Multispecific binding proteins that bind BCMA, NKG2D and CD16 and methods of use

Abstract

The present invention describes multispecific binding proteins that bind to and kill human cancer cells, as well as pharmaceutical compositions and methods of treating cancer. The cancer can be a cancer that expresses B cell maturation antigen (BCMA). Compared to anti-BCMA monoclonal antibodies, the multispecific binding proteins provided herein exhibit high efficacy and maximum lysis of target cells.

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Serial No. 62 / 716,207, filed August 8, 2018, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This specification is submitted together with a computer-readable copy of the sequence list (CRF). The CRF, created on August 8, 2019, entitled 14247-425-228_ST25.txt, is 137,008 bytes in size and is incorporated herein by reference in its entirety. Technical Field

[0005] This invention relates to multispecific binding proteins that bind to NKG2D, CD16, and B-cell maturation antigen (BCMA). Compared to anti-BCMA monoclonal antibodies, these multispecific binding proteins exhibit high efficiency and maximum lysis of target cells, and can be used to kill human cancer cells expressing BCMA. Background Technology

[0006] Despite substantial research findings and scientific advances in the literature regarding the treatment of this disease, cancer remains a significant health concern. Some of the most frequently diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains a leading cause of death in women. Current treatment options for these cancers are not effective for all patients and / or may have substantial adverse side effects. Treating other types of cancer using existing treatment options remains challenging.

[0007] Cancer immunotherapy is ideal because of its high specificity and ability to leverage the patient's own immune system to promote the destruction of cancer cells. Fusion proteins (such as bispecific T-cell conjugates) are described in the literature as cancer immunotherapies that bind to both tumor cells and T cells to promote the destruction of tumor cells. Antibodies that bind to certain tumor-associated antigens and certain immune cells have been described in the literature. See, for example, WO 2016 / 134371 and WO 2015 / 095412.

[0008] Natural killer (NK) cells are components of the innate immune system and comprise approximately 15% of circulating lymphocytes. NK cells infiltrate almost all tissues and are initially characterized by their ability to effectively kill tumor cells without prior sensitization. Activated NK cells kill target cells in a manner similar to cytotoxic T cells, via cytolytic granules containing perforin and granzymes, and via the death receptor pathway. Activated NK cells also secrete inflammatory cytokines, such as IFN-γ and chemokines, that promote the recruitment of other leukocytes to target tissues.

[0009] NK cells respond to signals through a variety of activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated via their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated via CD16 receptors on their surface through the homeostatic regions of some immunoglobulins. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0010] BCMA is a transmembrane protein belonging to the TNF receptor superfamily. It specifically binds to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B / TALL-1 / BAFF), leading to NF-κB and MAPK8 / JNK activation. Its expression is restricted to the B cell lineage and has been shown to be crucial for B cell development and autoimmune responses. BCMA also binds to various TRAF family members and can therefore transduce signals for cell survival and proliferation. BCMA is associated with a variety of cancers, such as multiple myeloma, lymphoma, and leukemia. This invention provides certain advantages for improving the treatment of cancers expressing BCMA. Summary of the Invention

[0011] This invention provides multispecific binding proteins that bind to BCMA (e.g., BCMA on cancer cells) and NKG2D and CD16 receptors expressed on, for example, natural killer cells. Such proteins can bind to more than one NK-activating receptor and can block the binding of natural ligands to NKG2D. In some embodiments, the protein can stimulate NK cells in humans and other species such as rodents and cynomolgus monkeys. In some embodiments, the protein can stimulate cytotoxic T cells in humans and other species such as rodents and cynomolgus monkeys. In some embodiments, the protein stimulates human NK cells. In some embodiments, the protein stimulates human cytotoxic T cells. Various aspects and embodiments of the invention are further described in detail below.

[0012] Therefore, one aspect of the present invention provides a protein comprising (a) a first antigen-binding site comprising a single-stranded variable fragment (scFv) binding NKG2D; (b) a second antigen-binding site binding B-cell maturation antigen (BCMA); and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16 or a third antigen-binding site binding CD16. In some embodiments, the protein of this disclosure further comprises an additional antigen-binding site binding BCMA. In some embodiments, the second antigen-binding site of the protein described in this disclosure is a Fab fragment binding BCMA. In some embodiments, the second antigen-binding site and the additional antigen-binding site of the protein described in this disclosure are both Fab fragments binding BCMA.

