Proteins that bind to NKG2D, CD16, and BAFF-R

Abstract

Multispecific binding proteins that bind to the NKG2D receptor, CD16, and B-cell activating factor receptor (BAFF-R) are described, as well as pharmaceutical compositions and methods of treatment of the multispecific binding proteins useful for the treatment of cancer and autoimmune inflammatory diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 250,160, filed September 29, 2021, the complete disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer-readable sequence listing submitted via the Patent Center in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence listing XML file submitted via the Patent Center is named "14247-700-228_seqlist.xml", was created on September 16, 2022, and is 305,046 bytes in size.

[0003] This application relates to multispecific binding proteins that bind to NKG2D, CD16, and B-cell activating factor receptor (BAFF-R) on cells, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including methods for the treatment of cancer. [Background technology]

[0004] Despite significant research efforts, cancer continues to pose a significant clinical and financial burden in countries around the world. According to the World Health Organization (WHO), cancer is the second leading cause of death. Surgery, radiation therapy, chemotherapy, biological therapy, immunotherapy, hormone therapy, stem cell transplantation, and precision medicine are among the existing treatments. Despite extensive research in these fields, highly effective and curative solutions, especially for the most aggressive forms of cancer, have yet to be identified. Furthermore, many of the existing anti-cancer treatments have substantial adverse side effects.

[0005] Cancer immunotherapies are desirable because they are highly specific and can use a patient's own immune system to promote the destruction of cancer cells. Fusion proteins, such as bispecific T cell engagers, are cancer immunotherapies that have been described in the literature to bind to tumor cells and T cells and facilitate tumor cell destruction.

[0006] Natural killer (NK) cells are components of the immune system, accounting for approximately 15% of circulating lymphocytes. NK cells were initially characterized by their ability to infiltrate virtually all tissues and effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells—i.e., via cytolytic granules containing perforin and granzymes, as well as via death receptor pathways. Activated NK cells also secrete proinflammatory cytokines and chemokines, such as IFN-γ, which promote the recruitment of other leukocytes to target tissues.

[0007] NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited through activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated through their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of several immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein expressed by essentially all natural killer cells, where it functions as an activating receptor. NKG2D is also found on T cells, where it acts as a costimulatory receptor. The ability to regulate NK cell function through NKG2D is useful in various therapeutic settings, including malignancies.

[0008] BAFF-R, also known as 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 in late transitional (T2) B cells and on all mature B cells, downregulated in germinal center B cells, re-expressed on memory cells, and absent on plasma cells (Non-Patent Document 1). BAFF-R is a receptor for B cell-activating factor (BAFF), a B cell survival factor. BAFF can associate with three receptors: BAFF-R, transmembrane activator and CMAL interactor (TACI), and B cell maturation antigen (BCMA). Of these three receptors, BAFF-R is the primary receptor involved in the development of follicular B cells and splenic marginal zone B cells (Non-Patent Document 2).

[0009] The BAFF / BAFF-R signaling axis may play a role in B cell hyperplasia. Increased expression of BAFF-R as well as elevated serum levels of BAFF have been observed in patients with non-Hodgkin's lymphoma (NHL) (Non-Patent Document 3). Single nucleotide polymorphisms (SNPs) in BAFF-R are 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 systemic lupus erythematosus (SLE) patients have elevated levels of BAFF in the serum (Non-Patent Document 6), and BAFF-R is consistently present 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 than protective B cells (Non-Patent Document 8), it has been proposed that unusually high levels of BAFF may contribute to the pathogenesis of autoimmune diseases by enhancing the survival of autoreactive B cells.

[0010] Thus, there remains a need in the art for new and useful proteins that bind to BAFF-R for use in the treatment of cancer and autoimmune inflammatory diseases. [Prior art documents]

Non-Patent Literature

[0011]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Non-Patent Literature 7

Non-Patent Literature 8

Summary of the Invention

Means for Solving the Problems

[0012] The present application provides multispecific binding proteins that bind to the NKG2D and GD16 receptors on natural killer cells, as well as BAFF-R. Such proteins can engage more than one NK-activating receptor and block the binding of natural ligands to NKG2D. In certain embodiments, the proteins can agonize NK cells in humans. In some embodiments, the proteins can agonize NK cells in humans and in other species, such as rodents and cynomolgus monkeys. Formulations containing any one of the proteins disclosed herein, cells containing one or more nucleic acids expressing the proteins, and methods of enhancing tumor cell death using the proteins are also provided.

[0013] Thus, in one aspect, the present application provides: (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to the B-cell activating factor receptor (BAFF-R); and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16.

[0014] In some embodiments of the proteins disclosed herein, the first antigen-binding site that binds to NKG2D is a Fab fragment and the second antigen-binding site that binds to BAFF-R is an scFv, hi some embodiments, the first antigen-binding site that binds to NKG2D is an scFv and the second antigen-binding site that binds to BAFF-R is a Fab fragment.

[0015] In some embodiments of the proteins disclosed herein, the protein further comprises an additional antigen-binding site that binds to BAFF-R. In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site that binds to BAFF-R and the additional antigen-binding site are Fab fragments. In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site that binds to BAFF-R and the additional antigen-binding site are each scFv. In certain embodiments, the amino acid sequences of the second antigen-binding site and the additional antigen-binding site are identical. In certain embodiments, the amino acid sequences of the second antigen-binding site and the additional antigen-binding site are different.

[0016] In some embodiments of the proteins disclosed herein, the scFvs that bind NKG2D are linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or portion thereof sufficient to bind to CD16, and the scFvs comprise a heavy chain variable domain and a light chain variable domain. In certain embodiments, each scFv that binds BAFF-R is linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or portion thereof sufficient to bind to CD16, and the scFvs comprise a heavy chain variable domain and a light chain variable domain. In certain embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

[0017] In some embodiments of the proteins disclosed herein, within an scFv that binds to NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, within each scFv that binds to BAFF-R, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In some embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain (numbered according to the Kabat numbering scheme). In some embodiments, within an scFv that binds to NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In some embodiments, within each scFv that binds to BAFF-R, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In certain embodiments, the flexible linker comprises (G4S)4. In certain embodiments, in an scFv that binds to NKG2D, the heavy chain variable domain is positioned C-terminal to the light chain variable domain. In certain embodiments, in each scFv that binds to BAFF-R, the heavy chain variable domain is positioned C-terminal to the light chain variable domain. In certain embodiments, in an scFv that binds to NKG2D, the heavy chain variable domain is positioned N-terminal to the light chain variable domain. In certain embodiments, in each scFv that binds to BAFF-R, the heavy chain variable domain is positioned N-terminal to the light chain variable domain. In certain embodiments, the Fab fragment that binds to NKG2D is not positioned between the antigen-binding site and Fc or a portion thereof. In certain embodiments, the Fab fragment that binds to BAFF-R is not positioned between the antigen-binding site and Fc or a portion thereof.

[0018] In another aspect, provided herein are (a) a first antigen-binding site comprising a Fab fragment that binds to NKG2D; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds to the B-cell activating factor receptor (BAFF-R); and (c) an Fc domain comprising a first antibody constant domain and a second antibody constant domain that form a heterodimer that binds to CD16, Proteins are provided in which the scFv is linked via a hinge to the N-terminus of a first antibody constant domain and the Fab is linked to the N-terminus of a second antibody constant domain.

[0019] In some embodiments, the hinge comprises Gly-Ser.

[0020] In some embodiments of the proteins disclosed herein, the first antigen-binding site binds to human NKG2D. In some embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NOs: 81, 82, and 112, respectively, and a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In some embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 81, 82, and 97, respectively, and a VL comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In some embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95, and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 85. In certain embodiments, the first antigen-binding site that binds to NKG2D comprises a VH comprising the amino acid sequence of SEQ ID NO: 95, and a VL comprising the amino acid sequence of SEQ ID NO: 85.

[0021] In some embodiments of the proteins disclosed herein, the second antigen-binding site comprises a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 260, 249, and 261, respectively, and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0022] In some embodiments of the proteins disclosed herein, the second antigen-binding site comprises a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 214, 233, and 248, respectively, and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 249, respectively. In some embodiments, the second antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 250 and a light chain variable domain at least 90% identical to SEQ ID NO: 251.

[0023] In some embodiments of the proteins disclosed herein, the second antigen-binding site comprises a VH having a G44C substitution relative to SEQ ID NO: 250 and a VL having a G100C substitution relative to SEQ ID NO: 251. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 253, or comprises a VH comprising the amino acid sequence of SEQ ID NO: 250 and a VL comprising the amino acid sequence of SEQ ID NO: 251. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 253. In some embodiments, the second antigen-binding site comprises a VH comprising the amino acid sequence of SEQ ID NO: 250 and a VL comprising the amino acid sequence of SEQ ID NO: 251.

[0024] In some embodiments of the proteins disclosed herein, the second antigen-binding site comprises a single-chain variable fragment (scFv), wherein the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 253. In some embodiments, the second antigen-binding site comprises an scFv, wherein the scFv comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 254 and 255. In some embodiments, the second antigen-binding site comprises an scFv, wherein the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 254. In some embodiments, the second antigen-binding site comprises an scFv, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 254.

[0025] In some embodiments of the proteins disclosed herein, the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 270. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 270. In some embodiments, the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 271. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 271.

[0026] In some embodiments of the proteins disclosed herein, the second 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.

[0027] In some embodiments of the proteins disclosed herein, the second 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).

[0028] In another aspect, provided herein are (a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO: 95 and the VL comprises the amino acid sequence of SEQ ID NO: 85; (b) a second antigen-binding site comprising a VH and a VL of an anti-BAFF-R antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO: 252 and the VL comprises the amino acid sequence of SEQ ID NO: 253; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16.

[0029] In another aspect, provided herein are (a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO: 95 and the VL comprises the amino acid sequence of SEQ ID NO: 85; (b) a second antigen-binding site comprising the amino acid sequence of SEQ ID NO: 254; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16.

[0030] In some embodiments of the proteins disclosed herein, the antibody Fc domain is a human IgG1 antibody Fc domain. In some embodiments, the antibody Fc domain, or a portion thereof, comprises an amino acid sequence at least 90% identical to SEQ ID NO: 118. In certain embodiments, at least one polypeptide chain of the antibody Fc domain comprises one or more mutations compared to SEQ ID NO: 118 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. In certain embodiments, at least one polypeptide chain of the antibody Fc domain comprises any one of the following amino acids: 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 ... 366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T411D, T411E, K439D, and K439E.In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations compared to SEQ ID NO: 118 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, The other polypeptide chain of the heavy chain constant region comprises one or more mutations relative to SEQ ID NO: 118 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). In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118, and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V, and F405T substitutions relative to SEQ ID NO: 118 (numbered according to the EU numbering system). In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises a F405L substitution relative to SEQ ID NO: 118, and another polypeptide chain of the antibody heavy chain constant region comprises a K409R substitution relative to SEQ ID NO: 118 (numbered according to the EU numbering system). In certain embodiments, one or more polypeptide chains of the antibody heavy chain constant region comprise a Y349C substitution relative to SEQ ID NO: 118, and another polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution relative to SEQ ID NO: 118 (numbered according to the EU numbering system).

[0031] In another aspect, the present application provides a method for manufacturing a method of a medical device comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0032] In another aspect, the present application provides a method for manufacturing a method of a medical device comprising: (a) a first polypeptide comprising an amino acid sequence at least 90% identical (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%) to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising an amino acid sequence at least 90% identical (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%) to the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide 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: 195.

[0033] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 195.

[0034] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 195.

[0035] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 195.

[0036] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0037] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0038] In some embodiments, the proteins provided herein are (a) a first polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 270; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195.

[0039] In some embodiments, the proteins provided herein include polypeptides 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: 270. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 270.

[0040] In some embodiments, the proteins provided herein include polypeptides 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: 194. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 194.

[0041] In some embodiments, the proteins provided herein include polypeptides 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: 195. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 195. In some embodiments, the proteins provided herein include polypeptides comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 195.

[0042] In another aspect, the present application provides a method for manufacturing a method of a medical device comprising: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 271; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 272; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 273.

[0043] In another aspect, the present application provides a pharmaceutical composition comprising a protein disclosed herein and a pharmaceutically acceptable carrier.

[0044] In another aspect, the present application provides a cell comprising one or more nucleic acids encoding the proteins disclosed herein.

[0045] In another aspect, the present application provides a method of enhancing tumor cell death, the method comprising exposing tumor cells and natural killer cells to an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0046] In another aspect, the application provides methods of treating cancer, the methods comprising administering to a subject in need thereof an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cancer is selected from the group consisting of 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 large B-cell lymphoma, and acute lymphocytic leukemia (ALL).

[0047] In another aspect, the present application provides a method of enhancing B cell death, the method comprising exposing B cells and natural killer cells to an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0048] In another aspect, the present application provides a method of treating an autoimmune inflammatory disease, the method comprising administering to a subject in need thereof an effective amount of a protein disclosed herein or a pharmaceutical composition disclosed herein.

[0049] In some embodiments of the proteins disclosed herein, the protein is a purified protein. In some embodiments, the protein is purified using 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. [Brief explanation of the drawings]

[0050] [Figure 1] 1 is a representation of a heterodimeric, multispecific antibody, e.g., a trispecific binding protein (TriNKET). Each arm can represent either an NKG2D-binding domain or a BAFF-R-binding domain. In some embodiments, the NKG2D-binding domain and the BAFF-R-binding domain can share a common light chain.

[0051] [Figure 2-1]Figures 2A-2E illustrate five exemplary formats of multispecific binding proteins, e.g., trispecific binding proteins (TriNKETs). As shown in Figure 2A, either the NKG2D-binding domain or the BAFF-R-binding domain can be in scFv format (left arm). An antibody containing an NKG2D-targeting scFv, a BAFF-R-targeting Fab fragment, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. An antibody containing a BAFF-R-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerized antibody constant region / domain that binds to CD16 is referred to herein as F3'-TriNKET (Figure 2E). As shown in Figure 2B, both the NKG2D-binding domain and the BAFF-R-binding domain can be in scFv format. Figures 2C-2D are diagrams of antibodies with three antigen-binding sites, including two antigen-binding sites that bind BAFF-R and an NKG2D-binding site fused to a heterodimerized antibody constant region. These antibody formats are referred to herein as F4-TriNKET. Figure 2C illustrates that the two BAFF-R-binding sites are in a Fab fragment format and the NKG2D-binding site is in an scFv format. Figure 2D illustrates that the BAFF-R-binding site is in an scFv format and the NKG2D-binding site is in an scFv format. Figure 2E depicts a trispecific antibody (TriNKET) containing a BAFF-R-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerized antibody constant region / domain ("CD domain") that binds CD16. The antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, heterodimerization mutations on the antibody constant regions include K360E and K409W on one constant domain and Q347R, D399V, and F405T on the opposing constant domain (shown as a triangular lock and key shape in the CD domains). The bold bar between the heavy and light chain variable domains of the Fab fragment represents a disulfide bond. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0052] [Figure 3] Figure 3 is a representation of TriNKET in triomab form, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain derived from two parent antibodies. The triomab form can be a heterodimeric construct consisting of one-half rat antibody and one-half mouse antibody.

[0053] [Figure 4] Figure 4 is a representation of a TriNKET in the KiH common light chain format involved in knobs-into-holes (KIH) technology. KiH is a heterodimer containing two Fab fragments that bind to targets 1 and 2 and an Fc that is stabilized by a heterodimerization mutation. A TriNKET in KiH format can be a heterodimeric construct with two Fab fragments that bind to target 1 and target 2, containing two different heavy chains and a common light chain that pairs with both heavy chains.

[0054] [Figure 5] Figure 5 is a representation of TriNKET in the form of a dual variable domain immunoglobulin (DVD-Ig™) in which the target binding domains of two monoclonal antibodies are joined via a flexible, naturally occurring linker to result in a tetravalent IgG-like molecule. The DVD-Ig™ is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of a Fab fragment targeting antigen 1. The DVD-Ig™ form contains a conventional Fc.

[0055] [Figure 6]Figure 6 is a representation of TriNKET in the orthogonal Fab fragment interface (Ortho-Fab) form, a heterodimeric construct containing two Fab fragments that bind target 1 and target 2 fused to an Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0056] [Figure 7] Figure 7 is a representation of TriNKET in 2-in-1Ig format.

[0057] [Figure 8] Figure 8 is a representation of the ES form of TriNKET, a heterodimeric construct containing two different Fab fragments that bind to target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0058] [Figure 9] Figure 9 is a representation of a TriNKET of an antibody that exchanges Fab fragments by replacing the heavy chain and attached light chain (cFae) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm exchanged form (cFae) is a heterodimer containing two Fab fragments that bind to targets 1 and 2 and an Fc stabilized by a heterodimerization mutation.

[0059] [Figure 10] FIG. 10 is a representation of the SEED form of TriNKET, a heterodimer containing two Fab fragments that bind to targets 1 and 2 and an Fc stabilized by a heterodimerization mutation.

[0060] [Figure 11]Figure 11 is a representation of the LuZ-Y form of TriNKET, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs that bind to target 1 and target 2 fused to an Fc. Heterodimerization is ensured through the leucine zipper motif fused to the C-terminus of the Fc.

[0061] [Figure 12] Figure 12 is a representation of TriNKET in Cov-X somatic form.

[0062] [Figure 13-1] Figures 13A-13B are representations of the κλ-Body form of TriNKET, a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by a dimerization mutation, where one Fab fragment targeting antigen 1 contains a kappa LC and the second Fab fragment targeting antigen 2 contains a lambda LC. Figure 13A is an exemplary representation of one form of κλ-Body, and Figure 13B is an exemplary representation of another κλ-Body. [Figure 13-2] Same as above.

[0063] [Figure 14] Figure 14 is a representation of an OAsc-Fab heterodimer construct containing a Fab fragment that binds target 1 and an scFab that binds target 2, both fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0064] [Figure 15] Figure 15 is a representation of DuetMab, a heterodimeric construct containing two different Fab fragments that bind antigens 1 and 2 and an Fc that is stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct light and heavy chain pairing.

[0065] [Figure 16] Figure 16 is a representation of CrossmAb, a heterodimeric construct with two different Fab fragments that bind to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. The CL and CH1 domains and the VH and VL domains are swapped, e.g., CH1 is fused in tandem with VL and CL is fused in tandem with VH.

[0066] [Figure 17] Figure 17 is a representation of Fit-Ig, a homodimeric construct in which a Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of a Fab fragment that binds antigen 1. The construct contains wild-type Fc.

[0067] [Figure 18-1] Figures 18A-18C are line graphs showing binding of BAFF-R-targeted TriNKET derived from hCOH-2 (Figure 18A), Genentech Hu9.1-73 (Figure 18B), and ianalumab-based antigen binding sites (three versions, F3', 2-Fab, and ianalumab-mAb, which do not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 18C) to BAFF-R-positive RAJI cells. [Figure 18-2] Same as above.

[0068] [Figure 19-1] Figures 19A-19C are line graphs showing NK cell-mediated lysis of BAFF-R positive RAJI cells by primary NK cells in the presence of BAFF-R-targeting TriNKET derived from hCOH-2 (Figure 19A), Genentech Hu9.1-73 (Figure 19B), and ianalumab-based antigen binding sites (three versions, F3', 2-Fab, and ianalumab-mAb, which do not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 19C). [Figure 19-2] Same as above.

[0069] [Figure 20-1]Figures 20A-20C are line graphs showing NK cell-mediated lysis of BAFF-R positive RAJI cells by KHYG-CD16V cells in the presence of BAFF-R-targeting TriNKET derived from hCOH-2 (Figure 20A), Genentech Hu9.1-73 (Figure 20B), and ianalumab-based antigen binding sites (three versions, F3', 2-Fab, and ianalumab-mAb, which do not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 20C). [Figure 20-2] Same as above.

