table 1). The BAFF-R-targeted CAR may comprise a Fab fragment or an scFv.

[0157] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct that contains at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that includes a functional signaling domain derived from a stimulatory molecule (also referred to herein as a "primary signaling domain").

[0158] Thus, in certain embodiments, a CAR comprises an extracellular antigen binding site that binds to BAFF-R as disclosed herein, a transmembrane domain, and an intracellular signaling domain that comprises a primary signaling domain. In certain embodiments, a CAR further comprises one or more functional signaling domains derived from at least one costimulatory molecule (also referred to herein as "costimulatory signaling domains").

[0159] In certain embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding site that binds to BAFF-R as disclosed herein (e.g., a BAFF-R binding scFv) as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding site that binds to BAFF-R as disclosed herein (e.g., a BAFF-R binding scFv) as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and a primary signaling domain. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding site that binds to BAFF-R as disclosed herein (e.g., a BAFF-R binding scFv) as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two costimulatory signaling domains and a primary signaling domain. In certain embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding site that binds to BAFF-R as disclosed herein (e.g., a BAFF-R-binding scFv) as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two costimulatory signaling domains and a primary signaling domain.

[0160] For example, in certain embodiments, the extracellular antigen-binding domain comprises an antigen-binding site (e.g., an scFv) comprising a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively; and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively. In certain embodiments, the antigen-binding site comprises a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 40 or 42; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 41 or 43. In certain embodiments, the antigen binding site comprises an scFv comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 44 or SEQ ID NO: 45. In certain embodiments, the antigen binding site comprises an scFv comprising an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 44.

[0161] Regarding the transmembrane domain, in various embodiments, the CAR is designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain is one that is naturally associated with one of the domains in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane protein and to minimize interaction with other members of the receptor complex. In another embodiment, the transmembrane domain is capable of homodimerizing with another CAR on the CAR T cell surface. In another embodiment, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a natural binding partner present in the same CAR T cell.

[0162] The transmembrane domain can be derived from any naturally occurring membrane-bound or transmembrane protein. In one embodiment, the transmembrane region is capable of transmitting a signal to the intracellular domain whenever the CAR is bound to a target. In one embodiment, the transmembrane domain comprises a transmembrane region of one or more proteins selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, BAFF-R, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is selected from the group consisting of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, IT The transmembrane domains include one or more proteins selected from the group consisting of GAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NKG2C.

[0163] The extracellular BAFF-R binding domain (e.g., BAFF-R binding scFv domain) domain can be linked to the transmembrane domain by a hinge region. Various hinges can be used, including, but not limited to, human Ig (immunoglobulin) hinges (e.g., IgG4 hinges, IgD hinges), Gly-Ser linkers, a(G4S)4 linkers, KIR2DS2 hinges, and CD8α hinges.

[0164] The intracellular signaling domain of the CAR of the present invention is involved in at least one of the specialized functions of the immune cell in which the CAR is placed (e.g., cytolytic activity or helper activity of T cells, including secretion of cytokines). Thus, as used herein, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0165] The intracellular signaling domain of a CAR comprises a primary signaling domain (i.e., a functional signaling domain derived from a stimulatory molecule) and one or more costimulatory signaling domains (i.e., a functional signaling domain derived from at least one costimulatory molecule).

[0166] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell, e.g., a T cell, a NK cell, or a B cell, that provides a cytoplasmic signaling sequence that regulates activation of the immune cell in a stimulatory manner for at least some aspects of an immune cell signaling pathway. In one embodiment, the signal is a primary signal that is initiated, e.g., by binding of the TCR / CD3 complex by a peptide-loaded MHC molecule, and results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc.

[0167] Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing cytoplasmic signaling sequences that are of particular use in the present invention include those derived from CD3zeta, common FcRgamma (FCER1G), FcgammaRIIa, FcRβ (FcεR1b), CD3gamma, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In one embodiment, the primary signaling domain in any one or more CARs of the present invention comprises a cytoplasmic signaling sequence derived from CD3-zeta.

[0168] In certain embodiments, the primary signaling domain is a functional signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, and / or CD3-ζ. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and / or DAP12. In a specific embodiment, the primary signaling domain is a functional signaling domain of the ζ chain associated with the T cell receptor complex.

[0169] As used herein, the term "costimulatory molecule" refers to a cognate binding partner in T cells that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by T cells, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, and a ligand that specifically binds to CD83. Further examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2R β, IL2R γ, IL7R. α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b , ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B 4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and ligands that specifically bind to CD83.In one embodiment, the costimulatory signaling domain of the CAR is a functional signaling domain of a costimulatory molecule described herein, e.g., OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), or any combination thereof.

[0170] As used herein, the term "signaling domain" refers to a functional portion of a protein that acts by transmitting information intracellularly to regulate cellular activity through a defined signaling pathway by generating second messengers or by functioning as an effector by responding to such messengers.

[0171] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other in a random or defined order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage.

[0172] Another aspect of the invention provides a nucleic acid encoding a BAFF-R-targeting CAR disclosed herein. The nucleic acid is useful for expressing the CAR in an effector cell (e.g., a T cell) by introducing the nucleic acid into the cell.

[0173] For example, modifications can be made in a sequence to generate equivalent or improved variants of the invention by changing one or more of the codons according to a codon degeneracy table. A DNA codon degeneracy table is shown in Table 3. [Table 13]

[0174] In certain embodiments, the nucleic acid is a DNA molecule (e.g., a cDNA molecule). In certain embodiments, the nucleic acid further comprises an expression control sequence (e.g., a promoter and / or enhancer) operably linked to the CAR coding sequence. In certain embodiments, the invention provides a vector comprising the nucleic acid. The vector can be a viral vector (e.g., an AAV vector, a lentiviral vector, or an adenoviral vector) or a non-viral vector (e.g., a plasmid).

[0175] In certain embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule). Methods for generating mRNA for use in transfection may include in vitro transcription of a template with specifically designed primers, followed by polyA addition to generate an RNA construct containing 3' and 5' untranslated sequences, a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases long. RNA molecules may be further modified to increase translation efficiency and / or stability, as disclosed, for example, in U.S. Pat. Nos. 8,278,036; 8,883,506, and 8,716,465. RNA molecules generated in this manner may efficiently transfect a variety of cell types.

[0176] In one embodiment, the nucleic acid encodes an amino acid sequence comprising a signal peptide at the amino terminus of the CAR. Such a signal peptide can promote cell surface localization of the CAR when it is expressed in an effector cell, and is cleaved from the CAR during cellular processing. In one embodiment, the nucleic acid encodes an amino acid sequence comprising a signal peptide at the N-terminus of the extracellular BAFF-R binding domain (e.g., BAFF-R binding scFv domain).