[0013] In some embodiments, the second antigen-binding site and additional antigen-binding site of the protein described in this disclosure are scFvs that bind BCMA. In some embodiments, the heavy chain variable domain of the scFv that binds NKG2D is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. In some embodiments, the light chain variable domain is located at the N-terminus of the heavy chain variable domain of the scFv that binds NKG2D.

[0014] In some embodiments, the NKG2D-binding scFv is linked to an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16. In some embodiments, the NKG2D-binding scFv is linked to an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16, via a hinge comprising Ala-Ser. In some embodiments, the NKG2D-binding scFv is linked to the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16, via a flexible linker comprising the amino acid sequence SEQ ID NO: 168. In some embodiments, the C-terminus of the Fc domain is linked to the V-terminus of the NKG2D-binding scFv. L The flexible linker at the N-terminus of the domain (e.g., SEQ ID NO: 98) has the amino acid sequence of SEQ ID NO: 168. In some embodiments, the C-terminus of the antibody Fc domain is linked to the N-terminus of the light chain variable domain of the scFv that binds NKG2D.

[0015] In some embodiments, within the scFv incorporating NKG2D, a disulfide bridge is formed between the heavy-chain variable structural domains and the light-chain variable structural domains of the scFv. In some embodiments, a disulfide bridge is formed between C44 from the heavy-chain variable structural domain and C100 from the light-chain variable structural domain.

[0016] In some embodiments, within the scFv incorporating NKG2D, the heavy chain variable structural domain is connected to the light chain variable structural domain via a flexible joint. In some embodiments, the flexible joint comprises (GlyGlyGlyGlySer). n (G4S) n (SEQ ID NO: 198), where n is an integer between 1 and 10. In some embodiments, the flexible connector has the amino acid sequence of SEQ ID NO: 167.

[0017] In some embodiments, the second antigen-binding site and the additional antigen-binding site scFv are connected via an Ala-Ser-containing hinge to an antibody Fc domain sufficient to bind CD16, or a portion thereof, or a third antigen-binding site sufficient to bind CD16. In some embodiments, the second antigen-binding site and the additional antigen-binding site scFv are connected to the antibody Fc domain via an Ala-Ser-containing hinge.

[0018] In some embodiments, a disulfide bridge is formed between the heavy chain variable domain and the light chain variable domain at the second antigen binding site and / or additional antigen binding sites. In some embodiments, a disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

[0019] In some embodiments, the NKG2D-bound scFv includes a light chain variable domain located at the N-terminus of the heavy chain variable domain, wherein the light chain variable domain is connected to the heavy chain variable domain of the scFv via a flexible linker containing the amino acid sequence of SEQ ID NO: 167, and the NKG2D-bound scFv is connected to the antibody Fc domain via a hinge containing Ala-Ser.

[0020] In some embodiments, the protein of the present invention comprises a first antigen-binding site containing scFv that binds NKG2D, the first antigen-binding site comprising:

[0021] (a) A heavy chain variable domain comprising the sequences of complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3), represented by the amino acid sequences of SEQ ID NO: 190, 96, and 191, respectively; and a light chain variable domain comprising the sequences of CDR1, CDR2, and CDR3, represented by the amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively;

[0022] (b) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96 and 193, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100 and 101, respectively.

[0023] (c) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 95, 96 and 97, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100 and 101, respectively.

[0024] (d) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 188, 88 and 189, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 91, 92 and 93, respectively.

[0025] (e) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 185, 104 and 192, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 107, 108 and 109, respectively.

[0026] (f) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 185, 72 and 159, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 75, 76 and 77, respectively.

[0027] (g) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 186, 80 and 187, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 83, 84 and 85, respectively.

[0028] (h) includes a heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96 and 194, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100 and 101, respectively.

[0029] (i) a heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96 and 195, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100 and 101, respectively.

[0030] (j) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96 and 196, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100 and 101, respectively.

[0031] (k) A heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 190, 96, and 197, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; or

[0032] (1) A heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 160, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; and

[0033] The second antigen-binding site and / or additional antigen-binding sites that bind to BCMA include:

[0034] (a) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 149, 150 and 151, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 153, 154 and 155, respectively.

[0035] (b) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 115, 116 and 117, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 120, 121 and 123, respectively.

[0036] (c) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 125, 126 and 127, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 129, 130 and 131, respectively.

[0037] (d) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 133, 134 and 135, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 137, 138 and 139, respectively.

[0038] (e) A heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 141, 142, and 143, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 145, 146, and 147, respectively; or

[0039] (f) A heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 115, 116 and 117, respectively; and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 120, 121 and 122, respectively.