[0070] [Figure 21] FIG. 21 is a graph showing the fluorescence output from a blocking assay of BAFF-biotin binding to hBAFF-R expressed on CHO cells by the indicated antibodies.

[0071] [Figure 22-1] Figures 22A-22D are graphs of the fluorescence output from binding assays on CHO cells showing binding of the indicated antibodies to hBAFF-R (Figures 22A, 22B) or the fluorescence output from blocking assays of BAFF-biotin binding to BAFF-R by the indicated antibodies (Figures 22C, 22D). [Figure 22-2] Same as above.

[0072] [Figure 23-1] Figures 23A-23E are flow cytometry plots showing binding of AB0369 scFv expressed in yeast to a no-antigen control (Figure 23A), h-BAFF-R-hFc (Figure 23B), irrelevant hFc (Figure 23C), hBAFF-R-GST (Figure 23D), or irrelevant GST (Figure 23E). The vertical axis shows scFv expression as measured by detection of the Flag epitope tag, and the horizontal axis shows binding of the biotinylated control scFv of the BAFF-R construct as measured by detection of streptavidin-PE. [Figure 23-2] Same as above.

[0073] [Figure 24]Figures 24A-24B are graphs showing the binding of AB0369 or the indicated controls to human BAFF-R (Figure 24A) or cynomolgus monkey BAFF-R (Figure 24B).

[0074] [Figure 25-1] Figures 25A-25G detail a multispecificity assay of a multispecific binding protein with the BAFF-R binding site derived from AB0369. Figure 25A is a schematic of the assay. Figures 25B-25G show graphs of AB0369 (left panel), a trastuzumab negative control (center panel), or an ixekizumab positive control (right panel) in the absence (top panel) or presence (bottom panel) of the multispecific reagent (PSR). [Figure 25-2] Same as above. [Figure 25-3] Same as above.

[0075] [Figure 26] FIG. 26 is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells induced by a multispecific binding protein with the BAFF-R binding site derived from AB0369.

[0076] [Figure 27] FIG. 27 is a graph showing the fluorescence output from a binding assay demonstrating blockade of BAFF-biotin binding to human BAFF-R expressed on CHO cells by AB0369 or as indicated.

[0077] [Figure 28-1]Figures 28A-28D are flow cytometry plots showing binding of hBAFF-R-hFc-His to the parental AB0369scFv or clones selected from the library produced by affinity maturation expressed in yeast after successive rounds of selection: Figure 28A shows binding to the parental AB0369scFv, Figure 28B shows binding to samples from the first round of clone selection, Figure 28C shows binding to samples from the second round of clone selection, and Figure 28D shows binding to the output from the second round of clone selection. [Figure 28-2] Same as above.

[0078] [Figure 29-1] Figures 29A-29E are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity-matured scFv clones expressed in yeast. Figure 29A shows binding to the parent AB0369, Figure 29B shows binding to AB0605, Figure 29C shows binding to AB0622, Figure 29D shows binding to AB0622, and Figure 29E shows binding to the ianalumab-based antigen-binding site. [Figure 29-2] Same as above.

[0079] [Figure 30-1] Figures 30A-30C are graphs demonstrating BAFF-R binding and cytotoxicity of multispecific binding proteins developed from affinity maturation of AB0369. Figure 30A 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 30B is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells induced by multispecific binding proteins with BAFF-R binding sites derived from the indicated clones. Figure 30C is a graph showing KHYG-1-CD16aV cytotoxicity assay of Ramos cells induced by multispecific binding proteins with BAFF-R binding sites derived from AB0622. [Figure 30-2] Same as above.

[0080] [Figure 31-1] Figures 31A-31E detail a multispecificity assay of multispecific binding proteins with BAFF-R binding sites derived from AB00605 and AB0606. Figure 31A is a schematic of the assay. Figures 31B-31E show graphs of AB0605 (left panel) or AB0606 (right panel) in the absence (top panel) or presence (bottom panel) of the multispecific reagent (PSR). [Figure 31-2] Same as above. [Figure 31-3] Same as above.

[0081] [Figure 32] Figures 32A-32C are flow cytometry plots showing binding of hBAFF-R-hFc-His to the parental AB0369scFv or clones selected from the library produced by affinity maturation and expressed in yeast after successive rounds of selection: Figure 32A shows binding to the parental AB0369scFv, Figure 32B shows binding to samples from the first round of clone selection, and Figure 32C shows binding to samples from the second round of clone selection.

[0082] [Figure 33-1] Figures 33A-33E are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity-matured scFv clones expressed in yeast. Figure 33A shows binding to the parent AB0369, Figure 33B shows binding to AB0679, Figure 33C shows binding to AB0681, Figure 33D shows binding to AB0682, and Figure 33E shows binding to the ianalumab-based antigen-binding site. [Figure 33-2] Same as above.

[0083] [Figure 34-1]Figures 34A-34C are graphs demonstrating BAFF-R binding to multispecific binding proteins developed from affinity maturation of AB0369. Figure 34A 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 34B is a graph showing binding of multispecific binding proteins with BAFF-R binding sites derived from the indicated clones to cynomolgus monkey BAFF-R expressed on CHO cells. Figure 34C is a graph showing fluorescence output from a binding assay demonstrating blockade of BAFF-biotin binding to BAFF-R expressed on CHO cells by the indicated antibodies. [Figure 34-2] Same as above.

[0084] [Figure 35] Figure 35 is a graph showing KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by multispecific binding proteins with BAFF-R binding sites from AB0679, AB0568, or Tool-F3' positive control.

[0085] [Figure 36-1] Figures 36A-36D are flow cytometry plots showing binding of hBAFF-R-hFc-His to parental AB0369scFv clones selected from a library produced by affinity maturation expressed in yeast after successive rounds of selection: Figure 36A shows binding to the parental AB0369scFv, Figure 36B shows binding to samples from the first round of clone selection, Figure 36C shows binding to samples from the second round of clone selection, and Figure 36D shows binding to samples from the third round of clone selection. [Figure 36-2] Same as above.

[0086] [Figure 37-1]Figures 37A-37F are flow cytometry plots showing binding of hBAFF-R-hFc-His to AB0369 and affinity-matured scFv clones expressed in yeast. Figure 37A shows binding to the parent AB0369, Figure 37B shows binding to AB0682, Figure 37C shows binding to AB0898, Figure 37D shows binding to AB0899, Figure 37E shows binding to AB0900, and Figure 37F shows binding to the ianalumab-based antigen-binding site. [Figure 37-2] Same as above.

[0087] [Figure 38] FIG. 38 is a graph showing KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by multispecific binding proteins with BAFF-R binding sites derived from AB0898, AB0899, or AB0900.

[0088] [Figure 39] Figures 39A-39C show graphs of the differential scanning calorimetry (DSC) profiles of AB0898 (Figure 39A), AB0899 (Figure 39B), and AB0900 (Figure 39C).

[0089] [Figure 40] FIG. 40 shows flow cytometry plots of binding of scFv clones expressed in yeast to biotinylated hBAFFR-Fc before (left) and after (right) challenge by incubation with 1 mM non-biotinylated hBAFFR-Fc.

[0090] [Figure 41-1] Figures 41A-41B show flow cytometry plots of binding of scFv clones expressed in yeast to biotinylated hBAFFR-Fc before (Figure 41A) and after (Figure 41B) challenge by incubation with 1 mM non-biotinylated hBAFFR-Fc. The clones tested (from left to right) are AB1080, AB1081, AB1084, AB1085, and ianalumab. [Figure 41-2] Same as above. [Figure 41-3] Same as above. [Figure 41-4] Same as above.

[0091] [Figure 42] Figures 42A-42B are graphs showing the binding of the indicated antibody clones to human BAFF-R (Figure 42A) or cynomolgus monkey BAFF-R (Figure 42B).

[0092] [Figure 43-1] Figures 43A-43I detail a multispecific assay of multispecific binding proteins with BAFF-R binding sites derived from AB1080 or AB1081. Figure 43A is a schematic of the assay. Figures 43B-I show graphs of AB1080 (left panel), AB1081 (center panel), a trastuzumab negative control (center right panel), or an ixekizumab positive control (right panel) in the absence (top panel) or presence (bottom panel) of the multispecific reagent (PSR). [Figure 43-2] Same as above. [Figure 43-3] Same as above.

[0093] [Figure 44] Figures 44A-44B show graphs of KHYG-1-CD16aV cytotoxicity assays of BJAB cells induced by multispecific binding proteins with BAFF-R binding sites derived from AB1080 (Figure 44A) or AB1085 (Figure 44B) compared to the Tool positive control.

[0094] [Figure 45] FIG. 45 is a graph showing the fluorescence output from a blocking assay of BAFF-biotin binding to human BAFF-R expressed on CHO cells by the indicated antibody clones.

[0095] [Figure 46-1]Figures 46A-46D show graphs of nano-dual scanning fluorimetry (nano-DSF) analysis of multispecific binding proteins with BAFF-R binding sites derived from AB1080 (Figure 46A), AB1081 (Figure 46B), AB1084 (Figure 46C), and AB1085 (Figure 46D). [Figure 46-2] Same as above.

[0096] [Figure 47] FIG. 47 shows a graph of hydrophobic interaction chromatography (HIC) analysis of multispecific binding proteins with BAFF-R binding sites derived from the indicated antibodies.

[0097] [Figure 48] FIG. 48 shows a graph of the HIC analysis of AB1612 compared to the indicated benchmark biologics.

[0098] [Figure 49] Figures 49A-49B are graphs showing binding of the indicated antibody clones to cynomolgus monkey BAFF-R (Figure 49A) or human BAFF-R (Figure 49B).

[0099] [Figure 50] FIG. 50 is a graph depicting the fluorescence output from a binding assay showing blockage of BAFF-biotin binding to human BAFF-R expressed on CHO cells by the indicated antibodies.

[0100] [Figure 51] Figures 51A-51C show the surface charge distribution of the BAFF-R binding arm of AB1424 / 1612 F3'TriNKET. Three orientations are shown: both faces (left panel: front view; center panel: back view) as well as the antigen-inducing surface (right panel: top view). Positively charged regions are colored blue, negatively charged regions are colored red, and hydrophobic surfaces are colored white.

[0101] [Figure 52-1]Figures 52A-52E are graphs showing the evaluation of the surface patches and CDR lengths of the BAFF-R binding arm of AB1424 / 1612 F3'TriNKET. The solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F3'TriNKET against a database of 377 late-stage therapeutic antibodies. In Figures 52A and 52B, the two inner dotted lines indicate 2 standard deviations (>95% of the reference molecules in this region), while the two outermost dotted lines indicate 3 standard deviations (>99.7% of the reference molecules in this region). In each plot in Figures 52C-52E, there are two dotted lines, one closer to the solid line and the other farther away. A dotted line closer to the solid line indicates 2 standard deviations (>95% of the reference molecules in this region), while a dotted line further from the solid line indicates 3 standard deviations (>99.7% of the reference molecules in this region). [Figure 52-2] Same as above.

[0102] [Figure 53] Figures 53A-53C show the surface charge distribution of the NKG2D-binding arm of AB1424 / 1612 F3'TriNKET. Three orientations are shown: both faces (left panel: front view; center panel: back view) as well as the antigen-induced surface (right panel: top view). Positively charged regions are colored blue, negatively charged regions are colored red, and hydrophobic surfaces are colored white.

[0103] [Figure 54-1]Figures 54A-54E are graphs showing the evaluation of the surface patches and CDR lengths of the NKG2D-R binding arm of AB1424 / 1612 F3'TriNKET. The solid lines and corresponding arrows indicate the scoring of the NKG2D-R binding arm of AB1424 / 1612 F3'TriNKET against a database of 377 late-stage therapeutic antibodies. In Figures 54A and 54B, the two inner dotted lines indicate 2 standard deviations (>95% of reference molecules within this region), while the two outermost dotted lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot in Figures 54C-54E, there are two dotted lines, one closer to the solid line and the other farther away. A dotted line closer to the solid line indicates 2 standard deviations (>95% of the reference molecules in this region), while a dotted line further from the solid line indicates 3 standard deviations (>99.7% of the reference molecules in this region). [Figure 54-2] Same as above.

[0104] [Figure 55-1] Figures 55A-55B are chromatograms showing HIC analysis of AB1424 / 1612 F3'TriNKET (Figure 55A) and a comparison with adalimumab and pembrolizumab (Figure 55B). [Figure 55-2] Same as above.

[0105] [Figure 56] Figure 56 is a graph showing capillary isoelectric focusing (cIEF) profiling of AB1424 / 1612 F3'TriNKET.

[0106] [Figure 57] Figures 57A-57B are graphs showing DSC profiling of AB1424 / 1612 F3'TriNKET in PBS pH 7.4 (Figure 57A) and HST pH 6.0 (Figure 57B).

[0107] [Figure 58]Figures 58A-58B are graphs showing n-curve analysis (Figure 58A) and confidence intervals (Figure 58B) of AB1424 / 1612 F3'TriNKET binding cell-based BAFF-R by Kinexa.

[0108] [Figure 59] Figures 59A-59B are graphs showing the binding of AB1424 / 1612 F3'TriNKET and the corresponding parental mAb to isogenic human BAFF-R-CHO cells (Figure 59A) and cynomolgus monkey BAFF-R-CHO cells (Figure 59B).

[0109] [Figure 60] Figures 60A-60F are graphs showing binding of AB1424 / 1612 F3'TriNKET to BAFF-R+ tumor cell lines. Titrations were performed in the presence of BJAB (Figure 60A), Raji (Figure 60B), RL (Figure 60C), Rs4;11 (Figure 60D), Jeko-1 (Figure 60E), and SUDHL-6 cells (Figure 60F). FOB = fold over background of stained vs. unstained samples.

[0110] [Figure 61-1] Figures 61A-61H are graphs showing surface plasmon resonance (SPR) of AB1424 / 1612 F3'TriNKET binding to human NKG2D. Colored lines represent raw data, and black traces represent 1:1 binding fits (top panels). The corresponding steady-state fits are shown (bottom panels). Vertical lines represent steady-state KD. [Figure 61-2] Same as above.

[0111] [Figure 62-1] Figures 62A-62H are graphs showing SPR of AB1424 / 1612 F3'TriNKET binding to cynomolgus monkey NKG2D. The colored lines represent the raw data, and the black traces represent the 1:1 binding fits (top panel). The corresponding steady-state fits are shown (bottom panel). The vertical lines represent the steady-state KD. [Figure 62-2] Same as above.

[0112] [Figure 63-1] Figures 63A-63H are graphs showing SPR of AB1424 / 1612 F3'TriNKET binding to human CD16a V158 (top panel) or trastuzumab (bottom panel). The colored lines represent the raw data, and the black traces represent the 1:1 binding fit. [Figure 63-2] Same as above. [Figure 63-3] Same as above. [Figure 63-4] Same as above.

[0113] [Figure 64-1] Figures 64A-64P are graphs showing SPR of AB1424 / 1612 F3'TriNKET (top panel) or trastuzumab (bottom panel) binding to human CD16a F158. The colored lines represent the raw data, and the black trace represents a 1:1 binding fit (top panel). [Figure 64-2] Same as above. [Figure 64-3] Same as above. [Figure 64-4] Same as above. [Figure 64-5] Same as above. [Figure 64-6] Same as above. [Figure 64-7] Same as above. [Figure 64-8] Same as above.

[0114] [Figure 65-1] Figures 65A-65H are graphs showing SPR of AB1424 / 1612 F3'TriNKET binding to cynomolgus monkey CD16. The colored lines represent the raw data, and the black traces represent the 1:1 binding fits (top panel). The corresponding steady-state fits (bottom panel). The vertical lines represent the steady-state KD. [Figure 65-2] Same as above. [Figure 65-3] Same as above. [Figure 65-4] Same as above.

[0115] [Figure 66] Figure 66 is a graph showing SPR of AB1424 / 1612 F3'TriNKET binding to NKG2D (brown), CD16a (purple), or mixed CD16a and NKG2D (blue) surfaces.

[0116] [Figure 67] Figures 67A-67B are graphs of sensorgrams depicting the binding of BAFF-R (800 nM) followed by hNKG2D (7 μM) to captured AB1424 / 1612 F3'TriNKET (Figure 67A) or the reverse target order of binding with human NKG2D (7 μM) followed by BAFF-R (800 nM) (Figure 67B).

[0117] [Figure 68] Figures 68A-68B are graphs showing SPR analysis of the binding of BAFF-R and TACI to immobilized AB1424 / 1612 F3'TriNKET (Figure 68A) and to specific anti-TACI mAb (Figure 68B).

[0118] [Figure 69] Figures 69A-69B are graphs showing binding of AB1424 / 1612 F3'TriNKET to parental cells that do not express BCMA (Figure 69A) and binding to isogenic BCMA+ cells compared to a control mAb specific anti-BCMA (Figure 69B).

[0119] [Figure 70] Figures 70A-70B are graphs showing the binding of AB1424 / 1612 F3'TriNKET to isogenic BAFFR+ CHO cells (Figure 70A) and lack of reactivity with the parental CHO line (Figure 70B).

[0120] [Figure 71-1]Figures 71A-71G detail the polyspecific assay of AB1424 / 1612 F3'TriNKET. Figure 71A is a schematic of the assay. Figures 71B-71G show graphs of AB1424 / 1612 F3'TriNKET (left panel), a trastuzumab negative control (center panel), or an ixekizumab positive control (right panel) in the absence (top panel) or presence (bottom panel) of the polyspecific reagent (PSR). [Figure 71-2] Same as above. [Figure 71-3] Same as above.

[0121] [Figure 72] Figures 72A-72C show graphs of RL cell cytotoxicity assays induced by AB1424 / 1612 F3'TriNKET (blue) or parental monoclonal antibody (red) using NK cells from three donors.

[0122] [Figure 73] Figures 73A-73D show a schematic representation of AB1424 / 1612 F3'TriNKET and controls to elucidate the mechanism of action.

[0123] [Figure 74] Figure 74 shows a graph of a KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F3'TriNKET lacking NKG2D binding (black), or silenced AB1424 / 1612 F3'TriNKET-Fc (red), or palivizumab F3'TriNKET (gray).

[0124] [Figure 75-1] Figures 75A-75H are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (top panel) and trastuzumab (bottom panel) to human CD64. The raw sensorgrams (colored) are overlaid with a 1:1 fit curve (black). [Figure 75-2] Same as above. [Figure 75-3] Same as above. [Figure 75-4] Same as above.

[0125] [Figure 76-1] Figures 76A-76H are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (top panel) and trastuzumab (bottom panel) to cynomolgus monkey CD64. Raw sensorgrams (colored) are overlaid with 1:1 fit curves (black). [Figure 76-2] Same as above. [Figure 76-3] Same as above. [Figure 76-4] Same as above.

[0126] [Figure 77-1] Figures 77A-77P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 77A-H) and trastuzumab (Figures 77I-P) to human CD32a H131. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 77-2] Same as above. [Figure 77-3] Same as above. [Figure 77-4] Same as above. [Figure 77-5] Same as above. [Figure 77-6] Same as above. [Figure 77-7] Same as above.

[0127] [Figure 78-1] Figures 78A-78P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 78A-H) and trastuzumab (Figures 78I-P) to human CD32a R131. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 78-2] Same as above. [Figure 78-3] Same as above. [Figure 78-4] Same as above. [Figure 78-5] Same as above.

[0128] [Figure 79-1] Figures 79A-79P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 79A-H) and trastuzumab (Figures 79I-P) to human CD32b. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 79-2] Same as above. [Figure 79-3] Same as above. [Figure 79-4] Same as above.

[0129] [Figure 80-1] Figures 80A-80P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 80A-H) and trastuzumab (Figures 80I-P) to human CD16b. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 80-2] Same as above. [Figure 80-3] Same as above. [Figure 80-4] Same as above.