[0177] RNA or DNA can be introduced into target cells using any of a number of different methods, including but not limited to electroporation, cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or commercial methods using biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0178] Another aspect of the invention provides immune effector cells expressing a BAFF-R-targeting CAR. Also provided are immune effector cells comprising a nucleic acid encoding a BAFF-R-targeting CAR. Immune effector cells include, but are not limited to, T cells and NK cells. In certain embodiments, T cells are CD8 + T cells, CD4 + The T cells or NK cells may be primary cells or cell lines.

[0179] Immune effector cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors, by methods known in the art. Immune effector cells can also be differentiated in vitro from pluripotent or multipotent cells (e.g., hematopoietic stem cells). In certain embodiments, the present invention provides pluripotent or multipotent cells (e.g., hematopoietic stem cells) that express a BAFF-R-targeting CAR (e.g., express a CAR on the cell membrane) or contain a nucleic acid disclosed herein.

[0180] In certain embodiments, immune effector cells are isolated and / or purified. For example, regulatory T cells can be removed from a T cell population using CD25-binding ligands. Effector cells expressing checkpoint proteins (e.g., PD-1, LAG-3, or TIM-3) can be removed by similar methods. In certain embodiments, effector cells are isolated by a positive selection step. For example, a population of T cells can be isolated by incubation with anti-CD3 / anti-CD28 conjugated beads. Other cell surface markers such as IFN-7, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin can also be used for positive selection.

[0181] Immune effector cells can be prepared using techniques described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; T cells may be activated and expanded generally using methods known in the art, as described in U.S. Pat. Nos. 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication Nos. 2006 / 0121005 and 2016 / 0340406. For example, in certain embodiments, T cells may be expanded and / or activated by contact with anti-CD3 and anti-CD28 antibodies under conditions appropriate to stimulate proliferation of the T cells. The cells may be grown in culture for a period of a few hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In one embodiment, the cells are grown for a period of 4-9 days. Multiple cycles of stimulation may be desirable for long-term cell culture (e.g., culture for periods of 60 days or more). In certain embodiments, the cell culture comprises serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, TNF-α, or combinations thereof. Other additives for cell growth known to those skilled in the art may also be included in the cell culture, such as detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. In certain embodiments, the immune effector cells of the present invention are cells obtained from in vitro expansion.

[0182] Further embodiments of BAFF-R-targeting CARs (e.g., regulatable CARs), nucleic acids encoding CARs, and effector cells expressing or comprising nucleic acids are set forth in U.S. Pat. Nos. 7,446,190 and 9,181,527, U.S. Patent Application Publication Nos. 2016 / 0340406 and 2017 / 0049819, and International Patent Application Publication No. WO 2018 / 140725.

[0183] BAFF-R / CD3-directed bispecific T cell engager In certain embodiments, the invention provides a BAFF-R / CD3-directed bispecific T cell engager comprising an antigen binding site that binds to BAFF-R as disclosed herein. In certain embodiments, the BAFF-R / CD3-directed bispecific T cell engager comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 44 and 45. In certain embodiments, the cytokine is attached to the Fc domain directly or via a linker.

[0184] In certain embodiments, the BAFF-R / CD3 directed bispecific T cell engager further comprises an antigen binding site that binds to CD3. Exemplary antigen binding sites that bind to CD3 are disclosed in International Patent Application Publication Nos. WO 2014 / 051433 and WO 2017 / 097723.

[0185] Another aspect of the invention provides nucleic acids encoding at least one polypeptide of a BAFF-R / CD3-directed bispecific T cell engager, where the polypeptide comprises an antigen binding site that binds to BAFF-R. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide that, when expressed, is at the N-terminus of one or more of the polypeptides of the BAFF-R / CD3-directed bispecific T cell engager. Also provided are nucleic acids, producer cells comprising the nucleic acids or vectors, and vectors (e.g., viral vectors) comprising the producer cells that express the BAFF-R / CD3-directed bispecific T cell engager.

[0186] Immune cytokines In certain embodiments, the present invention provides an immunocytokine comprising an antigen-binding site and a cytokine that binds to BAFF-R as disclosed herein. Any cytokine (e.g., proinflammatory cytokine) known in the art may be used, including, but not limited to, IL-2, IL-4, IL-10, IL-12, IL-15, TNF, IFNα, IFNγ, and GM-CSF. Further exemplary cytokines are disclosed in U.S. Pat. No. 9,567,399. In certain embodiments, the antigen-binding site is attached to the cytokine by chemical bonding (e.g., covalent or non-covalent chemical bonding). In certain embodiments, the antigen-binding site is attached to the cytokine by fusion of a polypeptide. The immunocytokine may further comprise an Fc domain attached to the antigen-binding site that binds to BAFF-R. In certain embodiments, the immunocytokine comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 44 and 45. In certain embodiments, the cytokine is linked to the Fc domain directly or via a linker.

[0187] Another aspect of the invention provides a nucleic acid encoding at least one polypeptide of an immunocytokine, wherein the polypeptide comprises an antigen binding site that binds to BAFF-R. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide, which when expressed is at the N-terminus of one or more of the polypeptides of the immunocytokine. Also provided are nucleic acids, production cells comprising the nucleic acids or vectors, and vectors (e.g., viral vectors) comprising the production cells that express the immunocytokine.

[0188] Antibody-drug conjugates In certain embodiments, the present invention provides an antibody-drug conjugate comprising an antigen-binding site that binds to BAFF-R as disclosed herein and a cytotoxic drug moiety. Exemplary cytotoxic drug moieties are disclosed in International Patent Application Publication Nos. WO 2014 / 160160 and WO 2015 / 143382. In certain embodiments, the cytotoxic drug moiety is selected from auristatins, N-acetyl-gamma calicheamicin, maytansinoids, pyrrolobenzodiazepines, and SN-38. The antigen-binding site may be attached to the cytotoxic drug moiety by chemical bonds (e.g., covalent or non-covalent chemical bonds). In certain embodiments, the antibody-drug conjugate further comprises an Fc domain attached to the antigen-binding site that binds to BAFF-R. In certain embodiments, the antibody-drug conjugate comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 44 and 45. In certain embodiments, the cytotoxic drug moiety is attached to the Fc domain directly or via a linker.