[0040] In some embodiments, the protein disclosed herein comprises the amino acid sequence of SEQ ID NO: 162.

[0041] In some embodiments, the protein of this disclosure comprises an amino acid sequence containing SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 165.

[0042] In some embodiments, the protein disclosed herein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 162.

[0043] In some embodiments, the protein disclosed herein comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 162.

[0044] In some embodiments, the protein disclosed herein comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 162.

[0045] In some embodiments, the protein of this disclosure comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 162, and also comprises SEQ ID NO: 163 and SEQ ID NO: 165.

[0046] In some embodiments, the protein of this disclosure includes a first antigen-binding site that binds to NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the amino acid sequence of SEQ ID NO: 94.

[0047] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 94 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 94 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 94 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 94 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 94 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0048] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 169 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 169 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 169 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 169 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99 identity with SEQ ID NO: 169 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0049] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 171 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 171 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 171 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 171 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 171 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0050] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 173 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 173 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 173 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 173 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 173 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0051] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 175 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 175 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 175 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 175 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 175 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0052] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 177 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 177 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 177 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 177 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 177 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0053] In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 179 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 179 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 179 and a light chain variable domain having at least 98% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 179 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding NKG2D, the first antigen-binding site comprising a heavy chain variable domain having at least 99 identity with SEQ ID NO: 179 and a light chain variable domain having at least 99% identity with SEQ ID NO: 98.

[0054] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 148 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 152. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 148 and a light chain variable domain having at least 95% identity with SEQ ID NO: 152. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 148 and a light chain variable domain having at least 98% identity with SEQ ID NO: 152. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 148 and a light chain variable domain having at least 99% identity with SEQ ID NO: 152. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 148 and a light chain variable domain having at least 99% identity with SEQ ID NO: 152.

[0055] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 119. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 119. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 114 and a light chain variable domain having at least 98% identity with SEQ ID NO: 119. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 114 and a light chain variable domain having at least 99% identity with SEQ ID NO: 119. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 114 and a light chain variable domain having at least 99% identity with SEQ ID NO: 119.

[0056] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 124 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 128. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 124 and a light chain variable domain having at least 95% identity with SEQ ID NO: 128. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 124 and a light chain variable domain having at least 98% identity with SEQ ID NO: 128. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 124 and a light chain variable domain having at least 99% identity with SEQ ID NO: 128. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 124 and a light chain variable domain having at least 99% identity with SEQ ID NO: 128.

[0057] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 132 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 136. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 132 and a light chain variable domain having at least 95% identity with SEQ ID NO: 136. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 132 and a light chain variable domain having at least 98% identity with SEQ ID NO: 136. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 132 and a light chain variable domain having at least 99% identity with SEQ ID NO: 136. In some embodiments, the protein of this disclosure includes a first antigen-binding site for binding BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 132 and a light chain variable domain having at least 99% identity with SEQ ID NO: 136.

[0058] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 140 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 144. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 140 and a light chain variable domain having at least 95% identity with SEQ ID NO: 144. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 140 and a light chain variable domain having at least 98% identity with SEQ ID NO: 144. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 140 and a light chain variable domain having at least 99% identity with SEQ ID NO: 144. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 140 and a light chain variable domain having at least 99% identity with SEQ ID NO: 144.

[0059] In some embodiments, the protein of this disclosure includes a second antigen-binding site for BCMA, the second antigen-binding site comprising a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) and a light chain variable domain having at least 90% identity with SEQ ID NO: 118. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 118. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 98% identity with SEQ ID NO: 114 and a light chain variable domain having at least 98% identity with SEQ ID NO: 118. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 114 and a light chain variable domain having at least 99% identity with SEQ ID NO: 118. In some embodiments, the protein of this disclosure includes a first antigen-binding site for BCMA, the first antigen-binding site comprising a heavy chain variable domain having at least 99% identity with SEQ ID NO: 114 and a light chain variable domain having at least 99% identity with SEQ ID NO: 118.

[0060] In some embodiments, the protein further comprises an additional antigen-binding site for binding BCMA. In some embodiments, the additional antigen-binding site comprises the same CDR1, CDR2, and CDR3 of the heavy chain variable domain and the same CDR1, CDR2, and CDR3 of the light chain variable domain of the second antigen-binding site for binding BCMA. In some embodiments, the additional antigen-binding site comprises a heavy chain variable domain having at least 90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the heavy chain variable domain of the second antigen-binding site for binding BCMA, and a light chain variable domain having at least 90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the light chain variable domain of the second antigen-binding site for binding BCMA. In some embodiments, the additional antigen-binding site comprises a heavy chain variable domain that is identical to the heavy chain variable domain of the second antigen-binding site that binds to BCMA, and a light chain variable domain that is identical to the light chain variable domain of the second antigen-binding site that binds to BCMA.