[0130] [Figure 81-1] Figures 81A-81H are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (top panel) and trastuzumab (bottom panel) to cynomolgus monkey CD16. [Figure 81-2] Same as above.

[0131] [Figure 82-1]Figures 82A-82P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 82A-H) and trastuzumab (Figures 82I-P) to human FcRn at pH 6.0. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 82-2] Same as above. [Figure 82-3] Same as above. [Figure 82-4] Same as above.

[0132] [Figure 83-1] Figures 83A-83P are graphs of sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (Figures 83A-H) and trastuzumab (Figures 83I-P) to cynomolgus monkey FcRn at pH 6.0. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 83-2] Same as above. [Figure 83-3] Same as above. [Figure 83-4] Same as above.

[0133] [Figure 84-1] Figures 84A-84H are graphs of raw sensorgrams showing the binding of AB1424 / 1612 F3'TriNKET (top panel) and trastuzumab (bottom panel) to human FcRn (left panel) and cynomolgus monkey FcRn (right panel) at pH 7.4. [Figure 84-2] Same as above. [Figure 84-3] Same as above. [Figure 84-4] Same as above.

[0134] [Figure 85] Figure 85 shows a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by two lots of AB1424 / 1612 F3'TriNKET (blue and red) or human IgG1k (gray).

[0135] [Figure 86-1] Figure 86A shows a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by two lots of AB1424 / 1612 F3'TriNKET (blue and red) or human IgG1k (gray).

[0136] [Figure 86-2] Figure 86B shows a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F3'TriNKET at 50% (red), 100% (blue), and 200% (green) of the nominal drug concentration (NDC).

[0137] [Figure 87] Figures 87A-87B show PEG precipitation Cm plots of AB1424 / 1612 F3'TriNKET in histidine (Figure 87A) and acetate (Figure 87B).

[0138] [Figure 88] Figures 88A-88B show PEG precipitation Cm plots of adalimumab in histidine (Figure 88A) and acetate (Figure 88B).

[0139] [Figure 89] Figures 89A-89C show kD plots of adalimumab in acetate (Figure 89A), histidine (Figure 89B), and phosphate (Figure 89C).

[0140] [Figure 90] Figures 90A-90C show kD plots of AB1424 / 1612 F3'TriNKET in acetate (Figure 90A), histidine (Figure 90B), and phosphate (Figure 90C).

[0141] [Figure 91] Figure 91 is a plot of viscosity versus concentration of AB1424 / 1612 F3'TriNKET at 25°C.

[0142] [Figure 92] Figure 92 is a chromatogram of size exclusion chromatography (SEC) analysis of AB1424 / 1612 F3'TriNKET after 4 weeks at 40°C in HST, pH 6.0 compared to the control.

[0143] [Figure 93] Figure 93 is a chromatogram of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of AB1424 / 1612 F3'TriNKET after 4 weeks at 40°C in HST, pH 6.0 compared to the control.

[0144] [Figure 94] Figure 94 is a graph showing the cIEF profiling of AB1424 / 1612 F3'TriNKET in HST, pH 6.0 compared to the control.

[0145] [Figure 95-1] Figures 95A-95C show binding of AB1424 / 1612 F3'TriNKET to hBAFF-R, hNKG2D, and hCD16aV after 4 weeks at 40°C in HST, pH 6.0, compared to controls. Figure 95A is a graph showing binding to BJAB cells (BAFF-R), and Figure 95B is a sensorgram showing binding to hNKG2D by SPR. Figure 95C is a sensorgram showing binding to hCD16a V158 by SPR. Colored sensorgrams represent raw data, and the black overlay represents a kinetic fit of the raw data. [Figure 95-2] Same as above.

[0146] [Figure 96] Figure 96 shows a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F3'TriNKET after 1 week (red), 2 weeks (green), and 3 weeks (purple) at 40°C in HST, pH 6.0 compared to control (blue).

[0147] [Figure 97] Figures 97A-97C show the surface charge distribution of the BAFF-R binding arm of AB1424 / 1612 F4 TriNKET. Three orientations are shown: both faces (left panel: front view; center panel: back view) as well as the antigen-inducing surface (right panel: top view). Positively charged regions are colored blue, negatively charged regions are colored red, and hydrophobic surfaces are colored white.

[0148] [Figure 98-1] Figures 98A-98E are graphs showing the evaluation of the surface patches and CDR lengths of the BAFF-R binding arm of AB1424 / 1612 F4 TriNKET. The solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F4 TriNKET against a database of 377 late-stage therapeutic antibodies. In Figures 98A and 98B, the two inner dotted lines indicate two standard deviations (>95% of the reference molecules in this region), while the two outermost dotted lines indicate three standard deviations (>99.7% of the reference molecules in this region). In each plot of Figures 98C-98E, there are two dotted lines—one closer to the solid line and one further from it. The dotted line closer to the solid line indicates two standard deviations (>95% of the reference molecules in this region), while the dotted line further from the solid line indicates three standard deviations (>99.7% of the reference molecules in this region). [Figure 98-2] Same as above.

[0149] [Figure 99] Figures 99A-99C show the surface charge distribution of the NKG2D-binding arm of AB1424 / 1612 F4 TriNKET. Three orientations are shown: both faces (left panel: front view; center panel: back view) as well as the antigen-induced surface (right panel: top view). Positively charged regions are colored blue, negatively charged regions are colored red, and hydrophobic surfaces are colored white.

[0150] [Figure 100-1]Figures 100A-100E are graphs showing the evaluation of the surface patches and CDR lengths of the NKG2D-R binding arm of AB1424 / 1612 F4 TriNKET. The solid lines and corresponding arrows indicate the scoring of the BAFF-R binding arm of AB1424 / 1612 F3' TriNKET against a database of 377 late-stage therapeutic antibodies. In Figures 100A and 100B, the two inner dotted lines indicate 2 standard deviations (>95% of reference molecules within this region), while the two outermost dotted lines indicate 3 standard deviations (>99.7% of reference molecules within this region). In each plot in Figures 100C-100E, there are two dotted lines—one closer to the solid line and one further from it. A dotted line closer to the solid line indicates 2 standard deviations (>95% of the reference molecules in this region), while a dotted line further from the solid line indicates 3 standard deviations (>99.7% of the reference molecules in this region). [Figure 100-2] Same as above.

[0151] [Figure 101] Figures 101A-101C are chromatograms of the SEC analysis of three lots of AB1424 / 1612 F4 TriNKET.

[0152] [Figure 102] Figure 102 is a graph showing the cIEF profiling of three lots of AB1424 / 1612 F4 TriNKET.

[0153] [Figure 103-1] Figures 103A-103B are: Figure 103A is a graph of HIC analysis of AB1424 / 1612 F4 TriNKET compared to the indicated benchmark commercial antibodies; Figure 103B is a graph of thermal stability analysis of AB1424 / 1612 F4 TriNKET by DSC. [Figure 103-2] Same as above.

[0154] [Figure 104]Figures 104A-104B show extracted ion chromatograms (XIC) for the engineered disulfide pair (non-reduced and reduced) in Fc and the most intense charge state for that peptide pair.

[0155] [Figure 105-1] Figures 105A-105B show the XIC for the engineered disulfide pair (non-reduced and reduced) in the scFv and the strongest charge state for that peptide pair. [Figure 105-2] Same as above.

[0156] [Figure 106] Figures 106A-106B are graphs showing the binding of AB1424 / 1612 F4 TriNKET, parental mAb, and F4-palivizumab to human BAFF-R+ isogenic CHO cells (Figure 106A) and cynomolgus monkey BAFF-R+ isogenic CHO cells (Figure 106B).

[0157] [Figure 107-1] Figures 107A-107L are graphs of SPR sensorgrams of AB1424 / 1612 F4 TriNKET binding to human NKG2D. [Figure 107-2] Same as above. [Figure 107-3] Same as above. [Figure 107-4] Same as above.

[0158] [Figure 108-1] Figures 108A-108P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 108A-H) and trastuzumab (Figures 108I-P) to human CD32a R131. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 108-2] Same as above. [Figure 108-3] Same as above. [Figure 108-4] Same as above.

[0159] [Figure 109-1] Figures 109A-109H are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (top panel) and trastuzumab (bottom panel) to human CD16a V158. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 109-2] Same as above.

[0160] [Figure 110-1] Figures 110A-110H are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (top panel) and trastuzumab (bottom panel) to human CD16a V158. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 110-2] Same as above.

[0161] [Figure 111-1] Figures 111A-111H are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (top panel) and trastuzumab (bottom panel) to human CD64. The raw sensorgrams (colored) are overlaid with a 1:1 fit curve (black). [Figure 111-2] Same as above.

[0162] [Figure 112-1] Figures 112A-112H are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (top panel) and trastuzumab (bottom panel) to cynomolgus monkey CD64. Raw sensorgrams (colored) are overlaid with 1:1 fit curves (black). [Figure 112-2] Same as above.

[0163] [Figure 113-1]Figures 113A-113P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 113A-H) and trastuzumab (Figures 113I-P) to human CD32a H131. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 113-2] Same as above. [Figure 113-3] Same as above. [Figure 113-4] Same as above.

[0164] [Figure 114-1] Figures 114A-114P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 114A-H) and trastuzumab (Figures 114I-P) to human CD32b. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 114-2] Same as above. [Figure 114-3] Same as above. [Figure 114-4] Same as above.

[0165] [Figure 115-1] Figures 115A-115P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 115A-H) and trastuzumab (Figures 115I-P) to human CD16b. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 115-2] Same as above. [Figure 115-3] Same as above. [Figure 115-4] Same as above.

[0166] [Figure 116-1]Figures 116A-116P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 116A-H) and trastuzumab (Figures 116I-P) to human FcRn at pH 6.0. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 116-2] Same as above. [Figure 116-3] Same as above. [Figure 116-4] Same as above.

[0167] [Figure 117-1] Figures 117A-117P are graphs of sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (Figures 117A-H) and trastuzumab (Figures 117I-P) to cynomolgus monkey FcRn at pH 6.0. For each molecule, the top panel represents the raw sensorgram, and the bottom panel represents the steady-state affinity fit. [Figure 117-2] Same as above. [Figure 117-3] Same as above. [Figure 117-4] Same as above.

[0168] [Figure 118-1] Figures 118A-118H are graphs of raw sensorgrams showing the binding of AB1424 / 1612 F4 TriNKET (top panel) and trastuzumab (bottom panel) to human FcRn (left panel) and cynomolgus monkey FcRn (right panel) at pH 7.4. [Figure 118-2] Same as above.

[0169] [Figure 119] Figure 119 is a graph showing SPR of AB1424 / 1612 F4 TriNKET binding to NKG2D (brown), CD16a (purple), or mixed CD16a and NKG2D (blue) surfaces.

[0170] [Figure 120]Figures 120A-120B are graphs showing the sequential saturation of BAFF-R and NKG2D by AB1424 / 1612 F4 TriNKET.

[0171] [Figure 121-1] Figures 121A-121I detail the polyspecific assay of AB1424 / 1612 F4 TriNKET. Figure 121A is a schematic of the assay. Figures 121B-121I show graphs of AB1424 / 1612 F4 TriNKET (left panel), trastuzumab (center left panel), rituximab (center right panel), or ixekizumab (right panel) in the absence (top panel) or presence (bottom panel) of the polyspecific reagent (PSR). [Figure 121-2] Same as above.

[0172] [Figure 122] Figure 122 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (blue) and human IgG1k (gray).

[0173] [Figure 123] Figure 123 is a graph of resting hNK-induced cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (blue) and parental mAb (red).

[0174] [Figure 124] Figure 124 is a chromatogram of the SEC analysis of AB1424 / 1612 F4 TriNKET after 4 weeks at 40°C in HST, pH 6.0 compared to the control.

[0175] [Figure 125] FIG. 125 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after 4 weeks at 40° C. in HST, pH 6.0 compared to control.

[0176] [Figure 126] Figure 126 is a graph showing the cIEF profiling of AB1424 / 1612 F4 TriNKET after 4 weeks at 40°C in HST, pH 6.0 compared to the control.

[0177] [Figure 127] Figure 127 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after 4 weeks at 40°C in HST, pH 6.0 compared to control.

[0178] [Figure 128] Figures 128A-128B are graphs of sensorgrams showing SPR of hCD16aV binding to AB1424 / 1612 F4 TriNKET after 4 weeks at 40°C in HST, pH 6.0 (Figure 128B) compared to the control (Figure 128A).

[0179] [Figure 129] Figure 129 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) after 4 weeks at 40°C in HST, pH 6.0 compared to control (blue).

[0180] [Figure 130] Figure 130 is a chromatogram of the SEC analysis of AB1424 / 1612 F4 TriNKET after forced oxidation compared to the control.

[0181] [Figure 131] Figure 131 is a chromatogram of reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after forced oxidation compared to the control.

[0182] [Figure 132] FIG. 132 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after forced oxidation.

[0183] [Figure 133] Figures 133A-133B are graphs of sensorgrams showing SPR of hCD16aV binding to AB1424 / 1612 F4 TriNKET control (Figure 133A) and after forced oxidation (Figure 133B).

[0184] [Figure 134] Figure 134 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) and control (blue) after forced oxidation.

[0185] [Figure 135] Figure 135 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after long-term low pH stress compared to the control.

[0186] [Figure 136] FIG. 136 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after long-term low pH stress compared to the control.

[0187] [Figure 137] Figure 137 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after long-term low pH stress compared to control.

[0188] [Figure 138] FIG. 138 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells after chronic low pH stress compared to control.

[0189] [Figure 139] Figures 139A-139B are graphs of sensorgrams showing SPR of hCD16aV binding to AB1424 / 1612 F4 TriNKET after prolonged low pH stress (Figure 139B) compared to the control (Figure 139A).

[0190] [Figure 140] Figure 140 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) and control (blue) after long-term low pH stress.

[0191] [Figure 141] FIG. 141 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after long-term high pH stress compared to the control.

[0192] [Figure 142] FIG. 142 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after long-term high pH stress compared to the control.

[0193] [Figure 143] Figure 143 is a graph showing cIEF profiling of AB1424 / 1612 F4 TriNKET after long-term high pH stress compared to control.

[0194] [Figure 144] FIG. 144 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells (red) after long-term high pH stress compared to control (blue).

[0195] [Figure 145] Figures 145A-145B are graphs of sensorgrams showing SPR of hCD16aV binding to AB1424 / 1612 F4 TriNKET after prolonged high pH stress (Figure 145B) compared to the control (Figure 145A).

[0196] [Figure 146]Figure 146 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) and control (blue) after long-term high pH stress.

[0197] [Figure 147] Figure 147 is a chromatogram of the SEC analysis of AB1424 / 1612 F4 TriNKET after six freeze / thaw cycles compared to the control.

[0198] [Figure 148] Figure 148 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after six freeze / thaw cycles compared to a control.

[0199] [Figure 149] Figure 149 is a graph showing the binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells (red) after six freeze / thaw cycles compared to the control (blue).

[0200] [Figure 150] Figure 150 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) and control (blue) after six freeze / thaw cycles.

[0201] [Figure 151] Figure 151 is a chromatogram of SEC analysis of AB1424 / 1612 F4 TriNKET after agitation stress compared to the control.

[0202] [Figure 152] FIG. 152 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after agitation stress compared to control.

[0203] [Figure 153]FIG. 153 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells (red) after agitation stress compared to control (blue).

[0204] [Fig. 154] Figure 154 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (red) and control (blue) after agitation stress.

[0205] [Figure 155-1] Figures 155A-155B are chromatograms of SEC analysis of AB1424 / 1612 F4 TriNKET Protein A eluate before (Figure 155A) and after (Figure 155B) the low pH hold. [Figure 155-2] Same as above.

[0206] [Figure 156] Figure 156 is a graph showing the cIEF profiling of AB1424 / 1612 F4 TriNKET after a low pH hold compared to the control.

[0207] [Figure 157] Figure 157 is a graph showing reduced CE-SDS analysis of AB1424 / 1612 F4 TriNKET after a low pH hold compared to a control.

[0208] [Figure 158] Figure 158 is a graph showing binding of AB1424 / 1612 F4 TriNKET to hBAFF-R+ cells (blue) after a low pH hold compared to the control (red).

[0209] [Figure 159] Figure 159 is a graph of KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F4 TriNKET (green) and control (red) after low pH hold.

[0210] [Figure 160] Figures 160A-160B are graphs showing binding of AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), and the parental mAb (black) to KHYG-1 (Figure 160A) and KHYG-1-CD16V (Figure 160B) cell lines.

[0211] [Figure 161] Figures 161A-161B are graphs showing the surface retention of BAFF-R on RL cells exposed to AB1424 / 1612 F3' TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), and parental mAb (black) (Figure 161A) and activation by IL-2 (Figure 161B).

[0212] [Figure 162] Figure 162 is a graph showing the surface retention of BAFF-R on Raji cells exposed to AB1424 / 1612 F3' TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), and parental mAb (black).

[0213] [Figure 163] Figure 163 is a graph of resting human NK cell-induced cytotoxicity assay of RL cells after incubation with AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), and human IgG1k (gray).

[0214] [Fig. 164] Figures 164A-164B are graphs of resting human NK cell-induced cytotoxicity assays of RL cells after incubation with AB1424 / 1612 F3' TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), F3' control (black), and F4 control (gray). Cells were co-cultured with control (Figure 164A) or IL-2 (Figure 164B).

[0215] [Figure 165] Figure 165 is a graph of a KHYG-1-CD16aV cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F3'TriNKET lacking NKG2D binding (black), or AB1424 / 1612 F3'TriNKET-Fc silenced (red), or palivizumab F3'TriNKET (gray).

[0216] [Figure 166] Figure 166 is a graph of resting human NK cell-induced cytotoxicity assay of BJAB cells induced by AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F3'TriNKET lacking NKG2D binding (black), or AB1424 / 1612 F3'TriNKET-Fc silenced (red), or palivizumab F3'TriNKET (gray).

[0217] [Figure 167] Figure 167 is a graph of resting human NK cell-induced cytotoxicity assay of RL cells after incubation with AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), AB1424 / 1612 F3'TriNKET plus soluble MICA (black), and AB1424 / 1612 F4 TriNKET plus soluble MICA (gray).

[0218] [Figure 168] Figure 168 is a graph of resting human NK cell-induced cytotoxicity assay of RL cells after incubation with AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), AB1424 / 1612 F3'TriNKET plus BAFF (black), and AB1424 / 1612 F4 TriNKET plus BAFF (gray).

[0219] [Figure 169] Figure 169 is a graph of interferon gamma (IFNγ) and CD107a production by BJAB cells after incubation with AB1424 / 1612 F3′ TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), F3′-palivizumab (light gray), and F4-palivizumab (dark gray).

[0220] [Figure 170] Figure 170 is a graph of phagocytosis of BJAB cells by M0 macrophages after incubation with AB1424 / 1612 F3'TriNKET (blue), AB1424 / 1612 F4 TriNKET (red), parental mAb (black), and Fc-silenced AB1424 / 1612 F3'TriNKET (pink).

[0221] [Figure 171] Figure 171 is a graph of a human serum-induced cytotoxicity assay of Raji cells after incubation with rituximab (black), AB1424 / 1612 F3'TriNKET (blue), or AB1424 / 1612 F3'TriNKET.

[0222] [Fig. 172] Figures 172A-172E are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3'TriNKET (blue) and F3'-palivizumab (red) in PBMCs to the indicated BAFF-R+ cells.

[0223] [Figure 173-1] Figures 173A-173F are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3'TriNKET (blue) and F3'-palivizumab (red) to the indicated cell types in human blood. [Figure 173-2] Same as above.

[0224] [Fig. 174]Figures 174A-174C are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3'TriNKET (blue) and F3'-palivizumab (red) to human erythrocytes.