[0189] immunotoxin In certain embodiments, the present invention provides immunotoxins comprising an antigen-binding site that binds to BAFF-R as disclosed herein and a cytotoxic peptide moiety. Any cytotoxic peptide moiety known in the art may be used, including, but not limited to, ricin, Diphtheria toxin, and Pseudomonas exotoxin A. Further exemplary cytotoxic peptides are disclosed in International Patent Application Publication Nos. WO 2012 / 154530 and WO 2014 / 164680. In certain embodiments, the cytotoxic peptide moiety is attached to the protein by chemical bonds (e.g., covalent or non-covalent chemical bonds). In certain embodiments, the cytotoxic peptide moiety is attached to the protein by fusion of the polypeptide. The immunotoxin may further comprise an Fc domain attached to the antigen-binding site that binds to BAFF-R. In certain embodiments, the immunotoxin comprises an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NOs: 44 and 45. In certain embodiments, the cytotoxic peptide moiety is attached to the Fc domain directly or via a linker.

[0190] Another aspect of the invention provides nucleic acids encoding at least one polypeptide of an immunotoxin, wherein the polypeptide comprises an antigen binding site that binds to BAFF-R. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide, which, when expressed, is at the N-terminus of one or more of the polypeptides of the immunotoxin. Also provided are vectors (e.g., viral vectors) comprising the nucleic acids, production cells comprising the nucleic acids or vectors, and production cells that express the immunotoxins.

[0191] II. Therapeutic Compositions and Their Uses The present invention provides methods for treating cancer or autoimmune disease using proteins, conjugates, or cells comprising the antigen-binding site disclosed herein and / or pharmaceutical compositions described herein. The methods may be used to treat a variety of cancers that express BAFF-R by administering to a patient in need thereof a therapeutically effective amount of a protein, conjugate, or cell comprising the antigen-binding site disclosed herein.

[0192] Therapeutic methods can be characterized according to the cancer to be treated. The cancer to be treated can be characterized according to the presence of a particular antigen, such as BAFF-R, expressed on the surface of the cancer cells.

[0193] Cancers characterized by expression of BAFF-R include, but are not limited to, B-cell non-Hodgkin's lymphoma (B-NHL), e.g., chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, acute lymphocytic leukemia (ALL); and autoimmune inflammatory diseases.

[0194] It is contemplated that the proteins, conjugates, cells, and / or pharmaceutical compositions described in this disclosure can be used to treat a variety of cancers, including but not limited to cancers where the cancer cells or cells within the cancer microenvironment express BAFF-R.

[0195] In certain embodiments, the cancer is a solid tumor. In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, or uterine cancer.In yet other embodiments, the cancer is angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid carcinoma, biliary tract carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract carcinoma, osteosarcoma, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, bile duct carcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, liver adenoma, hepatic adenomatosis, hepatobiliary carcinoma, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal Cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer of the penis, pharyngeal carcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal carcinoma, renal cell carcinoma, respiratory system carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinonasal carcinoma, skin cancer, small cell carcinoma, small intestine carcinoma, smooth muscle carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, spinal carcinoma, squamous cell carcinoma, rhabdomyosarcoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue carcinoma, undifferentiated carcinoma, ureteral carcinoma, urethral carcinoma, bladder carcinoma, urinary system carcinoma, cervical carcinoma, uterine carcinoma, uveal melanoma, vaginal carcinoma, verrucous carcinoma, vipoma, vulvar carcinoma, well-differentiated carcinoma, or Wilms' tumor.

[0196] In certain embodiments, the cancer is a hematological malignancy. In certain embodiments, the hematological malignancy is a leukemia. In certain embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, or acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloblast crisis of chronic myelogenous leukemia.

[0197] In certain embodiments, the present application provides methods for treating autoimmune inflammatory diseases using the proteins described herein and / or pharmaceutical compositions described herein, which can be used to treat a variety of BAFF-R expressing B cell associated autoimmune inflammatory diseases, including, but not limited to, multiple sclerosis, systemic lupus erythematosus, Graves' disease, Hashimoto's thyroiditis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, myasthenia gravis, and vasculitis.

[0198] III. Pharmaceutical Compositions Another aspect of the present application provides combination therapy: the proteins described herein can be used in combination with additional therapeutic agents to treat autoimmune diseases or to treat cancer.

[0199] Exemplary therapeutic agents that can be used as part of a combination therapy in the treatment of autoimmune inflammatory diseases are described in Li et al. (2017) Front. Pharmacol., 8:460 and include, for example, nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., cyclooxygenase-2 inhibitors), glucocorticoids (e.g., prednisone / prednisolone, methylprednisolone, and fluorinated glucocorticoids such as dexamethasone and betamethasone), disease-modifying antirheumatic drugs (DMARDs) (e.g., methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antimalarials, D-glucosamine, cyclosalicylates, cyclophosphamide, cyclosalicylates ... penicillamine, and cyclosporine), anti-TNF biologics (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab pegol, and their biosimilars), and other biologics that target CTLA-4 (e.g., abatacept), IL-6 receptor (e.g., tocilizumab), IL-1 (e.g., anakinra), Th1 immune responses (IL-12 / IL-23) (e.g., ustekinumab), Th17 immune responses (IL-17) (e.g., secukinumab), and CD20 (e.g., rituximab).

[0200] Exemplary therapeutic agents that may be used as part of a combination therapy in treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, , flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ (IFN-γ), colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor, as well as variants of the above agents that may exhibit different binding to their cognate receptors or increased or decreased serum half-lives.

[0201] Another class of drugs that can be used as part of combination therapy in treating cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3.The CTLA4 inhibitor ipilimumab has been approved by the United States Food and Drug Administration for the treatment of melanoma.

[0202] Still other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0203] Further categories of anti-cancer drugs include, for example: (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, ME (ii) an inhibitor selected from a K inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0204] The proteins of the present disclosure may also be used as an adjunct to surgical removal of the primary lesion.

[0205] The amount of protein and additional therapeutic agent and the relative timing of administration can be selected to achieve a desired combined therapeutic effect.For example, when administering a combination therapy to a patient who needs such administration, the therapeutic agents in the combination, or one or more pharmaceutical compositions containing the therapeutic agents, can be administered in any order, for example, sequentially, in parallel, together, simultaneously, etc.Furthermore, for example, the protein can be administered when the additional therapeutic agent exerts its prophylactic or therapeutic effect, or vice versa.

[0206] IV. Pharmaceutical Compositions The present disclosure also features a pharmaceutical composition containing a therapeutically effective amount of the protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for appropriate formulation. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0207] In one aspect, the disclosure provides a formulation of a protein containing a BAFF-R binding site described herein and a pharma- ceutically acceptable carrier.