[0061] The proteins disclosed herein comprise an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site for binding CD16. In some embodiments, the proteins disclosed herein comprise an antibody Fc domain. The antibody Fc domain can bind CD16. In some embodiments, the proteins disclosed herein comprise a portion of an antibody Fc domain that retains binding affinity (i.e., sufficient to bind CD16) for the antibody Fc domain. In some embodiments, the proteins disclosed herein comprise a third antigen-binding site for binding CD16. In some embodiments, the third antigen-binding site for binding CD16 comprises a Fab fragment. In some embodiments, the third antigen-binding site for binding CD16 comprises scFV.

[0062] In some embodiments, the scFv binding to NKG2D is linked to an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16. In some embodiments, the scFv binding to NKG2D is linked to an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16, via a hinge comprising Ala-Ser. In some embodiments, the scFv binding to NKG2D is linked to the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site sufficient to bind CD16, via a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind CD16, or the third antigen-binding site sufficient to bind CD16, is linked to the N-terminus of the light chain variable domain of the scFv binding NKG2D. In some embodiments, the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind CD16, or the third antigen-binding site sufficient to bind CD16, is linked to the V-terminus of the scFv binding NKG2D. L The flexible linker connected to the N-terminus of the structural domain (e.g., SEQ ID NO: 98) has the amino acid sequence of SEQ ID NO: 168.

[0063] In some embodiments, the protein disclosed herein comprises an antibody Fc domain. In some embodiments, an NKG2D-binding scFv is linked to the antibody Fc domain. In some embodiments, the NKG2D-binding scFv is linked to the antibody Fc domain via a hinge comprising Ala-Ser. In some embodiments, the NKG2D-binding scFv is linked to the C-terminus of the antibody Fc domain via a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the C-terminus of the antibody Fc domain is linked to the N-terminus of the light chain variable domain of the NKG2D-binding scFv. In some embodiments, the C-terminus of the Fc domain is linked to the V-terminus of the NKG2D-binding scFv. L The flexible linker connected to the N-terminus of the structural domain (e.g., SEQ ID NO: 98) has the amino acid sequence of SEQ ID NO: 168.

[0064] In some embodiments, the protein of this disclosure comprises an antibody Fc domain comprising a hinge and a CH2 domain of a human IgG1 antibody.

[0065] In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 90% identity with amino acids 234-332 of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 95% identity with amino acids 234-332 of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 98% identity with amino acids 234-332 of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 90% identity with the Fc domain of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 95% identity with the Fc domain of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 98% identity with the Fc domain of a human IgG1 antibody. In some embodiments, the protein of this disclosure comprises an Fc domain containing an amino acid sequence having at least 90% identity with the Fc domain of human IgG1, and differing at one or more positions selected from the group consisting of: Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0066] In some embodiments, the protein of this disclosure comprises an Fc domain of human IgG1 substituted with Q347R, D399V, and F405T. The protein of this disclosure comprises an Fc domain substituted with Q347R, D399V, and F405T, said Fc domain being linked to scFv which binds NKG2D.

[0067] In some embodiments, the protein disclosed herein comprises an Fc domain of human IgG1 containing K360E and K409W substitutions.

[0068] In some embodiments, the protein of this disclosure comprises an Fc domain containing K360E and K409W substitutions, the Fc domain being linked to a second antigen binding site.

[0069] In some embodiments, the first antigen-binding site binds to NKG2D with a KD of 2 nM to 120 nM, as measured by surface plasmon resonance. In some embodiments, the protein binds to NKG2D with a KD of 2 nM to 120 nM, as measured by surface plasmon resonance.

[0070] It also provides preparations containing at least one of these proteins; cells containing at least one or more nucleic acids expressing these proteins; and methods for using these proteins to enhance tumor cell death.

[0071] In another aspect of the invention, this disclosure provides a method for treating cancer, wherein a protein of this disclosure or a preparation comprising a protein of this disclosure is administered to a patient in need. In some embodiments, the cancer expresses BCMA. In some embodiments, at least 20% of the cancer cells express BCMA. In some embodiments, at least 50% of the cancer cells express BCMA. In some embodiments, at least 80% of the cancer cells express BCMA.