[0225] [Figure 175] Figures 175A-175F are graphs showing flow cytometry analysis of binding of (left to right) AB1424 / 1612 F3'TriNKET, F3'-palivizumab, AB1424 / 1612 F4 TriNKET, F4-palivizumab, and rituximab to PBMCs from the indicated human donors.

[0226] [Figure 176] Figures 176A-176F are histograms showing flow cytometry analysis of binding of AB1424 / 1612 F3'TriNKET (blue) and F3'-palivizumab (red) to the indicated PBMCs from cynomolgus monkey whole blood donor CYN317060.

[0227] [Figure 177] Figures 177A-177F are graphs showing flow cytometry analysis of binding of (left to right) AB1424 / 1612 F3'TriNKET, F3'-palivizumab, AB1424 / 1612 F4 TriNKET, F4-palivizumab, and rituximab to PBMCs from the indicated human donors.

[0228] [Figure 178] Figure 178 is a graph showing CD107a positivity of CD16+CD8+ NK cells in co-cultures of BJAB cells with PBMCs from cynomolgus monkey whole blood donor CYN317060. DETAILED DESCRIPTION OF THE INVENTION

[0229] The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and BAFF-R on cancer cells or B cells. In some embodiments, the multispecific proteins further comprise an additional antigen-binding site that binds to BAFF-R. The present application also provides pharmaceutical compositions comprising such multispecific binding proteins, as well as therapeutic methods using such multispecific binding proteins and pharmaceutical compositions for purposes such as treating autoimmune diseases and cancer. Various aspects of the multispecific binding proteins described in the present application are described in the following sections, although aspects of the multispecific binding proteins described in one particular section should not be limited to any particular section.

[0230] To facilitate the understanding of this application, a number of terms and phrases are defined below.

[0231] As used herein, the words "a" and "an" mean "one or more" and include plurals unless the context is inappropriate.

[0232] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions," which are sandwiched between adjacent, more conserved stretches known as "framework regions," or "FRs." 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 a 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 antigen-binding fragment of an antibody that retains the antigen-binding surface in an intact antibody, or in a recombinant polypeptide such as an scFv, using a peptide linker to join the heavy-chain variable domain to the light-chain variable domain in a single polypeptide.

[0233] As used herein, the term "tumor-associated antigen" refers to any antigen, including, but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, or lipid associated with cancer. Such antigens may be expressed on malignant cells or within the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stromal cells, or immune infiltrates. In certain embodiments of the present disclosure, the term "tumor-associated antigen" refers to BAFF-R, which is targeted by the second and / or additional antigen-binding sites present in the multispecific binding proteins of the present disclosure. However, it is understood that BAFF-R is also associated with diseases and disorders that are not tumors or cancers.

[0234] As used herein, the terms "subject" and "patient" refer to an organism 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 preferably humans.

[0235] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present application) sufficient to produce a benefit or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect that results in the improvement of a condition, disease, disorder, etc., e.g., reducing, modulating, ameliorating, or eliminating, or ameliorating the symptoms thereof.

[0236] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0237] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain 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] .

[0238] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound described herein that, upon administration to a subject, is capable of providing the compound described herein or its active metabolite or residue. As known to those skilled in the art, "salts" of the compounds described herein 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, while not themselves pharmaceutically acceptable, may be used in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0239] Exemplary bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein W is C 1~4 alkyl).

[0240] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. + , NH4 + , and NW4 + (Wherein W is C 1~4 Examples of suitable cations include the anions of the compounds described herein in combination with suitable cations such as alkyl groups.

[0241] For therapeutic use, the salts of the compounds described in this application are contemplated as pharmaceutically 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 pharmaceutically acceptable compound.

[0242] 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.

[0243] Throughout this specification, when compositions are described as having, comprising, or consisting of particular ingredients, or when processes and methods are described as having, comprising, or consisting of particular steps, it is also contemplated that there are compositions described herein that consist essentially of or consist of the recited ingredients, and that there are processes and methods according to this application that consist essentially of or consist of the recited processing steps.

[0244] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of the variable is followed.

[0245] I. Protein The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and BAFF-R on cancer cells. The multispecific binding proteins are useful in the pharmaceutical compositions and treatment methods described herein. Binding of the multispecific binding proteins to the NKG2D and CD16 receptors on natural killer cells enhances the activity of natural killer cells in destroying tumor cells that express the BAFF-R antigen. Binding of the multispecific binding proteins to BAFF-R-expressing cells brings cancer cells into close proximity with natural killer cells, which promotes the direct and indirect destruction of tumor cells by natural killer cells. Multispecific binding proteins that bind to NKG2D, CD16, and other targets are disclosed in International Publication Nos. 2018148445 and 2019157366 (which are not incorporated by reference herein). Further description of some exemplary multispecific binding proteins is provided below.

[0246] The first component of the multispecific binding protein is an antigen-binding site that binds to NKG2D receptor-expressing cells, including, but not limited to, NK cells, γδ T cells, and CD8 +Upon binding to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

[0247] The second component of the multispecific binding protein is an antigen-binding site that binds to BAFF-R. BAFF-R-expressing cells can be found, for example, in 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 large B-cell lymphoma, acute lymphocytic leukemia (ALL), and in autoimmune inflammatory diseases.

[0248] The third component of the multispecific binding protein is an antibody Fc domain or portion thereof, or an antigen binding site that binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0249] The additional antigen-binding site of the multispecific binding protein may bind to BAFF-R. In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second additional antigen-binding site that binds to BAFF-R is each a Fab fragment. In certain embodiments, the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site and additional antigen-binding site that bind to BAFF-R are each scFv. In certain embodiments, the first antigen-binding site that binds to NKG2D is a Fab fragment, and the second additional antigen-binding site that binds to BAFF-R is each an scFv. In certain embodiments, the first antigen-binding site that binds to NKG2D is a Fab, and the second additional antigen-binding site that binds to BAFF-R is each a Fab fragment.

[0250] The multispecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and optionally a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain comprises a second light chain variable domain and optionally a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen-binding site that binds to BAFF-R. In some embodiments, the first Fc domain and the second Fc domain, together, are capable of binding to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0251] The antigen-binding site may incorporate an antibody heavy chain variable domain and an antibody light chain variable domain each (e.g., arranged as found in an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may be a V chain variable domain, such as in a camelid antibody. H H antibodies or V antibodies such as those found in cartilaginous fishes NAR It may also be a single domain antibody such as an antibody.

[0252] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen-binding site.

[0253] Another exemplary format involves a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (e.g., FIG. 2A). In some embodiments, the first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused to a single-chain variable fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pairs with and binds to NKG2D, or binds to BAFF-R, either via a linker or antibody hinge. In some embodiments, the second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and a CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. In some embodiments, the second immunoglobulin heavy chain, when paired with the immunoglobulin heavy chain, either binds to NKG2D or binds to BAFF-R, provided that when the first Fc domain is fused to an scFv that binds to NKG2D, the second immunoglobulin heavy chain when paired with the immunoglobulin light chain binds to BAFF-R but not to NKG2D, or vice versa. In some embodiments, the scFv in the first immunoglobulin heavy chain binds to BAFF-R, and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain, when paired, bind to NKG2D (e.g., Figure 2E). In some embodiments, the scFv in the first immunoglobulin heavy chain binds to NKG2D, and the heavy chain variable domain in the second immunoglobulin heavy chain and the light chain variable domain in the immunoglobulin light chain, when paired, bind to BAFF-R. In some embodiments, the first Fc domain and the second Fc domain together are capable of binding to CD16 (e.g., Figure 2A). In some embodiments, the first Fc domain and the second Fc domain together are capable of binding to CD16 (e.g., Figure 2A).

[0254] Another exemplary format involves a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain and a second immunoglobulin heavy chain (e.g., Figure 2B). In some embodiments, the first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pairs with and binds to NKG2D or that binds to BAFF-R. In some embodiments, the second immunoglobulin heavy chain pairs with and binds to NKG2D, or comprises a second Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of a heavy chain variable domain and a light chain variable domain that binds BAFF-R, provided that when the first Fc domain is fused to an scFv that binds NKG2D, the second Fc domain fused to the scFv binds BAFF-R but not NKG2D, and vice versa. In some embodiments, the first Fc domain and the second Fc domain together are capable of binding to CD16 (e.g., Figure 2B).

[0255] In some embodiments, the single-chain variable fragment (scFv) described above is linked to an antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises the amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge comprises the amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge connecting the scFv (e.g., an scFv that binds to NKG2D or an scFv that binds to BAFF-R) to an antibody heavy chain constant domain comprises the amino acids Ala-Ser. In some embodiments, the hinge connecting the scFv (e.g., an scFv that binds to NKG2D or an scFv that binds to BAFF-R) to an antibody heavy chain constant domain comprises the amino acids Gly-Ser. In some other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence may provide flexibility in binding to the target antigen and a balance between flexibility and optimal shape.

[0256] In some embodiments, the single-chain variable fragment (scFv) described above comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain, improving the stability of the scFv. 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 (amino acid positions numbered according to Kabat). In some embodiments, 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, a (G4S)4 linker ((GlyGlyGlyGlySer)4 (SEQ ID NO: 119)). In some scFv embodiments, the heavy chain variable domain is located at the N-terminus of the light chain variable domain. In some scFv embodiments, the heavy chain variable domain is located at the C-terminus of the light chain variable domain.

[0257] The multispecific binding proteins described herein can further comprise one or more additional antigen-binding sites. The additional antigen-binding site can be fused, optionally via a linker sequence, to the N-terminus of the constant region CH2 domain or to the C-terminus of the constant region CH3 domain. In certain embodiments, the additional antigen-binding site takes the form of a single-chain variable region (scFv), optionally disulfide-stabilized, resulting in a tetravalent or trivalent multispecific binding protein. For example, the multispecific binding protein comprises a first antigen-binding site that binds NKG2D, a second antigen-binding site that binds BAFF-R, an additional antigen-binding site that binds BAFF-R, and a sufficient antibody constant region or portion thereof to bind CD16, or a fourth antigen-binding site that binds CD16. Any one of these antigen-binding sites can take the form of a Fab fragment or an scFv, such as any of the scFvs described above.

[0258] In some embodiments, the additional antigen-binding site binds to a different epitope of BAFF-R than the second antigen-binding site. In some embodiments, the additional antigen-binding site binds to the same epitope as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises the same heavy and light chain CDR sequences as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises the same heavy and light chain variable domain sequences as the second antigen-binding site. In some embodiments, the additional antigen-binding site has the same amino acid sequence as the second antigen-binding site. In some embodiments, the additional antigen-binding site comprises heavy and light chain variable domain sequences that are different from the heavy and light chain variable domain sequences of the second antigen-binding site. In some embodiments, the additional antigen-binding site has an amino acid sequence that is different from the sequence of the second antigen-binding site. In some embodiments, the second antigen-binding site and the additional antigen-binding site bind to different tumor-associated antigens. In some embodiments, the second antigen-binding site and the additional antigen-binding site bind to different antigens. Exemplary formats are shown in Figures 2C and 2D. Thus, the multispecific binding protein can provide bivalent engagement of BAFF-R. Bivalent engagement of BAFF-R by the multispecific protein can stabilize BAFF-R on the tumor cell surface and enhance NK cell cytotoxicity against tumor cells. Bivalent engagement of BAFF-R by the multispecific protein can confer stronger binding of the multispecific protein to tumor cells, thereby promoting a stronger NK cell cytotoxic response against tumor cells, particularly against tumor cells expressing low levels of BAFF-R.

[0259] Multispecific binding proteins can take additional formats. In some embodiments, the multispecific binding protein is in the form of a triomab, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain derived from two parent antibodies.

[0260] In some embodiments, the multispecific binding protein is in a KiH common light chain (LC) format that incorporates knobs-into-holes (KiH) technology (e.g., the multispecific binding protein displayed in Figure 21). The KiH common LC format is a heterodimer that includes a Fab that binds to a first target, a Fab that binds to a second target, and an Fc domain stabilized by a heterodimerization mutation. The two Fabs each include a heavy chain and a light chain, where the heavy chains of each Fab are different from each other and the light chain that pairs with each respective heavy chain is common to both Fabs.

[0261] In some embodiments, the multispecific binding protein is in KIH format, which involves knob-into-hole (KiH) technology. H This involves engineering the CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. The concept behind "knob-into-hole (KiH)" Fc technology is to introduce a "knob" into one CH3 domain (CH3A) by replacing a small residue with a bulky residue (e.g., T366W in EU numbering). CH3A To accommodate the "knob," a complementary "hole" surface was created on the other CH3 domain (CH3B) by replacing the adjacent residues closest to the knob with smaller residues (e.g., T366S / L368A / Y407V CH3BThe "hole" mutations were optimized by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(1):26-35). X-ray crystal structure of KiH Fc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9):1947-57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions caused by steric complementarity at the CH3-interdomain core interface, whereas the knob-knob and hole-hole interfaces disfavor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0262] In some embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies via a flexible, naturally occurring linker, resulting in a tetravalent IgG-like molecule.

[0263] In some embodiments, the multispecific binding proteins are based on an orthogonal Fab interface (ortho-Fab) configuration. In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191-8), structure-based domain design is used to separate the LC and HC domains of one Fab. VH-CH1 Complementary mutations are introduced only at the interface of the Fab fragment, while no changes are made to the other Fab fragment.

[0264] In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in an ES configuration, which is a heterodimerization construct containing two different Fabs that bind to target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0265] In some embodiments, the multispecific binding protein is in the form of a κλ-Body, a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by heterodimerization mutations. Fab fragment 1, which targets antigen 1, contains a kappa LC, and Fab fragment 2, which targets antigen 2, contains a lambda LC. Figure 13A is an exemplary representation of one form of a κλ-Body, and Figure 13B is an exemplary representation of another κλ-Body.

[0266] In some embodiments, the multispecific binding protein is a Fab arm exchanged form (an antibody in which the heavy chain and associated light chain (half molecule) have been swapped with a heavy chain-light chain pair from another molecule, resulting in an exchange of Fab fragment arms, resulting in a bispecific antibody).

[0267] In some embodiments, the multispecific binding protein is in the form of a SEED body. The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules that may broaden the therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins within the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while avoiding homodimerization of the AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

[0268] In some embodiments, the multispecific binding protein is in the LuZ-Y form, which uses a leucine zipper to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0269] In some embodiments, the multispecific binding protein is in the form of a Cov-X-Body. In bispecific CovX-Body, two different peptides are linked together using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. While the pharmacophores are responsible for functional activity, the antibody scaffold provides a long half-life and Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with another pharmacophore (Doppalapudi VR et al., PNAS (2010), 107(52); 22611-22616).

[0270] In some embodiments, the multispecific binding protein is in the form of an OAsc-Fab heterodimer, comprising a Fab that binds target 1 and an scFab that binds target 2, fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0271] In some embodiments, the multispecific binding protein is in the DuetMab format, a heterodimeric construct containing two different Fab fragments that bind antigens 1 and 2 and an Fc stabilized by a heterodimerization mutation. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct pairing of the LC and HC.

[0272] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, a heterodimeric construct with two different Fab fragments that bind to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains are switched with the VH and VL domains, e.g., CH1 is fused in frame with VL and CL is fused in frame with VH.

[0273] In some embodiments, the multispecific binding protein is in the Fit-Ig format, a homodimeric construct in which a Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of a Fab fragment that binds antigen 1. The construct contains a wild-type Fc.

[0274] The individual components of the multispecific binding proteins are described in more detail below.

[0275] NKG2D binding site Upon binding to the NKG2D and CD16 receptors and BAFF-R on natural killer cells, the multispecific binding protein can engage more than one NK activating receptor and block the binding of natural ligands to NKG2D. In certain embodiments, the protein can agonize NK cells in humans. In some embodiments, the protein can agonize NK cells in humans and in other species, such as rodents and cynomolgus monkeys. In some embodiments, the protein can agonize NK cells in humans and in other species, such as cynomolgus monkeys.

[0276] Table 1 lists peptide sequences of heavy and light chain variable domains that, in combination, can bind to NKG2D. In some embodiments, the heavy and light chain variable domains are arranged in a Fab format. In some embodiments, the heavy and light chain variable domains are fused together to form an scFv.

[0277] The NKG2D binding sites listed in Table 1 may vary in their binding affinity for NKG2D, but nevertheless, they all activate human NK cells.

[0278] Unless otherwise stated, the CDR sequences provided in Table 1 are determined by Kabat numbering. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

Table 7

Table 8

Table 9

Table 10

[0279] In certain embodiments, the first antigen-binding site that binds to NKG2D (e.g., human NKG2D) comprises an antibody heavy chain variable domain (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 VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (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 the VL of the same antibody disclosed in Table 1. In certain embodiments, the first antigen-binding site comprises heavy chain CHR1, CHR2, and CHR3 and light chain CHR1, CHR2, and CHR3 of the VH and VL sequences of an antibody disclosed in Table 1, 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. In certain embodiments, the first antigen-binding site comprises heavy chain CHR1, CHR2, and CHR3 and light chain CHR1, CHR2, and CHR3 of an antibody disclosed in Table 1.

[0280] In certain embodiments, a first antigen-binding site that binds to NKG2D comprises a heavy chain variable domain derived from SEQ ID NO: 1, e.g., by having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 1 and / or by incorporating amino acid sequences identical to CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4) of SEQ ID NO: 1. A heavy chain variable domain related to SEQ ID NO: 1 can be combined with a different light chain variable domain to form an NKG2D-binding site. For example, a first antigen binding site incorporating a heavy chain variable domain related to SEQ ID NO: 1 may further incorporate a light chain variable domain selected from a sequence derived from SEQ ID NOs: 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 46. For example, the first antigen-binding site incorporates a heavy chain variable domain having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of the sequences selected from SEQ ID NOs: 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 46.

[0281] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 26, 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: 32. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29, and 30, respectively, or SEQ ID NOs: 28, 29, and 31, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29, and 30, respectively, or SEQ ID NOs: 28, 29, and 31, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively.

[0282] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 36, 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: 42. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39, and 40, respectively, or SEQ ID NOs: 38, 39, and 41, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44, and 45, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39, and 40, respectively, or SEQ ID NOs: 38, 39, and 41, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 44, and 45, respectively.

[0283] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 47, 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: 49. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29, and 48, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34, and 51, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29, and 48, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 50, 34, and 51, respectively.

[0284] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 52, 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: 58. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55, and 56, respectively, or SEQ ID NOs: 54, 55, and 57, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 61, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55, and 56, respectively, or SEQ ID NOs: 54, 55, and 57, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 61, respectively.

[0285] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 62, 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: 68. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65, and 66, respectively, or SEQ ID NOs: 64, 65, and 67, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 69, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65, and 66, respectively, or SEQ ID NOs: 64, 65, and 67, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 69, respectively.

[0286] In certain embodiments, a first antigen-binding site that binds to NKG2D 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: 89, 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: 92. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55, and 90, respectively, or SEQ ID NOs: 54, 55, and 91, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44, and 94, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55, and 90, respectively, or SEQ ID NOs: 54, 55, and 91, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 93, 44, and 94, respectively.

[0287] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 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: 75. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72, and 73, respectively, or SEQ ID NOs: 115, 72, and 74, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77, and 78, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72, and 73, respectively, or SEQ ID NOs: 115, 72, and 74, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 76, 77, and 78, respectively.