[0208] In certain embodiments, the pharmaceutical composition comprises a protein that comprises an antigen-binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:40, and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:41. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 42, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:54, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:55. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:56, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:57.In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:59, and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:59. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:60, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:79. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 77, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:64, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63.In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:65, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:66, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:67, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:68, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63.In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:69, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:70, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:71, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 72, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 63.In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 73, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:74, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 75, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 76, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 63.

[0209] The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can be included in the composition for suitable formulation. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief overview of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0210] For example, the present disclosure may be present in an aqueous pharmaceutical formulation comprising a therapeutically effective amount of the protein in a buffer forming the formulation. The aqueous carrier may comprise sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffer (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. In certain embodiments, an aqueous formulation is prepared comprising the protein disclosed herein in a pH buffer. The pH of the formulation will typically be 3-11, more preferably 5-9 or 6-8, most preferably 7-8, e.g., 7-7.5. Intermediate pH ranges described above are also intended to be part of the present disclosure. For example, a range of values ​​using any combination of the values ​​described above as upper and / or lower limits is intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / mL citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system comprises 1-1.5 mg / mL citric acid, 0.25-0.5 mg / mL sodium citrate, 1.25-1.75 mg / mL disodium phosphate dihydrate, 0.7-1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / mL sodium chloride. The pH of the liquid formulation may be set by the addition of a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0211] In some embodiments, the formulation comprises an aqueous carrier that is pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of a liquid formulation. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffer (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0212] Polyols that can act as tonicifiers and stabilize the antibodies can also be included in the formulation. The polyol is added to the formulation in an amount that can vary with respect to the desired tonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also be modified with respect to the molecular weight of the polyol. For example, lesser amounts of monosaccharides (e.g., mannitol) can be added compared to disaccharides (such as trehalose). In certain embodiments, a polyol that can be used in the formulation as a tonicifier is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / mL. In certain embodiments, the mannitol concentration can be about 7.5 to about 15 mg / mL. In certain embodiments, the mannitol concentration can be about 10 to about 14 mg / mL. In certain embodiments, the mannitol concentration can be about 12 mg / mL. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0213] Detergents or surfactants may also be added to the formulation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces the aggregation of the antibody to be formulated and / or minimizes the formation of particulate matter in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain from about 0.1 mg / mL to about 10 mg / mL, or from about 0.5 mg / mL to about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0214] In certain embodiments, the liquid formulation of the present disclosure can be prepared as a solution with a concentration of 10 mg / mL in combination with sugar at a stabilizing level. In certain embodiments, the liquid formulation can be prepared in an aqueous carrier. In certain embodiments, the stabilizer can be added in an amount that is not more than the amount that can cause an undesirable or unsuitable viscosity for intravenous administration. In certain embodiments, the sugar can be a disaccharide, for example, sucrose. In certain embodiments, the liquid formulation can also include one or more of a buffer, a surfactant, and a preservative that is added to the formulation herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multi-dose) formulation.

[0215] In certain embodiments, the present disclosure provides a formulation having an extended shelf life comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0216] Deamidation is a common product mutation of peptides and proteins that can occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from a protein forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17 Dalton mass loss of the parent peptide. Subsequent hydrolysis results in an 18 Dalton mass gain. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is typically detectable as a 1 Dalton mass gain. Deamidation of asparagine results in either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide structure, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain affects the rate of deamidation. Gly and Ser following Asn in the protein sequence result in higher susceptibility to deamidation. In certain embodiments, the liquid formulations of the present disclosure may be stored under conditions of pH and humidity that prevent deamination of the protein product.

[0217] In certain embodiments, the formulation is a lyophilized formulation. In certain embodiments, the formulation is freeze-dried (lyophilized) and is contained in about 12 to 60 vials. In certain embodiments, the formulation is lyophilized and 45 mg of the freeze-dried formulation can be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation can be contained in one vial. In certain embodiments, 12, 27, or 45 vials of the freeze-dried formulation are combined to obtain a therapeutic dose of protein in an intravenous drug formulation. The formulation can be a liquid formulation. In certain embodiments, the liquid formulation is stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the liquid formulation is stored at about 600 mg / vial. In certain embodiments, the liquid formulation is stored at about 250 mg / vial.

[0218] In some embodiments, the lyophilized formulation comprises a protein as described herein and a lyophilization protectant. The lyophilization protectant can be a sugar, e.g., a disaccharide. In certain embodiments, the lyophilization protectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative. The amount of sucrose or maltose useful for stabilizing the lyophilized drug product can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be 1:2 to 1:5.

[0219] In certain embodiments, the pH of the formulation may be set by the addition of a pharma- ceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharma- ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharma- ceutically acceptable base may be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to 6-8. In certain embodiments, the pH range of the lyophilized drug product may be 7-8.

[0220] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., promotes the production of a substantially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents.

[0221] In certain embodiments, the lyophilized protein product is composed with an aqueous carrier. Aqueous carriers of interest herein are those that are pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either sterile water for injection, USP (SWFI) or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder dissolves into solution. In certain embodiments, the lyophilized protein product of the present disclosure is composed into about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0222] The protein composition can be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized formulation is combined with a sterile aqueous carrier before administration. The resulting composition in solid form can be packaged into a number of single-dose units, each containing a fixed amount of one or more of the above-mentioned drugs. The composition in solid form can also be packaged in a container for flexible amounts.

[0223] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without being toxic to the patient.

[0224] A particular dose may be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, a patient's dose may be adjusted to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those of skill in the art, especially in light of the dosage information and assays disclosed herein. Dosages may also be determined by the use of known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted as the course of the disease is monitored. Blood concentrations of the targetable construct or complex in the patient may be measured to see if dosages need to be adjusted to reach or maintain effective concentrations. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, and their dosages, are most likely to be effective for a given individual (Schmitz et al., Clinica. Chimica. Acta. 308:43-53, 2001; Steimer et al., Clinica. Chimica. Acta. 308:33-41, 2001).

[0225] In general, the dosage based on body weight is about 0.01 μg to about 100 mg / kg body weight, for example, about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0.1 μg g / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg~about 10μg / kg body weight, about 0.1μg~about 1μg / kg body weight, about 1μg~about 100mg / kg body weight, about 1μg~about 50mg / kg body weight, about 1μg~about 10mg / kg body weight, about 1μg~about 1mg / kg body weight, about 1μg~about 1 00 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight. Doses may be given daily, weekly, monthly or once or more than once a year, or once every 2 to 20 years. One skilled in the art can easily estimate the repetition rate of administration based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the present disclosure may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by perfusion through a catheter or by direct intralesional injection.It may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times yearly.