[0072] In some embodiments, the proteins disclosed herein are used to treat cancers selected from multiple myeloma, acute myelomonocytic leukemia, T-cell lymphoma, acute monocytic leukemia, and follicular lymphoma. Attached Figure Description

[0073] Figures 1A-1B An exemplary trispecific antibody (TriNKET) is shown, comprising a first antigen-binding site for NKG2D bound to scFv, a second antigen-binding site for BCMA bound to BCMA, an additional tumor-associated antigen-binding site for BCMA bound to scFv, and a heterodimeric antibody constant region bound to CD16. These antibody forms are referred to herein as F4-TriNKET. Figure 1A Two BCMA binding sites in Fab form are shown. Figure 1B Two BCMA binding sites in the form of scFv are shown.

[0074] Figure 2 An exemplary TriNKET is shown, comprising a first antigen-binding site for NKG2D bound to scFv, a second antigen-binding site for BCMA bound to scF2D, and a heterodimeric antibody constant region. This antibody form is referred to herein as F3-TriNKET.

[0075] Figure 3 This study demonstrated that, compared to anti-BCMA mAb, TriNKET (NKG2D-binding-F4-TriNKET-BCMA for short) targeting BCMA mediated more effective lysis of BCMA-positive KMS12-PE myeloma cells.

[0076] Figure 4 TriNKET, which targets BCMA, was shown to mediate more efficient lysis of BCMA-positive MM.1R myeloma cells compared to anti-BCMA mAb.

[0077] Figure 5 The study showed that incubation of the antibody targeting BCMA with TriNKET steadily increased total surface BCMA expression on KMS12-PE myeloma cells over time.

[0078] Figure 6 The study showed that incubation of the antibody targeting BCMA with TriNKET steadily increased total surface BCMA expression on MM.1R myeloma cells over time.

[0079] Figure 7 The study showed that prolonged incubation time with bivalent TriNKET significantly increased the amount of TriNKET binding to KMS12-PE myeloma cells.

[0080] Figure 8 The study showed that prolonged incubation time with bivalent TriNKET significantly increased the amount of TriNKET bound to MM.1R cells.

[0081] Figure 9 The results showed that in long-term purified NK killing assays, the bivalent TriNKET (F4 form) was superior to the bivalent mAb targeting BCMA and the monovalent TriNKET.

[0082] Figure 10 The efficacy of BCMA-TriNKET was demonstrated in a long-term cytotoxicity assay using fresh PBMC effector cells.

[0083] Figure 11 TriNKET, which targets BCMA, was shown to bind weakly (below the detection limit) to NKG2D expressed on KHYG-1 cells.

[0084] Figure 12 The binding of bivalent BCMA-targeting TriNKET (F4 form) was shown to be minimal beyond the binding of mAb Fc to KHYG-1 cells transduced to express CD16.

[0085] Figures 13A-13F This shows TriNKET (solid boundary, dark gray) targeting BCMA, extending beyond the background (dashed boundary, white), compared to NK cells in whole blood. Figure 13A ), CD8+ T cells ( Figure 13B ) and CD4+ T cells ( Figure 13C () does not bind significantly. Close to B cells ( Figure 13D), mononuclear cells ( Figure 13E ) and granulocytes ( Figure 13F The binding of IgG1 control (dotted boundary, light gray) is mainly mediated by Fc receptor.

[0086] Figure 14 The purity and target expression of CD8+ effector T cells are shown. As shown in the figure, CD8+ effector T cells generated using ConA stimulation and cultured with IL-15 have high purity (CD3+CD8+ cells > 99%) and all express NKG2D but not CD16.

[0087] Figures 15A-15B Cell lysis of KMS12-PE cells in the DELFIA assay is shown. The DELFIA cytotoxicity assay was performed using primary human CD8+ effector T cells and KMS12-PE target cells derived from two healthy donors. Figure 15A The results derived from cells from donor 1 were described and Figure 15B The results from cells derived from donor 2 are depicted. As shown in the figure, NKG2D-binding-F4-TriNKET-BCMA enhanced the lysis of KMS12-PE cells when co-cultured with activated CD8+ T cells, but not in the absence of effector cells. Parental anti-BCMA mAb or unrelated TriNKET did not enhance the lysis of CD8+ T cells from either donor.