[0288] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 79, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82, and 83, respectively, or SEQ ID NOs: 81, 82, and 84, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82, and 83, respectively, or SEQ ID NOs: 81, 82, and 84, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0289] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 95, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82, and 96, respectively, or SEQ ID NOs: 81, 82, and 97, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82, and 96, respectively, or SEQ ID NOs: 81, 82, and 97, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0290] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 98, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82, and 99, respectively, or SEQ ID NOs: 81, 82, and 100, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82, and 99, respectively, or SEQ ID NOs: 81, 82, and 100, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0291] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 101, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82, and 102, respectively, or SEQ ID NOs: 81, 82, and 103, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82, and 102, respectively, or SEQ ID NOs: 81, 82, and 103, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0292] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 104, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82, and 105, respectively, or SEQ ID NOs: 81, 82, and 106, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82, and 105, respectively, or SEQ ID NOs: 81, 82, and 106, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0293] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 107, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82, and 108, respectively, or SEQ ID NOs: 81, 82, and 109, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82, and 108, respectively, or SEQ ID NOs: 81, 82, and 109, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0294] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 110, 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: 85. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82, and 111, respectively, or SEQ ID NOs: 81, 82, and 112, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82, and 111, respectively, or SEQ ID NOs: 81, 82, and 112, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively.

[0295] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 113, 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: 114.

[0296] In certain embodiments, the first antigen-binding site that binds to NKG2D 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: 116, 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: 117.

[0297] Multispecific binding proteins include, but are not limited to, NK cells, γδ T cells, and CD8 + The multispecific binding proteins can bind to NKG2D-expressing cells, including αβ T cells. Upon binding to NKG2D, the multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

[0298] The multispecific binding protein binds to cells that express CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. The proteins of the present disclosure have a concentration of 2 nM to 120 nM, for example, 2 nM to 110 nM, 2 nM to 100 nM, 2 nM to 90 nM, 2 nM to 80 nM, 2 nM to 70 nM, 2 nM to 60 nM, 2 nM to 50 nM, 2 nM to 40 nM, 2 nM to 30 nM, 2 nM to 20 nM, 2 nM to 10 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2 ... .5nM, about 2nM, about 1.5nM, about 1nM, about 0.5nM to about 1nM, about 1nM to about 2nM, about 2nM to about 3nM, about 3nM to 4nM, about 4nM to about 5nM, about 5nM to about 6nM, about 6nM to about 7nM, about 7nM to about 8nM, about 8nM K of about 9nM, about 9nM to about 10nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, or about 8nM to about 10nM D In some embodiments, the NKG2D binding site binds to NKG2D with an affinity of 10-62 nM. D binds to NKG2D.

[0299] BAFF-R binding site The BAFF-R moiety of the multispecific binding proteins disclosed herein comprises a heavy chain variable domain and a light chain variable domain.

[0300] In one aspect, the present disclosure provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and BAFF-R. Table 2 lists some exemplary sequences of heavy and light chain variable domains that can combine to bind to BAFF-R.

[0301] CDR sequences are identified under Chothia numbering unless otherwise indicated.

Table 11

Table 12

Table 13

Table 14

Table 15

Table 17

Table 18

Table 19

[0302] In certain embodiments, the second antigen-binding site that binds to BAFF-R (e.g., human BAFF-R) comprises an antibody heavy chain variable domain (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 VH of an antibody disclosed in Table 2, and an antibody light chain variable domain (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 the VL of the same antibody disclosed in Table 2. In certain embodiments, the second antigen-binding site comprises heavy chain CHR1, CHR2, and CHR3 and light chain CHR1, CHR2, and CHR3 of the VH and VL sequences of the antigen-binding site disclosed in Table 2, as determined under the 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. In certain embodiments, the second 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 2.

[0303] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 145, 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: 146. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 157 or 135, 158 or 136, and 159 or 137, respectively (e.g., SEQ ID NOs: 157, 158, and 159, respectively, or SEQ ID NOs: 135, 136, and 137, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 160, 161, and 162, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 157 or 135, 158 or 136, and 159 or 137, respectively (e.g., SEQ ID NO: 157, 158, and 159, respectively, or SEQ ID NO: 135, 136, and 137, respectively), and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequence of SEQ ID NO: 160, 161, and 162, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises 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: 207 or 138.

[0304] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 147, 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: 148. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 166, 167, and 168, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 166, 167, and 168, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 139 or 140.

[0305] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 147, 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: 150. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 169, 170, and 168, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 163, 164, and 165, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 169, 170, and 168, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 141 or 142.

[0306] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 151, 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: 152. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 174, 175, and 176, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 174, 175, and 176, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 143 or 144.

[0307] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 153, 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: 154. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g., SEQ ID NOs: 177, 179, and 181, respectively, or SEQ ID NOs: 178, 180, and 182, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 184, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively, or SEQ ID NOs: 184, 186, and 187, respectively). In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g., SEQ ID NOs: 177, 179, and 181, respectively, or SEQ ID NOs: 178, 180, and 182, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 184, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively, or SEQ ID NOs: 184, 186, and 187, respectively).In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 149 or 190.

[0308] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 155, 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: 156. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g., SEQ ID NOs: 177, 179, and 181, respectively, or SEQ ID NOs: 178, 180, and 182, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 188, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively, or SEQ ID NOs: 188, 186, and 187, respectively). In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 177 or 178, 179 or 180, and 181 or 182, respectively (e.g., SEQ ID NOs: 177, 179, and 181, respectively, or SEQ ID NOs: 178, 180, and 182, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 183 or 188, 185 or 186, and 187, respectively (e.g., SEQ ID NOs: 183, 185, and 187, respectively, or SEQ ID NOs: 188, 186, and 187, respectively).In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 191 or 192.

[0309] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 260, 249, and 261, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 260, 249, and 261, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0310] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 310, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 216, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 216, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0311] In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 218, respectively. In certain embodiments, the second antigen-binding site that binds to BAFF-R comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 219, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 218, respectively.

[0312] In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 220, 215, and 221, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 222, respectively. In certain embodiments, the second antigen-binding site that binds to BAFF-R comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 220, 215, and 221, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 222, respectively.

[0313] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 226, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 226, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0314] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 277, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 223, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 223, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0315] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 278, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 224, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 224, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0316] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 279, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 225, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 215, and 225, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0317] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 231, 215, and 232, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 231, 215, and 232, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0318] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 280, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 227, 215, and 224, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 227, 215, and 224, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0319] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 281, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 228, 215, and 229, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 228, 215, and 229, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0320] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 282, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 215, and 224, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 215, and 224, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0321] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 236, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 236, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0322] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 283, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 242, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 242, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0323] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 284, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 234, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 234, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0324] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 285, 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: 276. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 235, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 230, 233, and 235, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 218, respectively.

[0325] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 245, 246, and 247, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 245, 246, and 247, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0326] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 286, 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: 253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 237, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 237, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0327] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 287, 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: 253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 238, 239, and 240, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 238, 239, and 240, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0328] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 288, 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: 253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 241, 233, and 242, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 241, 233, and 242, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0329] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 289, 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: 289. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 243, 215, and 244, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 243, 215, and 244, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively.

[0330] In certain embodiments, the VH of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 256, 257, and 258, respectively. In certain embodiments, the VL of the second antigen-binding site that binds to BAFF-R comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 256, 257, and 258, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 259, respectively.

[0331] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 250 or 252, 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: 251 or 253. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 248, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 233, and 248, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 217, 77, and 249, respectively. In certain embodiments, the second antigen-binding site is present as an scFv, and the scFv comprises 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: 254 or 255.

[0332] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 263, 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: 264. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 292, and 293, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 292, and 293, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively.

[0333] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 265, 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: 266. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 297, and 298, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 291, 297, and 298, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 294, 295, and 296, respectively.

[0334] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 267, 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: 268. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 299, 300, and 301, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 299, 300, and 301, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively.

[0335] In certain embodiments, the second antigen-binding site that binds to BAFF-R 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: 269, 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: 262. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 305, 306, and 307, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 308, 303, and 309, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 305, 306, and 307, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 308, 303, and 309, respectively.

[0336] In certain embodiments, the second antigen-binding site that binds to BAFF-R is an scFv. For example, in certain embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 207, 138, 139, 140, 141, 142, 143, 144, 149, 190, 191, 192, 254, or 255.

[0337] Alternatively, novel antigen-binding sites capable of binding to BAFF-R can be identified by screening for binding to the amino acid sequence defined by binding to the amino acid sequence defined in SEQ ID NO: 189, a variant thereof, a mature extracellular fragment thereof, or a fragment containing a domain of BAFF-R. SEQ ID NO: 189 [ka]

[0338] It is contemplated that in an scFv, the VH and VL can be connected by a linker, for example, a (GlyGlyGlyGlySer)4, i.e., a (G4S)4 linker (SEQ ID NO: 119). One of skill in the art will understand that any of the other disclosed linkers (see, e.g., Table 10) may be used in an scFv having the VH and VL sequences disclosed herein (e.g., in Table 2).

[0339] In each of the foregoing embodiments, it is contemplated herein that the VH and / or VL sequences of the scFv that bind to BAFF-R may contain amino acid modifications (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the VH and / or VL without affecting their ability to BAFF-R. For example, it is contemplated herein that the VH and / or VL sequences of the scFv that bind to BAFF-R may contain cysteine ​​heterodimerization mutations that promote the formation of disulfide bridges between the VH and VL of the scFv.

[0340] In certain embodiments, the second antigen-binding site competes with the corresponding antigen-binding site described above for binding to BAFF-R.

[0341] In certain embodiments, the second antigen-binding site blocks the interaction of BAFF-R with a BAFF ligand.

[0342] Fc domain Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, interaction with CD16 is primarily centered on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406(6793):267-273). Based on known domains, the binding affinity to CD16 can be enhanced or decreased, for example, 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. Thus, in certain embodiments, an antibody Fc domain or a portion thereof comprises a hinge and a CH2 domain.

[0343] The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as heterodimers. Promoting the preferential assembly of heterodimers can be achieved by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in U.S. Patent Application Publication Nos. 13 / 494870, 16 / 028850, 11 / 533709, 12 / 875015, 13 / 289934, 14 / 773418, 12 / 811207, 13 / 866756, 14 / 647480, 13 / 642253, and 14 / 830336. For example, mutations can be made in the CH3 domain of human IgG1, incorporating distinct pairs of amino acid substitutions in the first and second polypeptides, allowing the two chains to selectively heterodimerize with each other. The amino acid substitution positions exemplified below are all numbered according to the EU index by Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). Those skilled in the art of antibodies will understand that these conventions consist of non-contiguous numbering in certain regions of an immunoglobulin sequence, allowing for standardized reference to conserved positions in the immunoglobulin family. Thus, the positions of any given immunoglobulin defined by the EU index or by the Kabat numbering scheme do not necessarily correspond to its contiguous sequence.

[0344] Armed with knowledge of the residue numbers according to the Kabat or EU index numbering, one of skill in the art can apply the teachings of the art to identify amino acid sequence modifications within the scope of the present invention according to any commonly used numbering convention. It is understood that SEQ ID NOs provide consecutive numbering of the amino acids within a given polypeptide and therefore may not coincide with the corresponding amino acid numbers as provided by the Kabat or EU index.

[0345] In one scenario, amino acid substitutions in a first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces the original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), thereby allowing the larger amino acid substitution (bulge) to fit into the smaller amino acid substitution (cavity). For example, one polypeptide can incorporate a T366W substitution and another polypeptide can incorporate three substitutions including T366S, L368A, and Y407V.

[0346] The antibody heavy chain variable domains described in the present application can optionally be linked to an amino acid sequence at least 90% identical to an antibody constant region, e.g., an IgG constant region comprising the hinge, CH2, and CH3 domains (with or without the CH1 domain). In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, e.g., a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region. In one embodiment, an antibody Fc domain or portion thereof sufficient to bind to CD16 is linked to a wild-type human IgG1 Fc sequence [ka] In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, e.g., rabbit, dog, cat, mouse, or horse.

[0347] In some embodiments, the antibody constant domain linked to the scFv or Fab fragment is capable of binding to CD 16. In some embodiments, the protein incorporates a portion of an antibody Fc domain (e.g., a sufficient portion of an antibody Fc domain to bind to CD 16), where the antibody Fc domain comprises a hinge and CH2 domain (e.g., the hinge and CH2 domain of a human IgG1 antibody) and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0348] Compared to the human IgG1 constant region, one or more mutations can be incorporated into the constant region, for example 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, and L368E. , L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T411D, T411E, K439D, and K439E.

[0349] In certain embodiments, mutations that can be incorporated into CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into Cκ of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0350] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 3. [Table 24]

[0351] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 25]

[0352] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 5. [Table 26]

[0353] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6. [Table 27]

[0354] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 7, where the position indicated in the first polypeptide column is replaced with any known negatively charged amino acid and the position indicated in the second polypeptide column is replaced with any known positively charged amino acid. [Table 28]

[0355] Alternatively, at least one amino acid substitution can be selected from the following set in Table 8, where the position indicated in the first polypeptide column is replaced with any known positively charged amino acid and the position indicated in the second polypeptide column is replaced with any known negatively charged amino acid. [Table 29]

[0356] Alternatively, the amino acid substitutions may be selected from the following set in Table 9. [Table 30]

[0357] Alternatively, or in addition, the structural stability of a heteromultimeric protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, which forms an artificial disulfide bridge within the interface of the two polypeptides.

[0358] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0359] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at position T366.

[0360] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0361] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0362] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411.

[0363] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407.

[0364] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Q347, Y349, K360, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405.

[0365] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Y349, K360, Q347, and K409.

[0366] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of D356, E357, and D399.

[0367] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0368] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409.

[0369] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399.

[0370] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a Y349C substitution.

[0371] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a S354C substitution.

[0372] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by Q347R, D399V, and F405T substitutions.

[0373] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by Q347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by K360E and K409W substitutions.

[0374] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T366S, T368A, and Y407V substitutions.

[0375] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T366S, T368A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a T366W substitution.

[0376] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T350V, T366L, K392L, and T394W substitutions.

[0377] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T350V, T366L, K392L, and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by T350V, L351Y, F405A, and Y407V substitutions.

[0378] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a F405L substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 (e.g., human IgG1) constant region by a K409R substitution.

[0379] Exemplary Multispecific Binding Proteins Listed below are examples of TriNKETs that contain an antigen-binding site that binds to BAFF-R and an antigen-binding site that binds to NKG2D, each linked to an antibody constant region, where the antibody constant region contains a mutation that allows heterodimerization of the two Fc chains.

[0380] Exemplary BAFF-R-targeting TriNKETs are contemplated in F3', F4', and 2-Fab formats. As described above, in the F3' format, the antigen-binding site that binds to BAFF-R is an scFv, and the antigen-binding site that binds to NKG2D is a Fab. In the F4 format, the antigen-binding site that binds to BAFF-R is a Fab fragment, and the antigen-binding site that binds to NKG2D is an scFv. In each TriNKET, the scFv can contain Cys substitutions in the VH and VL regions, facilitating the formation of disulfide bridges between the VH and VL of the scFv. In the 2-Fab format, both the antigen-binding site that binds to BAFF-R and the antigen-binding site that binds to NKG2D are Fabs.

[0381] The VH and VL of an scFv can be connected via a linker, for example, a peptide linker. In certain embodiments, the peptide linker is a flexible linker. Regarding the amino acid composition of the linker, the peptide is selected for its properties of conferring flexibility, not interfering with the structure and function of other domains of the protein described herein, and resisting cleavage by proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, the VL is linked to the N-terminus or C-terminus of the VH via a (GlyGlyGlyGlySer)4 ((G4S)4) linker (SEQ ID NO: 119).

[0382] The length of the linker (e.g., flexible) 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.

[0383] In certain embodiments, the linker is (GS) n (SEQ ID NO: 120), (GGS) n (SEQ ID NO: 121), (GGGS) n (SEQ ID NO: 122), (GGSG) n (SEQ ID NO: 123), (GGSGG) n (SEQ ID NO: 124), and (GGGGS) n (SEQ ID NO: 125), 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 NO: 119 and SEQ ID NOs: 126-134, as listed in Table 10. [Table 31]

[0384] In F3'-TriNKET, the BAFF-R-binding scFv is linked to the N-terminus of the Fc via an Ala-Ser or Gly-Ser linker. The Ala-Ser or Gly-Ser linker is included in the elbow hinge region sequence to balance flexibility and optimal shape. In certain embodiments, the additional amino acid sequence Thr-Lys-Gly can be added N- or C-terminal to the Ala-Ser or Gly-Ser sequence in the hinge. In F4 TriNKET, the NKG2D-binding scFv is linked to the C-terminus of the Fc via a short linker containing the amino acid sequence SGSGGGGS (SEQ ID NO: 274).

[0385] As used herein to describe these exemplary TriNKETs, Fc includes the antibody hinge, CH2, and CH3. In each exemplary TriNKET, the Fc domain linked to the scFv contains the mutations Q347R, D399V, and F405T, and the Fc domain linked to the Fab contains the matching mutations K360E and K409W to form heterodimers. The Fc domain linked to the scFv further contains a S354C substitution in the CH3 domain, which forms a disulfide bond with the Y349C substitution on the Fc linked to the Fab. These substitutions are shown in bold in the sequences described in this subsection.

[0386] For example, a TriNKET described in the present disclosure is ianalumab-F3'. Ianalumab-F3' comprises (a) a BAFF-R-binding scFv comprising the VH and VL of ianalumab listed in Table 2 linked to an Fc domain, in an orientation in which the VH is C-terminal to the VL, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. Ianalumab-F3' comprises three polypeptides: scFv-ianalumab-VL-VH-Fc (SEQ ID NO: 193), A49MI-VH-CH1-Fc (SEQ ID NO: 194), and A49MI-VL-CL (SEQ ID NO: 195). scFv-ianalumab-VL-VH-Fc (SEQ ID NO: 193) ("S chain") [ka] A49MI-VH-CH1-Fc (SEQ ID NO: 194) ("H chain") [ka] A49MI-VL-CL (SEQ ID NO: 195) ("L chain") [ka]

[0387] scFv-ianalumab-VL-VH-Fc (SEQ ID NO: 193) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 207 and contains the heavy chain variable domain of ianalumab connected to the C-terminus of the light chain variable domain of ianalumab via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and Q100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0388] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-ianalumab-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-ianalumab-VL-VH-Fc.

[0389] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0390] Another TriNKET described in the present disclosure is Ianalumab-2-Fab, which comprises: (a) a BAFF-R-binding Fab fragment comprising the VH and VL sequences of Ianalumab as set forth in Table 2, which comprises a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain (this does not contain the antibody-dependent cellular cytotoxicity enhancing mutation present in commercially available Ianalumab antibodies); and (b) an NKG2D-binding Fab fragment derived from A49MI, which comprises a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. Ianalumab-2-Fab contains four polypeptides: ianalumab-VH-CH1-Fc-Genmab, ianalumab-VL-CL, A49MI-VH-CH1-Fc, and A49MI-VL-CL-Genmab. Ianalumab-VH-CH1-Fc-Genmab (SEQ ID NO: 196) [ka] Ianalumab-VL-CL (SEQ ID NO: 197) [ka] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) [ka] A49MI-VL-CL (SEQ ID NO: 195) [ka]

[0391] Ianalumab-VH-CH1-Fc-Genmab (SEQ ID NO: 196) represents the heavy chain portion of a Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 145) and CH1 domain of BAFF-R-binding ianalumab connected to the Fc domain. The Fc domain in ianalumab-VH-CH1-Fc-Genmab contains a F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0392] Ianalumab-VL-CL (SEQ ID NO: 197) represents the light chain portion of a Fab fragment containing the light chain variable domain and the light chain constant domain of BAFF-R-binding ianalumab (SEQ ID NO: 146).