[0226] The above description describes multiple aspects and embodiments of the present disclosure. The application specifically contemplates all combinations and permutations of the aspects and embodiments.

[0227] Throughout this specification, when compositions are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the present disclosure that consist essentially of, or consist of, the components recited, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the processing steps recited.

[0228] In this application, when an element or component is described as being included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0229] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or impliedly described herein, may be combined in various ways without departing from the spirit and scope of the present disclosure. For example, if a particular compound is mentioned, that compound may be used in various embodiments of the compositions of the present disclosure and / or in the methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments are described and illustrated to enable a clear and concise application to be written and drawn, but it is intended and understood that the embodiments may be combined or separated in various ways without departing from the teachings and disclosures of the present invention. For example, it will be understood that all features described and illustrated herein may be applicable to all aspects of the present disclosure described and illustrated herein.

[0230] The phrase "at least one of" should be understood to include each of the listed objects following the phrase individually, and various combinations of two or more of the listed objects, unless otherwise understood from context and usage. The phrase "and / or" in connection with three or more listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0231] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing" (including their grammatical equivalents) is generally to be understood as open-ended and non-limiting and not, for example, excluding additional, unrecited elements or steps, unless specifically stated otherwise or understood otherwise from the context.

[0232] When the use of the term "about" precedes a quantitative value, the present disclosure also includes the specific quantitative value itself, unless otherwise stated. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or implied.

[0233] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0234] For example, any and all instances of "such as" or "including" or use of exemplary language herein are intended solely to more fully illustrate the disclosure and do not pose limitations on the scope of the disclosure unless claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure. EXAMPLES

[0235] The disclosure having been generally described herein will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the disclosure and are not intended to limit the scope of the disclosure in any way.

[0236] Example 1. Generation and characterization of BAFF-R binding mAbs This example describes two antibody discovery campaigns carried out to identify binders of BAFF-R. Using yeast display technology, multiple rounds of affinity maturation (CDRH3 of interest, and CDRH1 / CDRH2 of interest), sequence liability correction, off-rate pressure optimization, and site-directed mutagenesis, one binder was selected for further expression and improved biological properties. These studies identified binder AB1612 / AB1424 as a binder that exhibited suitable properties for a biologic candidate and, more importantly, the ability to inhibit the BAFF-R-BAFF interaction (shown in Figure 1).

[0237] Immunization methods for recombinant proteins Antibodies specific for BAFF-R were generated by immunizing four different strains of mice (H2L2, NZBW, BALB-C, and SJL / J) with hBAFF-R-hFc-His fusion protein. A total of seven mice across the four different strains were selected for hybridoma fusions based on antiserum titers. For immune library generation, splenocytes from a subset of mice from each immunization arm were pooled; however, for yeast display mAb discovery, only splenocytes from H2L2 mice were used.

[0238] Sixteen 96-well plates per hybridoma fusion from five mouse fusions (spleen cells from two mice were pooled for H2L2 fusions and splenocytes from two mice were pooled for SJL / J fusions) were analyzed by specificity ELISA, where binding to human and cynomolgus BAFF-R-hFc-His was compared to binding to an unrelated hFc-His protein. Supernatants from 33 BAFF-R positive specific hybridomas were selected for further analysis. Supernatants were tested for binding to BAFF-R+ isogenic CHO cells and 16 positive hybridomas were further subcloned. Supernatants from the subclones were analyzed by specificity ELISA as above and 20 BAFF-R positive specific subclones were tested for binding to BAFF-R+ cells. Nine subclone mAbs showed strong binding to BAFF-R+ cells and were sequenced. Six unique sequences were obtained and the corresponding mAbs were further analyzed in cell-based assays for their ability to block the BAFF-R-BAFF interaction.

[0239] Binding of biotinylated BAFF to BAFF-R+ CHO cells was tested in the absence or presence of six BAFF-R specific mAbs or isotype control mAbs. A decrease in mean fluorescence intensity (MFI) in the presence of the antibodies suggested that the mAbs inhibited the association of BAFF binding to BAFF-R (hence named blocking antibodies). All clones tested did not inhibit BAFF binding to BAFF-R+ cells, hence all six were named non-blocking antibodies (Figure 2).

[0240] DNA immunization method Two groups of SWR / J mice were immunized with DNA, one with the full-length human BAFF-R cDNA construct, and the other with a mixture of the full-length human BAFF-R cDNA construct and the human BAFF-R extracellular domain cDNA construct. Based on the antiserum titers, the mice were pooled and then selected for single B cell sorting, and another pool was used for hybridoma fusion.

[0241] Focus on single B cell selection yielded 44 human and cynomolgus cross-reactive clones. These clones were sequenced, transiently expressed in 293 cells, and the specificity of the purified mAbs was confirmed by immunohistochemistry using hBAFF-R. + , cynoBAFF-R + Binding to isogenic CHO cells was analyzed by flow cytometry comparing binding to the parental cell line. Eight binders were purified and further analyzed for their ability to bind BAFF-R and block the BAFF-R-BAFF interaction. All eight clones were determined not to block and were not associated with hBAFF-R. + Weak affinity for cancer cells was demonstrated.

[0242] The specificity of clones obtained by traditional hybridoma techniques was analyzed by flow cytometry. The following assessments were performed: a) binding to cells expressing either full-length human BAFF-R or the human BAFF-R extracellular domain was compared to binding to non-transfected parental cells; b) binding to hBAFF-R + and cynoBAFF-R +Binding to isogenic cells was compared to binding to parental cells; c) hBAFF-R + Binding to cancer cells. 25 positive hybridoma fusions were identified and 14 hybridoma fusions were sequenced based on binding strength. Five unique sequences were obtained, which were consistent with BAFF-R. + They were analyzed for their ability to bind to cells and block the BAFF-R-BAFF interaction. All five clones were determined to be non-blocking clones (Figures 3A-3D), however, four of the five clones (clones 3A1, 1B3-A7, 7G4, and 10H7-C5) showed good affinity for hBAFF-R.

[0243] BAFF-R specific scFv discovered from yeast library Using yeast display, an scFv library was constructed from splenocytes obtained from humanized H2L2 mice immunized with recombinant human hBAFF-R-hFc-His protein as described above. Three rounds of selection were performed with 5 nM biotinylated hBAFF-R-hFc-His. Individual yeast colonies were picked, sequenced, and sequences analyzed. Negative selection was performed to remove non-specific binders. Sequence convergence indicated that the selection process was successful in enriching binders and therefore complete. Unique sequences were selected for further characterization. Three BAFF-R specific scFvs were found from one library (Table 4). However, these sequences were very similar to each other, therefore only sequence 1129_A01 (also referred to as AB0369scFv) was selected for further testing. [Table 14]

[0244] Flow cytometry was used to assess the specificity of binding of AB0369scFv to hBAFF-R-hFc-His, hBAFF-R-GST-His, and negative control proteins bearing hFc or GST tags but displayed on yeast. AB0369scFv showed moderate to weak affinity for hBAFF-R; however, it showed no binding to the negative control, suggesting high specificity for BAFF-R (Figures 4A-4E).