[0088] Figures 16A-16B The activation of human NK cells within 4 hours in the presence of BCMA-positive target cell lines or in the presence of anti-BCMA TriNKET or monoclonal antibodies is shown. Figure 16A The results were depicted using KMS12-PE cells (with low BCMA expression) as target cells. Figure 16B The results were depicted using H929 cells (high BCMA expression) as the target cell. As shown in the figure, F4-TriNKET triggered increased degranulation and IFNγ production at sub-nanomolar EC50 values ​​against both high and low BCMA expression cells. Compared to BCMA monoclonal antibodies, F4 TriNKET maximally stimulated a larger proportion of NK cells and enhanced efficacy against both cell lines. Detailed Implementation

[0089] This invention provides a multispecific binding protein that binds to BCMA, NKG2D receptors, and CD16 receptors. The multispecific binding protein can bind to BCMA on cancer cells and to NKG2D and CD16 receptors on natural killer cells to activate natural killer cells. The multispecific binding protein can also bind to BCMA on cancer cells and to NKG2D and CD16 receptors on cytotoxic T cells to activate cytotoxic T cells. This document also provides pharmaceutical compositions comprising such multispecific binding proteins, and treatment methods using such multispecific proteins and pharmaceutical compositions, including for treating cancer. Aspects of the invention are set forth in the following sections; however, the aspects of the invention described in a particular section are not limited to any particular section.

[0090] To facilitate understanding of the invention, several terms and phrases are defined below.

[0091] Unless the context is inappropriate, as used herein, the term "a (species)" means "one (species) or more (species)" and includes the plural.

[0092] As used herein, the term "antigen-binding site" refers to a portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") region of the heavy chain ("H") and the light chain ("L"). Three highly divergent extensions within the V region of the heavy and light chains are called "hypervariable regions," which are interposed between more conserved flanking segments called "frame regions" or "FRs." Therefore, the term "FR" refers to the amino acid sequence between and adjacent to the hypervariable regions naturally present in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface that binds the antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." In some animals such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single-domain antibody." Antigen-binding sites can be present in intact antibodies, in antigen-binding fragments of antibodies that retain the antigen-binding surface, or in recombinant peptides such as scFv, which use peptide linkers to connect the heavy chain variable domain to the light chain variable domain within a single peptide. All amino acid positions in the heavy or light chain variable regions disclosed herein are numbered according to Kabat numbering.

[0093] As used herein, the term "tumor-associated antigen" refers to any antigen associated with cancer, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, and lipids. Such antigens can be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal matrix, or immune infiltration.

[0094] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rats, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably include humans.

[0095] As used herein, the term "effective amount" refers to an amount of an agent (e.g., the protein of the present invention) sufficient to achieve a beneficial or desired result. When used in conjunction with a therapeutic agent, the term refers to an amount of such an agent sufficient to provide a therapeutic benefit in the treatment of a disease or condition, or sufficient to delay or minimize one or more symptoms associated with a disease or condition. An effective amount may be administered, applied, or given once or multiple times and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treatment" includes any effect that results in the improvement or modification of the symptoms of a symptom, disease, condition, etc., such as relief, reduction, modulation, modification, or elimination.

[0096] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0097] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard drug carriers, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA

[1975] .

[0098] As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt (e.g., acid or base) of the compounds of the present invention that, when administered to a subject, provides the compounds of the present invention or their active metabolites or residues. As is known to those skilled in the art, the "salts" of the compounds of the present invention may 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, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids, such as oxalic acid, although not pharmaceutically acceptable on their own, may be used to prepare salts used as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0099] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and NW4.+ Compound, where W is C 1-4 Alkyl groups, etc.

[0100] Exemplary salts include, but are not limited to: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, glucono-heptahydrates, glycerophosphates, hemisulfates, heptahydrates, hexanoates, hydrochlorides, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, palmitates, pectinates, persulfates, phenylpropionates, picrates, pentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc. Other examples of salts include those with suitable cations such as Na+. + NH4 + and NW4 + (where W is C) 1-4 The anions of the compounds of the present invention, such as alkyl groups.

[0101] For therapeutic purposes, salts of the compounds of the present invention are expected to be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, to prepare or purify pharmaceutically acceptable compounds.

[0102] Throughout this specification, where compositions are described as having, including, or containing specific components, or where processes and methods are described as having, including, or containing specific steps, it is also contemplated that compositions of the invention substantially consisting of or composed of the listed components, and processes and methods of the invention substantially consisting of or composed of the listed processing steps, are also possible.

[0103] Generally, unless otherwise specified, percentages of compositions are expressed by weight. Furthermore, if a variable is not defined, its previously defined meaning shall prevail.