[0393] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) comprises the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with ianalumab-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0394] As described above, A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0395] Another exemplary TriNKET described in the present disclosure is the hCOH-1-F3' TriNKET. hCOH-1-F3' comprises (a) a BAFF-R-binding scFv sequence from hCOH-1 in Table 2, in an orientation in which the VH linked to the Fc domain is positioned C-terminal to the VL, and (b) an NKG2D-binding Fab fragment from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. hCOH-1-F3' comprises three polypeptides: scFv-hCOH-1-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL. scFv-hCOH-1-VL-VH-Fc (SEQ ID NO: 198) ("S chain") [ka]

[0396] scFv-hCOH-1-VL-VH-Fc (SEQ ID NO: 198) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 149 and contains the heavy chain variable domain of hCOH-1 connected to the C-terminus of the light chain variable domain of hCOH-1 via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and G100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0397] As described above, A49MI-VH-CH1-Fc (SEQ ID NO: 194) comprises the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-hCOH-1-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-hCOH-1-VL-VH-Fc.

[0398] As described above, A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0399] Another TriNKET described in the present disclosure is hCOH-1-2-Fab. hCOH-1-2-Fab comprises (a) a BAFF-R-binding Fab fragment derived from hCOH-1, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. hCOH-1-2-Fab comprises four polypeptides: hCOH-1-VH-CH1-Fc-Genmab, hCOH-1-VL-CL, A49MI-VH-CH1-Fc-Genmab, and A49MI-VL-CL. hCOH-1-VH-CH1-Fc-Genmab (SEQ ID NO: 208) [ka] hCOH-1-VL-CL (SEQ ID NO: 209) [ka]

[0400] hCOH-1-VH-CH1-Fc-Genmab (SEQ ID NO: 208) represents the heavy chain portion of the Fab fragment, which contains the BAFF-R-binding hCOH-1 heavy chain variable domain (SEQ ID NO: 153) and CH1 domain connected to the Fc domain. The Fc domain in hCOH-1-VH-CH1-Fc-Genmab contains a F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0401] hCOH-1-VL-CL (SEQ ID NO: 209) represents the light chain portion of the Fab fragment containing the light chain variable domain (SEQ ID NO: 154) and the light chain constant domain of BAFF-R-binding hCOH-1.

[0402] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) comprises the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with the Fc in hCOH-1-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0403] A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0404] Another exemplary TriNKET described in this disclosure is hCOH-2-F3'. hCOH-2-F3' comprises (a) a BAFF-R-binding scFv derived from hCOH-2 of Table 2, in an orientation in which the VH linked to the Fc domain is positioned C-terminal to the VL, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. hCOH-2-F3' comprises three polypeptides: scFv-hCOH-1-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL. scFv-hCOH-2-VL-VH-Fc (SEQ ID NO: 210) ("S chain") [ka]

[0405] scFv-hCOH-2-VL-VH-Fc (SEQ ID NO: 210) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv comprises the amino acid sequence of SEQ ID NO: 191, which contains the heavy chain variable domain of hCOH-2 connected to the C-terminus of the light chain variable domain of hCOH-2 via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and G100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0406] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-hCOH-2-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-hCOH-2-VL-VH-Fc.

[0407] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0408] Another TriNKET described in the present disclosure is hCOH-2-2-Fab. hCOH-2-2-Fab comprises: (a) a BAFF-R-binding Fab fragment derived from hCOH-2, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain; and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. hCOH-2-2-Fab comprises four polypeptides: hCOH-2-VH-CH1-Fc-Genmab, hCOH-2-VL-CL, A49MI-VH-CH1-Fc-Genmab, and A49MI-VL-CL. hCOH-2-VH-CH1-Fc-Genmab (SEQ ID NO: 199) [ka] hCOH-2-VL-CL (SEQ ID NO: 200) [ka]

[0409] hCOH-2-VH-CH1-Fc-Genmab (SEQ ID NO: 199) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 155) and CH1 domain of BAFF-R-binding hCOH-2 connected to the Fc domain. The Fc domain in hCOH-2-VH-CH1-Fc-Genmab contains a F405L substitution for heterodimerization with the Fc in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0410] hCOH-2-VL-CL (SEQ ID NO: 200) represents the light chain portion of the Fab fragment containing the light chain variable domain (SEQ ID NO: 156) and the light chain constant domain of BAFF-R-binding hCOH-2.

[0411] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) comprises the heavy chain variable domain and CH1 domain of NKG2D-binding A49MI (SEQ ID NO: 95) connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with the Fc in hCOH-2-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0412] A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0413] Another exemplary TriNKET described in the present disclosure is V3-46s-F3'. V3-46s-F3' comprises (a) a BAFF-R-binding scFv sequence from V3-46s in Table 2, in an orientation in which the VH linked to the Fc domain is positioned C-terminal to the VL, and (b) an NKG2D-binding Fab fragment from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. V3-46s-F3' comprises three polypeptides: scFv-hCOH-1-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL. scFv-V3-46s-VL-VH-Fc (SEQ ID NO: 201) (“S chain”) [ka]

[0414] scFv-V3-46s-VL-VH-Fc (SEQ ID NO: 201) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 139 and comprises the heavy chain variable domain of V3-46s connected to the C-terminus of the light chain variable domain of V3-46s via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and Q100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0415] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-V3-46s-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-V3-46s-VL-VH-Fc.

[0416] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0417] Another TriNKET described in the present disclosure is V3-46s-2-Fab, which comprises: (a) a BAFF-R-binding Fab fragment derived from V3-46s, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain; and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. V3-46s-2-Fab contains four polypeptides: V3-46s-VH-CH1-Fc-Genmab, V3-46s-VL-CL, A49MI-VH-CH1-Fc-Genmab, and A49MI-VL-CL. V3-46s-VH-CH1-Fc-Genmab (SEQ ID NO: 202) [ka] V3-46s-VL-CL (SEQ ID NO: 203) [ka]

[0418] V3-46s-VH-CH1-Fc-Genmab (SEQ ID NO: 202) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 147) and CH1 domain of BAFF-R-binding V3-46s connected to the Fc domain. The Fc domain in V3-46s-VH-CH1-Fc-Genmab contains a F405L substitution for heterodimerization with the Fc domain in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0419] V3-46s-VL-CL (SEQ ID NO: 203) represents the light chain portion of a Fab fragment containing the light chain variable domain (SEQ ID NO: 148) and the light chain constant domain of BAFF-R-binding V3-46s.

[0420] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) comprises the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with the Fc in V3-46s-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0421] A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0422] Another exemplary TriNKET described in the present disclosure is V3-46s-42-F3'. V3-46s-42-F3' comprises (a) a BAFF-R-binding scFv sequence from V3-46s-42 in Table 2, in an orientation in which the VH linked to the Fc domain is positioned C-terminal to the VL, and (b) an NKG2D-binding Fab fragment from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. V3-46s-42-F3' comprises three polypeptides: scFv-V3-46s-42-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL. scFv-V3-46s-42-VL-VH-Fc (SEQ ID NO: 204) (“S chain”) [ka]

[0423] scFv-V3-46s-42-VL-VH-Fc (SEQ ID NO: 204) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 141 and contains the heavy chain variable domain of V3-46s-42 connected to the C-terminus of the light chain variable domain of V3-46s-42 via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and Q100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0424] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-V3-46s-42-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-V3-46s-42-VL-VH-Fc.

[0425] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0426] Another TriNKET described in the present disclosure is V3-46s-42-2-Fab, which comprises (a) a BAFF-R-binding Fab fragment derived from V3-46s-42, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. V3-46s-42-2-Fab contains four polypeptides: V3-46s-42-VH-CH1-Fc-Genmab, V3-46s-42-VL-CL, A49MI-VH-CH1-Fc-Genmab, and A49MI-VL-CL. V3-46s-42-VH-CH1-Fc-Genmab (SEQ ID NO: 202) [ka] V3-46s-42-VL-CL (SEQ ID NO: 206) [ka]

[0427] V3-46s-42-VH-CH1-Fc-Genmab (SEQ ID NO: 205) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain and CH1 domain of BAFF-R-binding V3-46s-42 (SEQ ID NO: 147) connected to the Fc domain. The Fc domain in V3-46s-42-VH-CH1-Fc contains a F405L substitution for heterodimerization with the Fc domain in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0428] V3-46s-42-VL-CL (SEQ ID NO: 206) represents the light chain portion of the Fab fragment containing the light chain variable domain and the light chain constant domain of BAFF-R-binding V3-46s-42 (SEQ ID NO: 150).

[0429] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with the Fc in V3-46s-42-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0430] A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0431] Another exemplary TriNKET described in the present disclosure is Hu9.1-73-F3' TriNKET. Hu9.1-73-F3' TriNKET comprises (a) a BAFF-R-binding scFv sequence from Hu9.1-73 in Table 2, in an orientation in which the VH linked to the Fc domain is positioned C-terminal to the VL, and (b) an NKG2D-binding Fab fragment from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. Hu9.1-73-F3' comprises three polypeptides: scFv-Hu9.1-73-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL. scFv-Hu9.1-73-VL-VH-Fc (SEQ ID NO: 211) ("S chain") [ka]

[0432] scFv-Hu9.1-73-VL-VH-Fc (SEQ ID NO: 211) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution for disulfide bond formation with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 143 and comprises the heavy chain variable domain of scFv-Hu9.1-73 connected to the C-terminus of the light chain variable domain of scFv-Hu9.1-73 via a (G4S)4 linker. The scFv contains Cys substitutions in the VH and VL regions at G44 and Q100, which facilitate the formation of disulfide bridges between the VH and VL of the scFv.

[0433] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-Hu9.1-73-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-Hu9.1-73-VL-VH-Fc.

[0434] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0435] Another TriNKET described in the present disclosure is Hu9.1-73-2-Fab, which comprises: (a) a BAFF-R-binding Fab fragment derived from Hu9.1-73, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain; and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to an Fc domain. Hu9.1-73-2-Fab contains four polypeptides: Hu9.1-73-VH-CH1-Fc-Genmab, Hu9.1-73-VL-CL, A49MI-VH-CH1-Fc-Genmab, and A49MI-VL-CL. Hu9.1-73-VH-CH1-Fc-Genmab (SEQ ID NO: 212) [ka] Hu9.1-73-VL-CL (SEQ ID NO: 205) [ka]

[0436] Hu9.1-73-VH-CH1-Fc-Genmab (SEQ ID NO: 212) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 151) and CH1 domain of BAFF-R-binding Hu9.1-73 connected to the Fc domain. The Fc domain in Hu9.1-73-VH-CH1-Fc contains a F405L substitution for heterodimerization with the Fc domain in A49MI-VH-CH1-Fc-Genmab, which contains a K409R substitution.

[0437] Hu9.1-73-VL-CL (SEQ ID NO: 205) represents the light chain portion of a Fab fragment containing the light chain variable domain (SEQ ID NO: 152) and light chain constant domain of BAFF-R-bound Hu9.1-73.

[0438] A49MI-VH-CH1-Fc-Genmab (SEQ ID NO: 213) contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to an Fc domain. The Fc domain in A49MI-VH-CH1-Fc-Genmab contains a K409R substitution for heterodimerization with the Fc in Hu9.1-73-VH-CH1-Fc-Genmab, which contains a F405L substitution.

[0439] A49MI-VL-CL (SEQ ID NO: 195) comprises the light chain variable domain (SEQ ID NO: 85) and the light chain constant domain of NKG2D-binding A49MI.

[0440] Another example of a TriNKET described in this disclosure is AB1424 / 1612-F3', which comprises (a) a BAFF-R-binding scFv sequence derived from AB1424 / 1612 in Table 2 (with cysteine ​​heterodimerization mutations for disulfide bridge formation) in an orientation in which the VH linked to the Fc domain is positioned N-terminal to the VL, and (b) an NKG2D-binding Fab fragment derived from A49MI, comprising a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain. AB1424 / 1612-F3' comprises three polypeptides: scFv-AB1424 / 1612-VL-VH-Fc (sequence number 193), A49MI-VH-CH1-Fc (sequence number 194), and A49MI-VL-CL (sequence number 195). scFv-AB1424 / 1612-VH-VL-Fc (SEQ ID NO: 270) ("S chain") [ka] A49MI-VH-CH1-Fc (SEQ ID NO: 194) ("H chain") [ka] A49MI-VL-CL (SEQ ID NO: 195) ("L chain") [ka]

[0441] scFv-AB1424 / 1612-VH-VL-Fc (SEQ ID NO: 270) represents the complete sequence of a BAFF-R-binding scFv linked to an Fc domain via an Ala-Ser hinge. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution in A49MI-VH-CH1-Fc, described below. The scFv has the amino acid sequence of SEQ ID NO: 254 and contains the light chain variable domain of AB1424 / 1612 connected to the C-terminus of the light chain variable domain of AB1424 / 1612 via a (G4S)4 linker. The scFv contains cysteine ​​substitutions in the VH and VL regions at G44 and G100 to facilitate disulfide bridge formation between the VH and VL of the scFv.

[0442] A49MI-VH-CH1-Fc (SEQ ID NO: 194) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 95) and CH1 domain of the NKG2D-binding A49MI connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc in scFv-AB1424 / 1612-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc in scFv-AB1424 / 1612-VL-VH-Fc.

[0443] A49MI-VL-CL (SEQ ID NO: 195) represents the light chain portion of a Fab fragment comprising the light chain variable domain and the light chain constant domain of the NKG2D-binding A49MI (SEQ ID NO: 85).

[0444] Another example of a TriNKET described in this disclosure is AB1424 / 1612-F4, which comprises (a) two BAFF-R-binding Fab fragments derived from AB1424 / 1612 in Table 2, each comprising a heavy chain comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is connected to the Fc domain, and (b) an NKG2D-binding scFv sequence derived from A49MI linked to the C-terminus of the Fc domain in an orientation such that the VH is positioned C-terminal to the VL. AB1424 / 1612-F4 comprises four polypeptides: a first polypeptide comprising AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv (sequence number 271), a second polypeptide comprising AB-1424 / 1612-VH-CH1-CH2-CH3 (sequence number 272), and third and fourth polypeptides, each comprising AB1424 / 1612-VL-CL (sequence number 273). AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv (SEQ ID NO: 271) ("M" chain) [ka] AB-1424 / 1612-VH-CH1-CH2-CH3 (SEQ ID NO: 272) ("H" chain) [ka] AB1424 / 1612-VL-CL (SEQ ID NO: 273) ("L" chain) [ka]

[0445] AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv (SEQ ID NO: 271) represents the heavy chain portion of a Fab fragment, comprising the heavy chain variable domain (SEQ ID NO: 250) and CH1 domain of BAFF-R-binding AB1424 / 1612 connected to an Fc domain and further connected to an scFv. The scFv has the amino acid sequence of SEQ ID NO: 275, which comprises the heavy chain variable domain (SEQ ID NO: 95) of NKG2D-binding A49MI connected via a (G4S)4 linker to the C-terminus of the light chain variable domain (SEQ ID NO: 85) of A49MI. The scFv also contains substitutions of Cys in the VH and VL regions at G44 and G100, facilitating disulfide bridge formation between the VH and VL of the scFv. The scFv of AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv is linked to the C-terminus of the CH3 domain by a short SGSGGGGS (SEQ ID NO: 274) linker. The Fc domain in AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv contains Q347R, D399V, and F405T substitutions for heterodimerization, and a S354C substitution for disulfide bond formation with the Y349C substitution in AB-1424 / 1612-VH-CH1-CH2-CH3, as described below.

[0446] AB1424 / 1612-VH-CH1-CH2-CH3 (SEQ ID NO: 272) represents the heavy chain portion of the Fab fragment, which contains the heavy chain variable domain (SEQ ID NO: 250) and CH1 domain of BAFF-R-binding AB1424 / 1612 connected to the Fc domain. The Fc domain in A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc domain in AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv. In AB1424 / 1612-VH-CH1-CH2-CH3, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc domain in AB1424 / 1612-VH-CH1-CH2-CH3-A49MI-scFv.

[0447] AB1424 / 1612-VL-CL (SEQ ID NO: 273) represents the light chain portion of the Fab fragment containing the light chain variable domain (SEQ ID NO: 251) and the light chain constant domain of BAFF-R-binding AB1424 / 1612.

[0448] In certain embodiments, an F3'TriNKET described in the present disclosure is identical to one of the exemplary TriNKETs described above, except that (a) the Fc domain linked to the NKG2D-binding Fab fragment contains Q347R, D399V, and F405T substitutions in the CH3 domain for heterodimerization, and the Fc domain linked to the BAFF-R-binding scFv contains matching K360E and K409W substitutions in the CH3 domain, and / or (b) the Fc domain linked to the NKG2D-binding Fab fragment contains a S354C substitution in the CH3 domain, and the Fc domain linked to the BAFF-R-binding scFv contains a matching Y349C substitution in the CH3 domain for disulfide bond formation.

[0449] In certain embodiments, a 2-Fab TriNKET described in this disclosure is identical to one of the exemplary TriNKETs described above, except that the Fc domain linked to the NKG2D-binding Fab fragment contains an F405L substitution in the CH3 domain for heterodimerization, and the Fc domain linked to the BAFF-R-binding Fab fragment contains a matching K409R substitution in the CH3 domain.

[0450] Those skilled in the art will understand that during protein production and / or storage, an N-terminal glutamic acid (E) or glutamine (Q) can cyclize to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Thus, in some embodiments in which the N-terminal residue of a polypeptide's amino acid sequence is E or Q, the corresponding amino acid sequence with E or Q replaced by pyroglutamic acid is also contemplated herein.

[0451] Those skilled in the art will also understand that during protein production and / or storage, a lysine (K) at the C-terminus of a protein may be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed in proteins that include an Fc domain at their C-terminus. Thus, in some embodiments in which the C-terminal residue of a polypeptide's amino acid sequence (e.g., an Fc domain sequence) is K, the corresponding amino acid sequence in which the K is removed is also contemplated herein.

[0452] The multispecific proteins described above can be produced using recombinant DNA techniques 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, and a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector, and the first, second, and third expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0453] To achieve the highest yield of the multispecific protein, various ratios of the first, second, and third expression vectors can be explored to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix technology.

[0454] The clones can be cultured under conditions suitable for bioreactor scale-up to maintain expression of the multispecific protein. The multispecific protein 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.

[0455] II. Properties of Multispecific Proteins The multispecific proteins described herein comprise an NKG2D-binding site, a BAFF-R-binding site, and an antibody Fc domain or portion thereof sufficient to bind CD 16, or an antigen-binding site that binds to CD 16. In some embodiments, the multispecific protein contains an additional antigen-binding site that binds to BAFF-R, as exemplified in the F4-TriNKET format (e.g., Figures 2C and 2D).

[0456] In some embodiments, the multispecific proteins exhibit similar thermostability to the corresponding monoclonal antibody, i.e., a monoclonal antibody that contains the same BAFF-R binding site as that incorporated into the multispecific protein.

[0457] In some embodiments, a multispecific protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and cells expressing BAFF-R, such as certain tumor cells. Binding of a multispecific protein to NK cells can enhance the activity of NK cells toward the destruction of BAFF-R-expressing cells (e.g., BAFF-R-expressing tumor cells). It has been reported that NK cells exhibit stronger cytotoxicity toward stressed target cells (see Chan et al., (2014) Cell Death Differ. 21(1):5-14). Without wishing to be bound by theory, it has been hypothesized that when NK cells engage a population of cells via TriNKET, the NK cells may selectively kill stressed target cells (e.g., malignant cells and cells in the tumor microenvironment). This mechanism may contribute to the increased specificity and reduced toxicity of TriNKET, allowing for the selective elimination of stressed cells even when BAFF-R expression is not restricted to the desired target cells.

[0458] In some embodiments, the multispecific protein binds to BAFF-R with similar affinity to a corresponding anti-BAFF-R monoclonal antibody (i.e., a monoclonal antibody containing the same BAFF-R binding site as that incorporated into the multispecific protein). In some embodiments, the multispecific protein is more effective at killing tumor cells expressing BAFF-R than the corresponding monoclonal antibody.