[0245] 1129_A01 (AB0369scFv) was converted to a multispecific binding protein containing scFv and two non-BAFF-R binders to obtain AB0369. AB0369 was bound to human (hBAFF-R-CHO) BAFF-R. + Cells (Figure 5A) and cynomolgus monkey (cBAFF-R-CHO)BAFF-R + BAFF-R binds to cells (Figure 5B), lacks nonspecific interactions by polyspecific reagent (PSR) assay (Figures 6A and 6B), and + It was further analyzed for its ability to lyse Ramos cancer cells (Figure 7 and Table 5) and block the BAFF-BAFF-R interaction (Figure 8). AB0369 bound to both human and cynomolgus BAFF-R on the surface of isogenic CHO cells and inhibited EC 50 was about 10 nM, making it a good choice for further expression. [Table 15]

[0246] The ability of AB0369 to block BAFF-R-BAFF interaction was tested in a cell-based blocking assay. Briefly, CHO cells expressing human BAFF-R were harvested, washed with cold FACS buffer, and seeded at a density of 100,000 cells / well. Test substances were diluted in FACS buffer, and 50 μL of diluted multispecific binding protein or mAb was added to the cells, incubated on ice for 60 minutes, and then washed with FACS buffer. 12 nM BAFF-biotin was diluted in FACS buffer, and 100 μL was added per well, incubated on ice for 60 minutes, and then washed with FACS buffer. Cells were incubated with 100 μL of 1:200 streptavidin-PE diluted in FACS buffer, incubated on ice for 30 minutes, and then washed with FACS buffer. Cells were then incubated in 100 μL of 1:1,000 diluted live / dead dye in PBS for 15 minutes, then washed with FACS buffer, and fixed. After incubation, cells were washed with FACS buffer and resuspended in FACS buffer and prepared for analysis by flow cytometry. Median fluorescence intensity (MFI) was calculated for each sample and secondary alone control. Maximum MFI was calculated for BAFF-biotin alone and minimum MFI was calculated for streptavidin-phycoerythrin alone. Data were fitted to a 4-parameter nonlinear regression curve using GraphPad Prism.

[0247] These studies demonstrated that AB0369 could partially block the BAFF-R-BAFF interaction. However, blocking was significantly less potent than the ianalumab-based reference control, which does not contain any antibody-dependent cellular cytotoxicity-enhancing mutations unlike the parent antibody, likely due to the low affinity of AB0369 (Figure 8 and Table 6). Since AB0369 scFv was the only blocking antibody identified from all the above discovery efforts, it underwent further expression by affinity maturation of CDRH3 and CDRH1 / CDRH2, as well as additional amino acid changes to facilitate protein production and stability. [Table 16]

[0248] Affinity maturation of AB0369 Randomized affinity maturation of featured CDRH3 As described above, AB0369 was shown to specifically bind to BAFF-R expressing cells. To search for mutants with improved binding affinity, a yeast display affinity maturation library was created by mutating the CDRH3 residues of AB0369 (RFTMLRGLIIEDYGMDV (SEQ ID NO: 3)). To enrich for scFvs with higher affinity to hBAFF-R, two rounds of selection were performed using biotinylated hBAFF-R-hFc-His at 1 nM (Figures 9A-9D). The affinity between the parental clone AB0369 and representative individual library clones was compared. Three rounds of FACS sorting revealed that the parental clone [ka] Nine clones were obtained that contained one or two amino acid differences compared to hBAFF-R and showed higher binding affinity to hBAFF-R than the parental clones and parent-derived scFvs (with the ianalumab-based scFv used as a reference control) (Figures 10A-10E).

[0249] The scFv with the highest hBAFF-R binding affinity was converted into a multispecific binding protein containing the scFv and two non-BAFF-R binders expressed in Expi293 cells and further analyzed for their ability to bind BAFF-R expressing cells (Figure 11A) and to lyse Ramos cancer cells expressing BAFF-R (Figure 11B, Figure 11C). All multispecific binding proteins scored negative in the multispecific assay, suggesting that the improved binding affinity was specific to BAFF-R (Figures 12A-12B). Further testing showed a greater than three-fold improvement in BAFF-R binding, indicating a significant improvement in EC 50 This translated into a 6-10-fold improvement in potency as measured by β-glucose (Table 7). Maximum lysis remained unchanged, suggesting that improved binding affinity of BAFF-R was the primary driver of this improvement in potency. [Table 17]

[0250] Featured CDRH1 and CDRH2 combined affinity maturation Results from affinity maturation studies of the featured CDRH3 demonstrated improved affinity, with further improvement highly desirable. Therefore, CDRH1 and CDRH2 sequences were selected for affinity maturation using the mature CDRH3 scaffold (CDRH1: GFTFSSY (SEQ ID NO: 1) and CDRH2: WYDGSN (SEQ ID NO: 2)). The goal was to design and select binders with improved affinity over the parent clone (AB0369scFv) or the optimized variants of CDRH3 described above. This created a library with randomized CDRH1 and CDRH2, while retaining the optimized CDRH3. Two rounds of FACS were performed to enrich for high affinity binders (Figures 13A-13C).

[0251] After FACS, 24 clones were identified. Several clones with CDRH1 (RFTMLRGWYIEDYGMDV (SEQ ID NO: 14); RFTMLRGQYIEDYGMDV (SEQ ID NO: 13); RFTMLRGWIIEDYGMDV (SEQ ID NO: 15)) changes to the optimized CDRH3 backbone were found to show significant improvement in affinity for hBAFF-R compared to the parental AB0369 scFv (1129_A01) (Figures 14A-14D) or the ianalumab-based scFv reference control (Figure 14E).