[0104] I. Protein

[0105] This invention provides a multispecific binding protein that binds to BCMA on cancer cells and NKG2D and CD16 receptors on natural killer cells to activate natural killer cells. The multispecific binding protein is used in the pharmaceutical compositions and treatment methods described herein. The binding of the multispecific binding protein to NKG2D and CD16 receptors on natural killer cells enhances the destructive activity of natural killer cells against cancer cells. The binding of the multispecific binding protein to BCMA on cancer cells brings cancer cells into proximity to natural killer cells, which promotes both direct and indirect destruction of cancer cells by natural killer cells.

[0106] The multispecific binding protein described herein can also bind to BCMA on cancer cells and NKG2D and CD16 receptors on cytotoxic T cells to activate cytotoxic T cells. The multispecific binding protein can be used in the pharmaceutical compositions and treatment methods described herein. The binding of the multispecific binding protein to NKG2D and CD16 receptors on cytotoxic T cells enhances the destructive activity of cytotoxic T cells against cancer cells. The binding of the multispecific binding protein to BCMA on cancer cells brings cancer cells closer to cytotoxic T cells, which promotes the destruction of cancer cells by cytotoxic T cells.

[0107] Further descriptions of exemplary multispecific binding proteins are provided below.

[0108] The first component of the multispecific binding protein binds to cells expressing the NKG2D receptor, which may include, but is not limited to, NK cells, NKT cells, γδT cells, and CD8 cells. + αβ T cells. After NKG2D binding, multispecific binding proteins can block the binding of natural ligands (such as ULBP6 and MICA) to NKG2D and activate the NKG2D receptor.

[0109] The second component of the multispecific binding protein binds to cells expressing BCMA, which may include, but is not limited to, multiple myeloma and B-cell malignancies.

[0110] The third component of the multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes (including natural killer cells, cytotoxic T cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells).

[0111] The multispecific binding proteins described in this article can take various forms. Figure 1A The image shows F4 TriNKET with two BCMA-binding antigen-binding sites, both of which are Fab fragments. F4 TriNKET (Fab) contains a first antigen-binding site for NKG2D containing scFv; a second antigen-binding site for BCMA; an additional antigen-binding site for BCMA; and a heterodimeric antibody constant region for binding CD16. F4 TriNKET (Fab) is a heterodimeric multispecific antibody containing four peptides: a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and two immunoglobulin light chains (…). Figure 1AThe first immunoglobulin heavy chain, from its N-terminus to its C-terminus, includes a heavy chain variable domain (VH) connected to a heavy chain constant region 1 (CH1) forming a first (VH-CH1) domain and a first Fc (hinge-CH2-CH3) domain, wherein the first (VH-CH1) domain pairs with a first light chain to form a first Fab that binds BCMA, and wherein the (VH-CH1) domain is connected to the first Fc (hinge-CH2-CH3) domain via a linker or hinge. Figure 1A The second immunoglobulin heavy chain, from its N-terminus to its C-terminus, includes a second (VH-CH1) domain, a second Fc (hinge-CH2-CH3) domain, and a single-chain variable fragment (scFv) consisting of VH and VL cells that pair and bind NKG2D. The second Fc domain is connected at its N-terminus to the second (VH-CH1) domain via a linker or hinge, and at its C-terminus to the NKG2D-binding scFV via a linker or hinge. The second (VH-CH1) domain pairs with the second light chain to form a second Fab cell that binds BCMA. Figure 1A ).

[0112] F4 TriNKET (scFv) is a heterodimeric multispecific antibody containing two peptides: the first immunoglobulin heavy chain and the second immunoglobulin heavy chain. Figure 1B The first immunoglobulin heavy chain includes a first scFv that binds BCMA and a first Fc (hinge-CH2-CH3) domain from the N-terminus to the C-terminus, wherein the first scFv that binds BCMA is connected to the first Fc (hinge-CH2-CH3) domain via a linker or hinge. Figure 1B The second immunoglobulin heavy chain, from its N-terminus to its C-terminus, includes a second scFv that binds BCMA, a second Fc (hinge-CH2-CH3) domain, and an scFv that binds NKG2D. The second Fc domain is connected at its N-terminus to the second scFv domain that binds BCMA via a linker or hinge, and at its C-terminus to the scFv that binds NKG2D via a linker or hinge. Figure 1B ).