[0459] In certain embodiments, the multispecific proteins described herein, which contain a binding site for BAFF-R, activate human primary NK cells when co-cultured with cells expressing BAFF-R. NK cell activation is characterized by an increase in CD107a degranulation and IFN-γ cytokine production. Furthermore, compared to the corresponding anti-BAFF-R monoclonal antibody, the multispecific proteins can exhibit superior activation of human NK cells in the presence of cells expressing BAFF-R.

[0460] In some embodiments, multispecific proteins described herein that contain a binding site for BAFF-R enhance the activation of resting and IL-2-activated human NK cells when co-cultured with cells expressing BAFF-R.

[0461] In some embodiments, the multispecific proteins offer advantages in targeting tumor cells that express intermediate and low levels of BAFF-R compared to corresponding monoclonal antibodies that bind to BAFF-R.

[0462] In some embodiments, the bivalent F4 format of TriNKET (i.e., TriNKET containing an additional antigen-binding site that binds to BAFF-R) improves the avidity of TriNKET to bind to BAFF-R, an effect that stabilizes the expression and maintenance of high levels of BAFF-R on the surface of tumor cells. In some embodiments, F4-TriNKET mediates more potent killing of tumor cells than the corresponding F3-TriNKET or F3'-TriNKET.

[0463] III. Therapeutic uses The present application also describes methods for treating autoimmune diseases or cancer using the multispecific binding proteins described herein and / or pharmaceutical compositions described herein. The methods can be used to treat a variety of cancers or autoimmune diseases that express BAFF-R.

[0464] Therapeutic methods can be characterized according to the cancer being treated. The cancer being treated can be characterized according to the presence of a particular antigen, e.g., BAFF-R, expressed on the surface of the cancer cells.

[0465] Cancers characterized by expression of BAFF-R include, but are not limited to, B-cell non-Hodgkin's lymphoma (B-NHL), such as 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 large B-cell lymphoma, acute lymphocytic leukemia (ALL), and autoimmune inflammatory diseases.

[0466] It is contemplated that the proteins, conjugates, cells, and / or pharmaceutical compositions described in this disclosure may be used to treat various cancers, including but not limited to, cancers in which the cancer cells or cells in the cancer microenvironment express BAFF-R.

[0467] 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, stomach cancer, testicular cancer, or uterine cancer.In yet other embodiments, the cancer is an angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid gland carcinoma, bile duct 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 tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract carcinoma, bone cancer, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus mammary gland carcinoma, Cephaloma / cytoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, yolk sac carcinoma, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, epithelial carcinoma, epithelial cell carcinoma, Ewing's sarcoma, cancer of the eye and orbit, cancer of the female genital tract, focal nodular hyperplasia, gallbladder cancer, cancer of the gastric cardia, cancer of the gastric fundus, gastrinoma, glioblastoma, glycogenoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary carcinoma, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, Sarcoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelial carcinoma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, serous papillary adenocarcinoma, penile cancer , pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cell carcinoma, sinonasal cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous melanoma, VIP-secreting tumor, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0468] In certain embodiments, the cancer is a malignant hematological disease. In certain embodiments, the malignant hematological disease is a leukemia. In certain embodiments, it is selected from the group consisting of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, T-lymphoblastic acute leukemia, or acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myelogenous leukemia.

[0469] In some embodiments, the present application provides methods for treating autoimmune inflammatory diseases using the multispecific binding proteins described herein and / or pharmaceutical compositions described herein, which may 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 1 diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, myasthenia gravis, and vasculitis.

[0470] IV. Combination Therapy Another aspect of the present disclosure provides combination therapies. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat autoimmune diseases or to treat cancer.

[0471] Exemplary therapeutic agents that may be used as part of a combination therapy in treating 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., COX-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-penicillin, etc.). These include 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).

[0472] 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, lantitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinone, streptozocin, nimustine, vindesine, flutamide, hydroxybenzoates, benzodiazepines ...

[0473] An additional class of drugs that can be used as part of a combination therapy in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents 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.

[0474] 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).

[0475] Still other 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-deoxyadenine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors (ii) an inhibitor selected from an inhibitor 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.

[0476] The proteins of the present application may also be used as an adjunct to surgical removal of the primary lesion.

[0477] The amounts of the multispecific binding protein and additional therapeutic agent, as well as the relative timing of administration, can be selected to achieve a desired combination therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the combined therapeutic agents, or pharmaceutical compositions comprising the therapeutic agents, can be administered in any order, e.g., sequentially, concomitantly, together, simultaneously, etc. Further, for example, the multispecific binding protein can be administered for the time period during which the additional therapeutic agent exerts its prophylactic or therapeutic effect, or vice versa.

[0478] V. Pharmaceutical Compositions The present disclosure also describes pharmaceutical compositions containing a therapeutically effective amount of the proteins described herein. The compositions can be formulated for use in a variety of drug delivery systems. For appropriate formulation, one or more physiologically acceptable excipients or carriers can also be included in the composition. 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).

[0479] The intravenous drug delivery formulations described herein may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing tubing and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and may be contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried, and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulations from 12, 27, or 45 vials may be combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg per vial.

[0480] The protein can be present in a liquid, aqueous pharmaceutical formulation that includes a therapeutically effective amount of the protein in a buffer solution that forms the formulation.

[0481] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3 to 11, e.g., 5 to 9 or 6 to 8, and in certain embodiments, 7 to 8, e.g., 7 to 7.5. The resulting solid composition may be packaged in a plurality of single-dose units, each containing a fixed amount of the above-described agent. The solid composition may also be packaged in flexible-quantity containers.

[0482] In certain embodiments, the present application describes formulations with extended shelf life comprising a multispecific binding protein described herein in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0483] In certain embodiments, aqueous formulations are prepared comprising a protein of the present disclosure in a pH buffered solution. The formulation buffer may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. pH ranges intermediate to those listed above are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination of the values ​​listed above as upper and / or lower limits are 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.

[0484] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range may be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about pH 5.0 to about 5.2. In certain embodiments, the buffer system includes 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 mg / mL), and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system comprises about 1 to about 1.5 mg / mL citric acid, about 0.25 to about 0.5 mg / mL sodium citrate, about 1.25 to about 1.75 mg / mL disodium phosphate dihydrate, about 0.7 to about 1.1 mg / mL sodium dihydrogen phosphate dihydrate, and about 6.0 to about 6.4 mg / mL sodium chloride. In certain embodiments, the pH of the formulation is adjusted using sodium hydroxide.

[0485] Polyols, which can act as tonicifiers and stabilize 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 isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added can also vary with respect to the molecular weight of the polyol. For example, a small amount of a monosaccharide (e.g., mannitol) may be added as compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that can be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the concentration of mannitol can be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol can be about 7.5 to about 15 mg / mL. In certain embodiments, the concentration of mannitol can be about 10 to about 14 mg / mL. In certain embodiments, the concentration of mannitol can be about 12 mg / mL. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0486] 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 aggregation of the formulated antibody and / or minimizes the formation of particulates 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 ed., 1996). In certain embodiments, the formulation may contain from about 0.1 mg / mL to about 10 mg / mL of polysorbate 80, 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.

[0487] In embodiments, the multispecific binding proteins described herein are formulated as liquid formulations. The liquid formulations may be provided at a concentration of 10 mg / mL in USP / Ph Eur Type I 50R vials closed with rubber stoppers and sealed with aluminum crimp seal closures. The stoppers may be made from USP and Ph Eur compliant elastomers. In certain embodiments, the vials may be filled with 61.2 mL of protein product solution to allow for a draw volume of 60 mL. In certain embodiments, the liquid formulations may be diluted with 0.9% saline.

[0488] In certain embodiments, the liquid formulations described herein may be prepared as a 10 mg / mL solution in combination with a sugar at a stabilizing level. In certain embodiments, the liquid formulations may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount that does not result in an undesirable or inappropriate viscosity for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0489] In certain embodiments, the pH of the liquid formulation can be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base is sodium hydroxide.

[0490] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from proteins to form succinimide intermediates that can undergo hydrolysis. The succinimide intermediate results in a 17-dalton mass loss from 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. Therefore, deamidation is typically detectable as a 1-dalton mass gain. Deamidation of asparagine produces 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 conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain affects the deamidation rate. Gly and Ser following Asn in the protein sequence are more susceptible to deamidation.

[0491] In certain embodiments, the liquid formulations described in this application may be stored under conditions of pH and humidity to prevent deamination of the protein product.

[0492] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0493] Preservatives may optionally be added to the formulations described herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0494] Intravenous (IV) formulations may be the preferred route of administration in certain cases, such as when a patient is hospitalized after transplant and receives all medications via the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0495] In certain embodiments, salts or buffer components can be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions can serve as counterions.

[0496] The multispecific binding proteins described herein can be present as a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant can be a sugar, e.g., a disaccharide. In certain embodiments, the lyoprotectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0497] The amount of sucrose or maltose useful for stabilizing a lyophilized drug product can be a weight ratio of protein to sucrose or maltose of at least 1:2, in certain embodiments, the weight ratio of protein to sucrose or maltose can be between 1:2 and 1:5.

[0498] In certain embodiments, the pH of the formulation before lyophilization can be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base is sodium hydroxide.

[0499] Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range for the lyophilized drug product may be between 7 and 8.

[0500] In certain embodiments, salts or buffer components can be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions can serve as counterions.

[0501] In certain embodiments, a "bulking agent" can 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., facilitating the production of an essentially uniform lyophilized cake that maintains an open-pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the multispecific binding proteins described herein can contain such bulking agents.

[0502] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0503] In certain embodiments, the lyophilized drug product may be comprised of an aqueous carrier. Aqueous carriers of interest herein are those that are pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffer solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0504] In certain embodiments, the lyophilized drug product 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.

[0505] In certain embodiments, the lyophilized protein product is constituted in about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0506] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the multispecific binding proteins described in this application may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient.

[0507] The specific volume may be a uniform dose for each patient, e.g., 50-5000 mg of protein. Alternatively, the patient's dose may be tailored 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 patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment can be routinely made by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages can 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 disease progression is monitored. Blood levels of the targetable construct or complex in the patient may be measured to determine whether dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, and their dosages, are 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).

[0508] Generally, dosages based on body weight are from about 0.01 μg to about 100 mg / kg body weight, e.g., from about 0.01 μg to about 100 mg / kg body weight, from about 0.01 μg to about 50 mg / kg body weight, from about 0.01 μg to about 10 mg / kg body weight, from about 0.01 μg to about 1 mg / kg body weight, from about 0.01 μg to about 100 μg / kg body weight, from about 0.01 μg to about 50 μg / kg body weight, from about 0.01 μg to about 10 μg / kg body weight, from about 0.01 μg to about 1 μg / kg body weight, from 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, and about 50 mg to about 100 mg / kg body weight.

[0509] Doses may be given one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly, or even once every 2 to 20 years. One of skill in the art can readily estimate repetition rates for dosing based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the multispecific binding proteins described herein may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intraoral, by perfusion via a catheter, or by direct intralesional injection. It may be administered one or more times daily, one or more times weekly, one or more times monthly, and one or more times yearly.

[0510] The above description provides multiple aspects and embodiments of the multispecific binding proteins described in the present application. This patent application specifically contemplates all combinations and permutations of aspects and embodiments. Any and all examples or exemplary language herein, such as "such as" or "including," is intended merely to better describe the multispecific binding proteins described in the present application and does not pose a limitation on the scope of the disclosure unless expressly so stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the multispecific binding proteins described in the present application. [Example]

[0511] The following examples are illustrative only and are not intended to limit in any way the scope or content of the multispecific binding proteins described in this application.

[0512] Example 1 - Evaluation of TriNKET binding to human BAFF-R expressed in cells TriNKET binding to cell surface BAFF-R was assessed using the BAFF-R-positive human B-lymphoblastoid RAJI cell line. Certain BAFF-R TriNKETs in 2-Fab and F3' formats, as described in the "Exemplary Multispecific Binding Proteins" subsection above, were diluted and incubated with Raji cells. The binding patterns of TriNKET and the parent monoclonal antibody were detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were then incubated with a fluorophore-conjugated anti-human IgG secondary antibody and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values ​​were normalized to a secondary antibody-only control to obtain fold-over-background (FOB) values.

[0513] As shown in Figures 18A-18C, BAFF-R TriNKETs containing BAFF-R binding sites derived from hCOH-2 (Figure 18A), Hu9.1-73 (Figure 18B), and ianalumab-based antigen binding sites (three versions, F3', 2-Fab, and ianalumab-mAb, which do not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 18C) bind at subnanomolar concentrations and with maximum MFIs similar to or higher than the corresponding parental control antibodies that do not contain the ADCC-enhancing mutations used in ianalumab. The EC values ​​of these TriNKET-bound BAFF-R were 50 Values ​​are shown in Table 11. Similar results were obtained with a second BAFF-R positive cell line, Ramos (data not shown). [Table 32]

[0514] Example 2 - Human NK cell cytotoxicity assay Lysis of BAFF-R-expressing target cells by immune effector cells in the presence of TriNKET was measured by the DELFIA cytotoxicity assay. Briefly, the BAFF-R-expressing human cancer cell line RAJI was harvested from culture, washed with HBS, and diluted to 10% in growth medium for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were resuspended at 0.5–1.0 × 10 cells / mL. The manufacturer's instructions were followed for labeling of target cells. After labeling, the cells were washed three times with HBS and resuspended at 0.5–1.0 × 10 cells / mL in culture medium. 5 The cells were resuspended at 100 μl / mL. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Monoclonal antibodies against BAFF-R or TriNKET were diluted in culture medium, and 50 μl of the diluted mAb or TriNKET was added to each well.

[0515] To prepare NK cells, PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation, washed, and prepared for NK cell isolation. NK cells were isolated using a negative selection technique using magnetic beads. The purity of isolated NK cells was typically >90% CD3 - CD56 + The isolated NK cells were rested overnight and harvested from the culture. The cells were then washed and resuspended in 10 ml of culture medium for an effector-to-target (E:T) ratio of 5:1. 5 ~2.0×10 6 The NK cells were resuspended at a concentration of 1 / mL. 50 μl of NK cells were added to each well of the plate for a total culture volume of 200 μl. The plate was incubated at 37°C with 5% CO for 2-3 hours.

[0516] After incubation, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 × g for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate, and 200 μl of room temperature europium solution (Perkin Elmer C135-100) was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes, then read using a SpectraMax i3X instrument.

[0517] The spontaneous release of substances capable of forming fluorescent chelates with europium was measured in target cells incubated in the absence of NK cells. The maximal release of such substances was measured in target cells lysed with 1% Triton-X. The % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) x 100%.

[0518] Figures 19A-19C show NK cell-mediated lysis of BAFF-R-positive RAJI cells by primary NK cells in the presence of BAFF-R-targeting TriNKET derived from hCOH-2 (Figure 19A), Genentech Hu9.1-73 (Figure 19B), and ianalumab-based antigen binding sites (three versions, F3', 2-Fab, and ianalumab-mAb, which do not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 19C). The parental BAFF-R-targeting monoclonal antibody showed little enhancement of NK cell-mediated lysis of RAJI target cells. All BAFF-R-targeting TriNKETs (hCOH-2-F3', hCOH-2-2-Fab, Hu9.1-73-F3', Hu9.1-73-2-Fab, ianalumab-F3', and ianalumab-2-Fab) showed superior lysis of target cells compared to the respective BAFF-R-targeting monoclonal antibodies. 50 The values ​​are shown in Table 12. [Table 33]

[0519] To confirm the cytotoxicity findings, the NK cell line KHYG-1-CD16aV was engineered to stably express CD16aV and NKG2D and assayed as described above. EC was measured for TriNKET and parental mAbs derived from hCOH-2 (Figure 20A), Hu9.1-73 (Figure 20B), and ianalumab-based antigen-binding sites (three versions: F3', 2-Fab, and ianalumab-mAb, which does not contain the antibody-dependent cellular cytotoxicity-enhancing mutations present in the commercially available ianalumab antibody) (Figure 20C). 50 EC50 and maximal lysis values ​​were derived from the cytolysis curves by GraphPad Prizm software using a four-parameter logistic nonlinear regression curve-fitting model (Figure 13). All BAFF-R TriNKETs tested exhibited subnanomolar EC50 and highly efficient maximal lysis in the KHYG-1 CD16aV-mediated cytotoxicity assay. Similar results were obtained with a second BAFF-R-positive cell line, Ramos (data not shown). [Table 34]

[0520] Example 3 - Generation and characterization of BAFF-R binding mAbs Recombinant protein immunization method BAFF-R-specific antibodies were generated by immunizing four different strains of mice (H2L2, NZBW, BALB-C, and SJL / J) with hBAFF-R-hFc-His fusion protein. Based on the antiserum titer, a total of seven mice across the four different strains were selected for hybridoma fusion. Spleen cells from a subset of mice from each immunization arm were reserved for immune library generation, but only splenocytes from H2L2 mice were used for yeast-display mAb discovery.

[0521] From five mouse fusions (spleen cells from two mice were pooled for the H2L2 fusion and splenocytes from two mice were pooled for the SJL / J fusion), 16 96-well plates per hybridoma fusion were analyzed by specificity ELISA, in which binding to human and cynomolgus BAFF-R-hFc-His and binding to an unrelated hFc-His protein were compared. Supernatants from 33 BAFF-R-positive and 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 the specificity ELISA described above, and 20 BAFF-R-positive and specific subclones were tested for binding to BAFF-R+ cells. Nine subclone mAbs demonstrated strong binding to BAFF-R+ cells and were sequenced. Six unique sequences were obtained and the corresponding mAbs were further analyzed for their ability to block the BAFF-R-BAFF interaction in a cell-based assay.

[0522] Binding of biotinylated BAFF to BAFF-R+ CHO cells was tested in the absence or presence of six BAFF-R-specific mAbs or an isotype control mAb. A reduction in mean fluorescence intensity (MFI) in the presence of the antibody indicated that the mAb inhibited BAFF binding to BAFF-R and was therefore designated a blocking antibody. All clones tested did not inhibit BAFF binding to BAFF-R+ cells; therefore, all six were designated non-blocking (Figure 21).

[0523] DNA immunization method Two groups of SWR / J mice were immunized with DNA. One group was immunized with a full-length human BAFF-R cDNA construct, and the other group was immunized with a mixture of full-length human BAFF-R and a human BAFF-R extracellular domain cDNA construct. Based on the antiserum titer, mice were pooled and then selected for single B cell sorting, and another pool was used for hybridoma fusion.

[0524] A single B cell sorting effort yielded 44 human and cynomolgus monkey cross-reactive clones. These clones were sequenced, transiently expressed in 293 cells, and the specificity of the purified mAbs was confirmed using hBAFF-R. + , cynomolgus monkey BAFF-R + Binding to isogenic CHO cells and the parental cell line was analyzed by flow cytometry, where binding was compared. Eight binders were purified and further analyzed for their ability to bind to BAFF-R and block the BAFF-R-BAFF interaction. All eight clones were determined to be non-blocking and blocked hBAFF-R. + Weak affinity for cancer cells was demonstrated.

[0525] The specificity of clones obtained by the traditional hybridoma approach was analyzed by flow cytometry to assess: a) binding to cells expressing either full-length human BAFF-R or the human BAFF-R extracellular domain compared to binding to non-transfected parental cells; + and cynomolgus monkey BAFF-R + c) binding to isogenic cells compared to binding to parental cells; + Binding to cancer cells was performed. 25 positive hybridoma fusions were identified, and 14 hybridoma fusions were sequenced based on binding intensity. Five unique sequences were obtained, and 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 (Figure 22), but four of the five clones (clones 3A1, 1B3-A7, 7G4, and 10H7-C5) demonstrated good affinity for hBAFF-R.