[0252] The scFv with the highest hBAFF-R binding affinity was converted into a multispecific binding protein containing the scFv and two non-BAFF-R binders expressed in Expi293 cells and their ability to bind to human BAFF-R expressing cells (Figure 15A), cynomolgus monkey BAFF-R, and cynomolgus monkey BAFF-R were also expressed. +The multispecific binding proteins were further analyzed for their ability to bind to cells (Figure 15B) and to inhibit the BAFF-R-BAFF interaction (Figure 15C and Table 8). Testing of the multispecific binding proteins showed improvement in all three of these criteria, inhibiting BAFF-R in the KHYG-1-CD16a-mediated cytotoxicity assay. + Efficient killing of BJAB cells was demonstrated (Figure 16, Table 9). [Table 18] [Table 19]

[0253] Correcting potential sequence vulnerabilities Because affinity matured clones had amino acids in their CDRs that could negatively affect protein expression, stability, or immunogenicity, an additional library was constructed to select clones that did not have these amino acids. Three rounds of selection were performed using 1 nM biotinylated hBAFF-R-hFc-His protein to cause enrichment of high affinity binders (Figures 17A-17D). In total, 23 binders were identified, 12 of which were predicted to be free of undesirable amino acids ("disorder-fixed").

[0254] Preferred clones from these libraries lacking potential sequence sensitivities were successfully identified, including AB0898, (a sensitivity-corrected version of AB0682 described above), AB0899, and AB0900, which were then tested for their binding to hBAFF-R displayed on yeast. All clones showed higher affinity for hBAFF-R than the parental AB0369scFv (Figures 18A-18F).

[0255] Characterization of a multispecific binding protein with modified hindrance Three of the lesion-corrected clones were converted into multispecific binding proteins containing scFv and two non-BAFF-R binders expressed in Expi293 cells, purified by a two-step purification process, and characterized by size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), binding to BAFF-R-expressing cells, and ability to lyse BJAB cells in a KHYG-1-CD16aV-mediated cytotoxicity assay. The characterization of these clones, summarized in Table 10, indicates that lesion-correction was successful. No negative effect on cell binding was observed, and all three clones showed potent killing of tumor cells expressing BAFF-R (Figure 19). However, the thermostability of the molecules was Tm1>65°C, as shown in Figure 20. [Table 20]

[0256] As mentioned above, substitution of potential sequence-sensitive residues with specific amino acids in the CDRs had minimal effect on binding affinity, but binding and thermostability data from BAFF-R expressing cells suggested that further improvements were desirable. Therefore, CDRH1 and CDRH2 sequences (CDRH1: GFTFSSY (SEQ ID NO: 1) and CDRH2: WYDGSN (SEQ ID NO: 2)) were affinity matured into a sensitive CDRH3 scaffold and off-rate pressure was applied to select for high affinity clones. Briefly, clones were pre-incubated with biotinylated hBAFF-R-hFc-His at a concentration of 100 pM and then loaded with 1 μM non-biotinylated hBAFF-R-hFc-His for 2 hours. Yeast-displayed anti-BAFF-R scFv that remained bound to biotinylated hBAFF-R-hFc-His were selected and the process was repeated three times to enrich for high affinity binders with slower off-rates. As shown in FIG. 21, the clones remained bound to biotinylated hBAFF-R-hFc-His even after off-rate pressure challenge, whereas the ianalumab-based scFv reference control lost binding to biotinylated hBAFF-R-hFc-His under these conditions, suggesting a slower dissociation rate.

[0257] Analysis of individual clones showed high affinity for hBAFF-R-hFc-His (Figure 22) and importantly, the clones remained bound to biotinylated hBAFF-R-hFc-His. Notably, the ianalumab-based benchmark scFv showed reduced binding to biotinylated hBAFF-R-hFc-His after loading (Figure 22). Upon further consideration, some of the clones were removed because they contained additional undesirable amino acids or properties. The sequences of selected clones from the above studies are shown in Table 11. [Table 21]

[0258] Selected clones from the off-rate loading study above were generated as multispecific binding proteins containing scFvs of individual binders and two non-BAFF-R binders expressed in Expi293 cells and characterized by binding to hBAFF-R-expressing cells and cynomolgus monkey BAFF-R-expressing cells, ability to lyse BAFF-R-expressing cancer cells in a KHYG-1-CD16aV-mediated cytotoxicity assay, ability to block BAFF-BAFF-R interactions, thermal stability (differential scanning fluorometry, DSF) and hydrophobicity (HIC) (results are summarized in Table 12). BAFF-R of AB1080, AB1081, and AB1085 +Binding affinity to cells was improved compared to the parental clone (Figures 23A-23B compared to Table 12). In addition, binding affinity to cynoBAFF-R was similar to that to hBAFF-R (Figures 23A-23B). Lack of multispecificity was confirmed by PSR assay (Figures 24A-24B). AB1084 was excluded from further studies due to its long retention time during HIC and subsequent higher tendency to aggregate. The improved multispecific binding proteins showed much higher potency than the multispecific binding proteins based on the ianalumab sequence (Figures 25A-25B). Furthermore, a greater than 10-fold improvement in potency was observed compared to the original AB0369 multispecific binding protein. Importantly, the ability to block BAFF-BAFF-R binding was significantly improved compared to the parental AB0369 multispecific binding protein (Figure 26). [Table 22]

[0259] These multispecific binding proteins met acceptable thermal stability criteria compared to controls adalimumab (Humira) and pembrolizumab (Keytruda) (Figure 27). HIC chromatograms showed that AB1080 and AB1081 had retention times of 11.4 and 11.5 min, respectively. AB1085 showed a retention time of 9.5 min, which is at the lower end of the range among approved late-stage therapeutic antibodies, indicating very favorable hydrophobic behavior (Figure 27).

[0260] AB1080 and AB1081 showed improved binding to BAFF-R and did not contain any sequence barriers within the CDR sequences, but their hydrophobicity was higher compared to a panel of benchmark therapeutic antibodies. AB1085 showed the desired hydrophobicity and affinity, but contained potential sequence barriers within the CDRH2 and CDRH3 sequences (Figure 28). The sequences of AB1080, AB1081 and AB1085 were compared, and the AB1080 sequence was analyzed and further corrected to remove the hydrophobic generated W to Q (CDRH3: [ka] from [ka] The resulting AB1424 / AB1612 multispecific binding protein exhibited favorable low hydrophobicity within the range of functional biologics (Figure 29), while maintaining the same high affinity for BAFF-R (Table 13, Figures 30A-30B), potent blocking of BAFF-R-BAFF binding (Figure 1), and contained sequences that did not contain the lesions characteristic of the parent AB1080 (Table 14). [Table 23] [Table 24]

[0261] In conclusion, two antibody discovery campaigns utilizing recombinant protein and DNA immunization were performed. In the first campaign, four moderate affinity BAFF-R-BAFF non-blocking antibodies were identified. A single binder, AB0369scFv, discovered from the second campaign, displayed the ability to block the BAFF-R-BAFF interaction. Extensive expression of AB0396scFv through multiple rounds of affinity maturation, modification of hindrance liability, and rational sequence design yielded binders AB1612 / AB1424, which displayed desirable properties as therapeutic candidates.

[0262] Incorporation by Reference Unless stated to the contrary, the entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0263] Equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all respects as illustrative rather than limiting the present disclosure described herein. The various components of the different embodiments and the various disclosed method steps may be utilized in various combinations and permutations, and all such variations should be considered as aspects of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. An antigen-binding site that binds to BAFF-R, (a) a heavy chain variable domain (VH) comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences identical to the amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively, and a light chain variable domain (VL) comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 49, respectively; (b) a VH comprising a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 50, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 51, and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 52, and a VL comprising a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 49; (c) a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively, and a VL comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 49, respectively; (d) a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 1, 2, and 16, respectively, and a VL comprising sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (e) a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 21, 2, and 22, respectively, and a VL comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; or (f) an antigen-binding site comprising a VH comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 20, 23, and 26, respectively, and a VL comprising CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

2. 2. The antigen-binding site of claim 1, wherein the VH comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 1, 23, and 38, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences identical to the amino acid sequences of SEQ ID NOs: 4, 5, and 39, respectively.

3. wherein the VH comprises an amino acid sequence 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 SEQ ID NO: 40; and the VL comprises an amino acid sequence 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 SEQ ID NO: 41; 2. The antigen-binding site of claim 1, wherein optionally, the VH comprises a G44C substitution compared to SEQ ID NO: 40; and the VL comprises a G100C substitution compared to SEQ ID NO:

41.

4. The antigen-binding site of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 40 and the VL comprises the amino acid sequence of SEQ ID NO: 41, or the VH comprises the amino acid sequence of SEQ ID NO: 42 and the VL comprises the amino acid sequence of SEQ ID NO:

43.

5. The method of claim 1, wherein the antigen-binding site is present as a single chain fragment variable (scFv), a Fab fragment, or a monoclonal antibody; Optionally, the antigen-binding site is present as an scFv comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 44 or SEQ ID NO: 45, or is present as an scFv comprising an amino acid sequence that is identical to the sequence of SEQ ID NO: 44 or SEQ ID NO:

45. (i) the antigen-binding site has a dissociation constant (K) of 5 nM or less when measured by surface plasmon resonance (SPR). Ｄ ) binds to human BAFF-R; and / or (ii) The antigen-binding site of claim 1, wherein the antigen-binding site inhibits binding of BAFF-R to BAFF.

7. 2. A protein comprising an antigen-binding site according to claim 1, wherein the antigen-binding site further comprises a human IgG heavy chain constant region or a human IgG1 heavy chain constant region, and optionally: (a) each polypeptide chain of the antibody heavy chain constant region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a wild-type human IgG1 Fc region of SEQ ID NO: 61; (b) at least one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations relative to the amino acid sequence of SEQ ID NO: 61 at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system; (c) at least one polypeptide chain of the antibody heavy chain constant region is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366V, T366I, T366V ... and / or comprising one or more mutations to the amino acid sequence of SEQ ID NO: 61 selected from: 6S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E; and / or one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations relative to the amino acid sequence of SEQ ID NO: 61 at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439; and the antibody heavy chain constant region wherein the other polypeptide chain of said protein comprises one or more mutations relative to the amino acid sequence of SEQ ID NO: 61 at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system. (a) one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to the amino acid sequence of SEQ ID NO: 61; and another polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to the amino acid sequence of SEQ ID NO: 61, numbered according to the EU numbering system; and / or (b) one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to the amino acid sequence of SEQ ID NO: 61; and the other polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution relative to the amino acid sequence of SEQ ID NO: 61, numbered according to the EU numbering system.

9. 10. An antibody-drug conjugate comprising the protein of claim 7 and a drug moiety, optionally wherein the drug moiety is selected from the group consisting of an auristatin, an N-acetyl-gamma calicheamicin, a maytansinoid, a pyrrolobenzodiazepine, and SN-38.

10. 10. An immunocytokine comprising the antigen-binding site of claim 1 and a cytokine, optionally wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-10, IL-12, IL-15, TNF, and IFNα.

11. 10. A bispecific T cell engager comprising the antigen-binding site of claim 1 and an antigen-binding site that binds to CD3.

12. (a) an antigen-binding site according to claim 1; (b) a transmembrane domain; and (c) intracellular signaling domain A chimeric antigen receptor (CAR) comprising, optionally, (i) the transmembrane domain is selected from the transmembrane region of the α, β, or ζ chain of the T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, BAFF-R, CD37, CD64, CD80, CD86, CD134, CD137, CD152, and CD154; (ii) the intracellular signaling domain comprises a primary signaling domain comprising the functional signaling domains of CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12; and / or (iii) A CAR, wherein the intracellular signaling domain further comprises a costimulatory signaling domain comprising a functional signaling domain of a costimulatory receptor, and optionally the costimulatory receptor is selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS and 4-1BB (CD137), or any combination thereof.

13. 13. An immune effector cell expressing a CAR according to claim 12, optionally comprising: (i) a T cell, optionally wherein the T cell is a CD8 + T cell, a CD4 + T cell, a γδ T cell, or a NKT cell; or (ii) Immune effector cells, which are NK cells.

14. 14. A pharmaceutical composition comprising the protein of claim 7 or 8, the antibody-drug conjugate of claim 9, the immunocytokine of claim 10, the bispecific T cell engager of claim 11, or the immune effector cell of claim 13; and a pharmaceutically acceptable carrier.

15. 15. The pharmaceutical composition of claim 14 for use in a method for treating cancer, said method comprising administering to a subject in need thereof an effective amount of said protein, said antibody-drug conjugate, said immunocytokine, said bispecific T cell engager, said immune effector cell, or said pharmaceutical composition, and optionally (a) the cancer is B-cell non-Hodgkin's lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal B-cell lymphoma, and acute lymphocytic leukemia (ALL); and / or (b) the cancer expresses BAFF-R.

16. 15. The pharmaceutical composition of claim 14 for use in a method for treating an autoimmune inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of the protein, the antibody-drug conjugate, the immunocytokine, the bispecific T cell engager, the immune effector cell, or the pharmaceutical composition.

17. 12. The antigen-binding site of any one of claims 1 to 6, the protein of claim 7 or 8, the antibody-drug conjugate of claim 9, the immunocytokine of claim 10, or the bispecific T cell engager of claim 11, which is a purified antigen-binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager, optionally purified by a method selected from the group consisting of centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

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