[0526] BAFF-R-specific scFv discovered from a yeast library Using yeast display, an scFv library was constructed from splenocytes obtained from humanized H2L2 mice immunized with the recombinant human hBAFF-R-hFc-His protein described above. Three rounds of selection were performed using biotinylated hBAFF-R-hFc-His at 5 nM. Individual yeast colonies were picked, sequenced, and analyzed. Sequence convergence indicated that the selection process was successful and complete in enriching binders. Unique sequences were selected for further characterization. Three BAFF-R-specific scFvs were discovered from one library (Table 14). However, because these sequences were very similar to each other, only sequence 1129_A01 (also referred to as AB0369scFv) was selected for further study. [Table 35]

[0527] Flow cytometry was used to assess the specificity of AB0369scFv binding to hBAFF-R-hFc-His, hBAFF-R-GST-His, and negative control proteins bearing hFc or GAT tags while displayed on yeast. AB0369scFv demonstrated moderate to weak affinity for hBAFF-R, but showed no binding to the negative control, thus suggesting high specificity for BAFF-R (Figure 23).

[0528] To generate AB0369, 1129_A01 (AB0369 scFv) was converted into a multispecific binding protein containing the scFv and two non-BAFF-R binders. AB0369 binds human (hBAFF-R-CHO) and cynomolgus monkey (cBAFF-R-CHO) BAFF-R. + Its ability to bind to cells (Figures 24A and 24B), lack of nonspecific interactions by polyspecific reagent (PSR) assay (Figures 25A-G), BAFF-R +AB0369 was further analyzed for its ability to lyse Ramos cancer cells (Figure 26 and Table 15) and block BAFF-BAFF-R interaction (Figure 27). AB0369 binds to both human and cynomolgus monkey BAFF-R on the surface of isogenic CHO cells, with BAFF-R binding occurring with an EC 50 This made it a good choice for further development. [Table 36]

[0529] 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 in cold FACS buffer, and seeded at a density of 100,000 cells per well. Test substances were diluted in FACS buffer, and 50 μL of the 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, added at 100 μL 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. The cells were then incubated in 100 μL of a 1:1,000 dilution of live / dead dye in PBS for 15 minutes, then washed with FACS buffer and fixed. After incubation, the cells were washed with FACS buffer and resuspended in FACS buffer for analysis by flow cytometry. The median fluorescence intensity (MFI) of each sample and the secondary-only control was calculated. The maximum MFI was calculated for BAFF-biotin alone, and the minimum MFI was calculated for streptavidin-phylcoerythrin alone. The data were fitted to a four-parameter linear regression curve using GraphPad Prism.

[0530] These studies revealed that AB0369 could partially block the BAFF-R-BAFF interaction. However, this blocking was significantly less potent than the ianalumab-based benchmark control, which, unlike the parent antibody, does not contain the antibody-dependent cellular cytotoxicity-enhancing mutations, likely due to the lower affinity of AB0369 (Figure 27 and Table 16). Because AB0369 scFv was the only blocking antibody identified from all of the above drug discovery studies, it was further developed by affinity maturation of CDRH3 and CDRH1 / CDRH2, as well as additional amino acid changes to facilitate protein production and stability. [Table 37]

[0531] Affinity maturation of AB0369 Randomized affinity maturation focused on CDRH3 As described above, AB0369 demonstrated specific binding to BAFF-R-expressing cells. To search for variants with improved binding affinity, a yeast display affinity maturation library was generated by mutating the CDRH3 residues of AB0369 (RFTMLRGLIIEDYGMDV (SEQ ID NO: 216)). To enrich for scFvs with higher affinity for hBAFF-R, two rounds of selection were performed using biotinylated hBAFF-R-hFc-His at 1 nM (Figures 28A-28D). The affinity between the parental clone AB0369 and representative individual library clones was compared. Three rounds of FACS sorting, using an ianalumab-based scFv as a benchmark control, resulted in nine clones containing one or two amino acid differences (bold) compared to the parental clone. [ka] It showed higher binding affinity to hBAFF-R than the parental clone and parent-derived scFv (Figs. 29A to 29D).

[0532] 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 to BAFF-R-expressing cells (Figure 10A) and lyse BAFF-R-expressing Ramos cancer cells (Figures 30B, 30C). All multispecific binding proteins scored negative in the multispecific assay, suggesting that the improved binding affinity was BAFF-R specific (Figures 31A-31E). Further studies demonstrated a greater than three-fold improvement in BAFF-R binding, which was consistent with the EC 50 This led to a 6- to 10-fold improvement in potency as measured by BAFF-R binding affinity (Table 17). Maximum lysis remained unchanged, suggesting that improvements in BAFF-R binding affinity were a key driver of this improvement in potency. [Table 38]

[0533] Combinatorial affinity maturation focusing on CDRH1 and CDRH2 Results from affinity maturation studies focused on CDRH3 demonstrated improvements in affinity, but further improvement is highly desirable. Therefore, using the matured CDRH3 scaffold, CDRH1 and CDRH2 sequences were selected for affinity maturation (CDRH1: GFTFSSY (SEQ ID NO: 214) and CDRH2: WYDGSN (SEQ ID NO: 215)). The goal was to engineer and select binders with improved affinity over the parent clone (AB0369 scFv) or the CDRH3-optimized variants described above. This generated 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 32A-C).

[0534] After FACS, 24 clones were identified. Several clones with changes in CDRH1 on the optimized CDRH3 scaffold (RFTMLRGWYIEDYGMDV (SEQ ID NO: 224); RFTMLRGQYIEDYGMDV (SEQ ID NO: 223); RFTMLRGWIIEDYGMDV (SEQ ID NO: 225)) were observed to show significant improvements in hBAFF-R affinity compared to the parental AB0369 scFv (1129_A01) (Figures 33A-33D) or compared to the ianalumab-based scFv benchmark control (scFv containing VH and VL sequences based on the ianalumab VH and VL sequences but not containing the ADCC-enhancing mutations used in the parent antibody) (Figure 33E).

[0535] The scFv with the highest hBAFF-R binding affinity was converted into a multispecific binding protein containing the scFv and two BAFF-R binders, expressed in Expi293 cells, and their ability to bind to human BAFF-R-expressing cells (Figure 34A), cynomolgus monkey BAFF-R, and BAFF-R-expressing cells was assessed. + They were further analyzed for their ability to bind to cells (Figure 34B) and to inhibit the BAFF-R-BAFF interaction (Figure 34C and Table 18). The multispecific binding proteins tested 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 35, Table 19). [Table 39] [Table 40]

[0536] Repairing potential sequence liabilities Because affinity-matured clones contained amino acids in their CDRs that could adversely affect protein expression, stability, or immunogenicity, additional libraries were constructed to select clones that did not contain these amino acids. Three rounds of selection were performed with 1 nM biotinylated hBAFF-R-hFc-His protein, resulting in high-affinity enrichment (Figures 36A-36D). A total of 23 binders were identified, 12 of which were predicted to be free of undesirable amino acids ("liability-corrected").

[0537] Preferred clones from these libraries, including AB0898 (a liability-corrected version of AB0682 described above), AB0899, and AB0900, were successfully identified and tested for their binding to hBAFF-R while displayed on yeast. All clones showed higher affinity for hBAFF-R than the parental AB0369 scFv (Figures 37A-F).

[0538] Characterization of a Liability-Modified Multispecific Binding Protein Three of the liability-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 the ability to lyse BJAB cells in a KHYG-1-CD16aV-mediated cytotoxicity assay. The characterization of these clones is summarized in Table 20 and demonstrates successful liability correction. No adverse effects on cell binding were observed, and all three clones demonstrated potent killing of BAFF-R-expressing tumor cells (Figure 38). However, the thermostability of the molecules was not significantly affected by T, as shown in Figures 39A-C. m1 It was >65℃. [Table 41]

[0539] As described above, substitution of potential sequence liability residues in the CDRs with specific amino acids had minimal impact on binding affinity, but binding and thermal stability data from BAFF-R-expressing cells suggested further improvement was desirable. Therefore, the CDRH1 and CDRH2 sequences (CDRH1: GFTFSSY (SEQ ID NO: 214) and CDRH2: WYDGSN (SEQ ID NO: 215)) were affinity matured into a liability-corrected CDRH3 backbone, and off-rate pressure was applied to select for high-affinity clones. Briefly, clones were preincubated with biotinylated hBAFF-R-hFc-His at a concentration of 100 pM and then challenged with 1 μM non-biotinylated hBAFF-R-hFc-His for 2 hours. Yeast displaying anti-BAFF-R scFv that remained bound to biotinylated hBAFF-R-hFc-His was selected, and this process was repeated three times to enrich for high-affinity binders with slower off-rates. As shown in Figure 40, even after the off-rate pressure challenge, the clone remained bound to biotinylated hBAFF-R-hFc-His, whereas the ianalumab-based scFv benchmark control failed to bind to biotinylated hBAFF-R-hFc-His under these conditions, suggesting a slower dissociation rate.

[0540] Analysis of individual clones demonstrated high affinity for hBAFF-R-hFc-His (Figure 41), and importantly, the clones remained bound to biotinylated hBAFF-R-hFc-His. Notably, the ianalumab-based benchmark scFv showed failure to bind to biotinylated hBAFF-R-hFc-His after challenge (Figures 41A and 41B). Some of these clones were excluded from further consideration because they contained additional undesirable amino acids or properties. The sequences of selected clones from the above study are shown in Table 21. [Table 42]

[0541] Potential sequence liabilities are underlined in bold, and residues showing variability between clones are displayed in bold.

[0542] Selected clones from the off-rate challenge study described above were produced as multispecific binding proteins containing scFv of each binder 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 fluorimetry, DSF), and hydrophobicity (HIC) (results are summarized in Table 22). + Binding affinity to cells was improved compared to the parent clone (Figures 42A and 42B, compared to Table 20). Furthermore, binding affinity to cynomolgus monkey BAFF-R was similar to that to hBAFF-R (Figures 42A and 42B). Lack of multispecificity was confirmed by PSR assay (Figures 43A-I). AB1084 was excluded from further studies due to its longer retention time in HIC and subsequent higher tendency to aggregate. The improved multispecific binding protein demonstrated much higher potency than the multispecific binding protein based on the ianalumab sequence (Figures 44A and 44B). Additionally, a more 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 parent AB0369 multispecific binding protein (Figure 45). [Table 43]

[0543] These multispecific binding proteins met the criteria for acceptable thermal stability compared to the controls adalimumab (Humira) and pembrolizumab (Keytruda) (Figure 46). HIC chromatograms revealed that AB1080 and AB1081 had retention times of 11.4 and 11.5 minutes, respectively. AB1085 demonstrated a retention time of 9.5 minutes, which is at the low end of approved late-stage therapeutic antibodies and suggests highly favorable hydrophobic behavior (Figures 46A-D).

[0544] AB1080 and AB1081 showed improved binding to BAFF-R and did not contain any sequence liability in the CDR sequences, but their hydrophobicity was higher compared to a panel of benchmarked therapeutic antibodies. AB1085 demonstrated the desired hydrophobicity and affinity but contained potential sequence liability in the CDRH2 and CDRH3 sequences (Figure 47). The sequences of AB1080, AB1081, and AB1085 were compared, and the AB1080 sequence was analyzed and further modified to generate a W to Q hydrophobicity-reducing mutation (Figure 47). [ka] from [ka] The resulting AB1424 / AB1612 multispecific binding protein maintained the same high affinity for BAFF-R (Table 23, Figures 49A and 49B), potent blocking of BAFF-R-BAFF binding (Figure 50), and demonstrated favorable low hydrophobicity (Table 24) within the range of well-performing biologics (Figure 48), while containing the liability-free sequence characteristic of the parent AB1080. [Table 44] [Table 45]

[0545] In conclusion, two antibody discovery campaigns utilizing recombinant protein and DNA immunization were completed. The first campaign identified four moderate-affinity, non-blocking antibodies. A single binder, AB0369scFv, discovered from the second campaign displayed the ability to block the BAFF-R-BAFF interaction. Extensive development of AB0396scFv through multiple rounds of affinity maturation, liability modification, and rational sequence design led to binders AB1612 / AB1424, which demonstrated desirable properties for therapeutic candidates.

[0546] Example 4 - Molecular analysis of AB1424 / AB1612 F3'TriNKET format In this example, the molecular format, design, structure, and properties of AB1424 / AB1612 F3'TriNKET were analyzed. These studies a) provided basic biochemical and biophysical properties of the molecule, b) demonstrated the affinity of AB1424 / AB1612 F3'TriNKET for BAFF-R, NKG2D, and CD16a (V and F allele variants), c) con...

Claims

1. A protein, (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to B-cell activating factor receptor (BAFF-R); and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16. (i) the first antigen-binding site that binds to NKG2D is a Fab fragment, and the second antigen-binding site that binds to BAFF-R is an scFv; or (ii) The protein of claim 1, wherein the first antigen-binding site that binds to NKG2D is an scFv and the second antigen-binding site that binds to BAFF-R is a Fab fragment.

3. The protein of claim 2, wherein the protein further comprises an additional antigen-binding site that binds to BAFF-R; (i) the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site that binds to BAFF-R and the additional antigen-binding site are each Fab fragments; or (ii) the first antigen-binding site that binds to NKG2D is an scFv, and the second antigen-binding site that binds to BAFF-R and the additional antigen-binding site are each scFv; Optionally, the amino acid sequences of the second antigen-binding site and the additional antigen-binding site are identical. (a) the scFv that binds to NKG2D is linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or a portion thereof sufficient to bind to CD16, the scFv comprising a heavy chain variable domain and a light chain variable domain; and / or each scFv that binds to BAFF-R is linked via a hinge comprising Ala-Ser or Gly-Ser to an antibody constant domain or a portion thereof sufficient to bind to CD16, the scFv comprising a heavy chain variable domain and a light chain variable domain; Optionally, the hinge further comprises the amino acid sequence Thr-Lys-Gly; (b) in the scFv that binds to NKG2D, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv, and / or In scFvs that bind to BAFF-R, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv; Optionally, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, numbered under the Kabat numbering scheme; (c) within the scFv that binds to NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker, and / or In scFvs that bind to BAFF-R, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker; Optionally, the flexible linker comprises (G 4 S) 4 (SEQ ID NO: 119); (d) (i) within the scFv that binds NKG2D, the heavy chain variable domain is positioned C-terminal to the light chain variable domain; and / or In scFvs that bind to BAFF-R, the heavy chain variable domain is positioned C-terminal to the light chain variable domain; or (ii) within the scFv that binds to NKG2D, the heavy chain variable domain is positioned N-terminal to the light chain variable domain, and / or In scFvs that bind to BAFF-R, the heavy chain variable domain is positioned N-terminal to the light chain variable domain; and / or (e) the Fab fragment that binds to NKG2D is not located between the antigen-binding site and the Fc or portion thereof; or The protein of claim 2, wherein the Fab that binds to BAFF-R is not located between the antigen binding site and the Fc or portion thereof.

5. The protein (a) a first antigen-binding site comprising a Fab fragment that binds to NKG2D; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds to B-cell activating factor receptor (BAFF-R); and (c) an Fc domain comprising a first antibody constant domain and a second antibody constant domain, which form a heterodimer that binds to CD16; the scFv is linked via a hinge to the N-terminus of the first antibody constant domain, and the Fab is linked to the N-terminus of the second antibody constant domain; Optionally, the hinge comprises Gly-Ser. (i) the first antigen-binding site that binds to NKG2D comprises a VH comprising a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) having the amino acid sequences of SEQ ID NOs: 81, 82, and 112, respectively, and a VL comprising a CDR1, a CDR2, and a CDR3 having the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively; (ii) the first antigen-binding site that binds to NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 81, 82, and 97, respectively, and a VL comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 86, 77, and 87, respectively; (iii) the first antigen-binding site that binds to NKG2D comprises a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 95, and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO: 85; or (iv) The protein of claim 1, wherein the first antigen-binding site that binds to NKG2D comprises a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO:

85.

7. the second antigen-binding site comprises: (i) a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 260, 249, and 261, respectively, and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 259, respectively; (ii) a heavy chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 214, 233, and 248, respectively, and a light chain variable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 217, 77, and 249, respectively; Optionally, the second antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 250 and a light chain variable domain at least 90% identical to SEQ ID NO: 251; and / or wherein the second antigen-binding site comprises a VH having a G44C substitution compared to SEQ ID NO: 250 and a VL having a G100C substitution compared to SEQ ID NO: 251; and / or (iii) The protein of claim 1, comprising a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 253, or a VH comprising the amino acid sequence of SEQ ID NO: 250 and a VL comprising the amino acid sequence of SEQ ID NO:

251.

8. the second antigen-binding site comprises a single-chain variable fragment (scFv); (i) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 253; and / or (ii) The protein of claim 1, wherein the scFv comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 254 and 255. (i) the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:270, optionally, the protein comprises the amino acid sequence of SEQ ID NO:270; or (ii) The protein of claim 1, wherein the protein comprises an amino acid sequence at least 90% identical to SEQ ID NO: 271, and optionally, the protein comprises the amino acid sequence of SEQ ID NO:

271. (i) the second 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 second antigen-binding site inhibits the binding of BAFF-R to BAFF;

11. The protein (a) a first antigen-binding site comprising a VH and a VL of an anti-NKG2D antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO: 95 and the VL comprises the amino acid sequence of SEQ ID NO: 85; (b) a second antigen-binding site comprising a VH and a VL of an anti-BAFF-R antibody, wherein the VH comprises the amino acid sequence of SEQ ID NO: 252 and the VL comprises the amino acid sequence of SEQ ID NO: 253; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16.

12. The protein of claim 11, wherein the second antigen-binding site comprises the amino acid sequence set forth in SEQ ID NO:

254.

13. The protein of claim 1 , wherein the antibody Fc domain is a human IgG1 antibody Fc domain.

14. (a) the antibody Fc domain, or a portion thereof, comprises an amino acid sequence at least 90% identical to SEQ ID NO:118; (b) at least one polypeptide chain of the antibody Fc domain comprises one or more mutations compared to SEQ ID NO: 118 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 Fc domain 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, T366S, L and / or comprises one or more mutations compared to SEQ ID NO: 118 selected from: 368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, F405L, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T411D, T411E, K439D, and K439E; and / or (d) one polypeptide chain of the antibody heavy chain constant region has a sequence similar to or different from SEQ ID NO: 118 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, numbered according to the EU numbering system.

14. The protein of claim 13, wherein the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations compared to SEQ ID NO: 118 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. (i) one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118, numbered according to the EU numbering system, and another polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to SEQ ID NO: 118; or one polypeptide chain of said antibody heavy chain constant region comprises a F405L substitution compared to SEQ ID NO: 118, numbered according to the EU numbering system, and the other polypeptide chain of said antibody heavy chain constant region comprises a K409R substitution compared to SEQ ID NO: 118; and / or (ii) one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO: 118, numbered according to the EU numbering system, and the other polypeptide chain of the antibody heavy chain constant region comprises a S354C substitution relative to SEQ ID NO:

118.

16. The protein (A) (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 270; and (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 194; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 195; or (B) (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 271; and (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 272; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:

273.

17. A pharmaceutical composition comprising the protein according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. A cell comprising one or more nucleic acids encoding a protein according to any one of claims 1 to 16.

19. 17. An in vitro method for enhancing tumor cell death or B cell death, the method comprising exposing (i) the tumor cells or the B cells, and (ii) natural killer cells to an effective amount of a protein according to any one of claims 1 to 16 or a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.

20. 18. The pharmaceutical composition of claim 17 for use in a method for treating cancer, said method comprising administering to a subject in need thereof an effective amount of said protein or said pharmaceutical composition; The pharmaceutical composition, wherein the cancer is selected from the group consisting of 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 large B-cell lymphoma, and acute lymphocytic leukemia (ALL).

21. 18. The pharmaceutical composition of claim 17 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 or the pharmaceutical composition.

22. 17. The protein of any one of claims 1 to 16, wherein the protein is a purified protein, and the protein is purified using 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.