[0113] The TriNKET referred to as "NKG2D-Combination-F4-TriNKET-BCMA" can refer to... Figure 1A (NKG2D-Combined-F4(Fab)-TriNKET-BCMA) or Figure 1B TriNKET as described in (NKG2D-binding-F4(scFv)-TriNKET-BCMA). For example, TriNKET "A49-F4-TRINKET-BCMA" refers to a TriNKET of the form "NKG2D-binding-F4-TriNKET-BCMA" with NKG-2D binding domains containing VH and VL of A49 (see Table 1 below).

[0114] In some embodiments, the single-chain variable fragment (scFv) described above is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises the amino acid Ala-Ser. In other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence provides flexibility for binding to the target antigen and a balance between flexibility and optimal geometry.

[0115] 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 to enhance the stability of the scFv. For example, a disulfide bridge may be formed between the C44 residue of the heavy-chain variable domain and the C100 residue of the light-chain variable domain. In some embodiments, the heavy-chain variable domain is connected to the light-chain variable domain via a flexible linker. Any suitable linker, such as a (G4S)4 linker, can be used. In some embodiments of the scFv, the heavy-chain variable domain is located at the N-terminus of the light-chain variable domain. In some embodiments of the scFv, the heavy-chain variable domain is located at the C-terminus of the light-chain variable domain.

[0116] Multispecific binding proteins can provide bivalent or monovalent binding to BCMA. Bivalent binding of multispecific proteins to BCMA stabilizes BCMA on the surface of cancer cells and enhances NK cell cytotoxicity against cancer cells. Bivalent binding of multispecific proteins to BCMA confers a stronger binding to cancer cells, thereby promoting a stronger cytotoxic response of NK cells against cancer cells, especially those expressing low levels of BCMA. The bivalent binding of multispecific proteins to BCMA described in this article can also enhance the cytotoxicity of cytotoxic T cells against cancer cells. Bivalent binding of multispecific proteins to BCMA confers a stronger binding to cancer cells, thereby promoting a stronger cytotoxic response of cytotoxic T cells against cancer cells.

[0117] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is primarily concentrated in the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406(6793): 267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using phage display libraries or yeast surface-displayed cDNA libraries, or mutations can be designed based on the known three-dimensional structure of the interaction.

[0118] In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, such as a human IgG1 antibody. In some embodiments, a mutation is introduced into the antibody constant domain to achieve heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from the constant domain of human IgG1, the antibody constant domain may comprise an amino acid sequence having at least 90% identity with amino acids 234-332 of the human IgG1 antibody, and differing at one or more positions selected from the group consisting of: Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. All amino acid positions in the Fc domain or hinge region disclosed in this paper are numbered according to EU designations.

[0119] In some embodiments, the antibody constant domain may comprise an amino acid sequence having at least 90% identity with amino acids 234-332 of the human IgG1 antibody, differing in that it contains one or more substitutions 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, T366 V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T3 94F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[0120] The following are examples of scFvs linked to antibody constant regions, which also include mutations that cause heterodimerization of the two polypeptide chains. These include heavy chain variable domains (V) from NKG2D. H ) and light chain variable structural domain (V L The scFv of this disclosure is used to prepare the multispecific protein. Each sequence represents a V L -(G4S)4-V H - Heterodimerization mutation of hinge (AS)-Fc (underlined). V L and V H Includes 100V L -44V H The SS bridge (underlined) can be derived from any antibody targeting tumors or binding to NKG2D. The bent hinge region sequence includes Ala-Ser (AS, bold and underlined) to balance flexibility with optimal geometry. In some embodiments, an additional Thr-Lys-Gly sequence may be added to the AS sequence at the hinge. The (G4S)4 connector is underlined in the sequence listed below.

[0121] The TriNKET disclosed herein is NKG2D-binding-F4-TriNKET-BCMA, A49-F4-TriNKET-BCMA, comprising a first polypeptide (F4-BCMAFc-AJ chain B-NKG2D binding scFv) containing the sequence of SEQ ID NO: 162, a second polypeptide (anti-BCMA HC hinge Fc) containing the sequence of SEQ ID NO: 163, and a third polypeptide and a fourth polypeptide (anti-BCMA-full LC) containing the sequences of SEQ ID NO: 165, respectively.

[0122] The first polypeptide (i.e., F4-BCMAFc-AJ chain B-NKG2D binding scFv (SEQ ID NO: 162)) and the third polypeptide (i.e. anti-BCMA-full LC) form the first BCMA-targeting Fab fragment (including a heavy chain variable domain (V)). H The F4-BCMAFc-AJ chain B-NKG2D-binding scFv comprises a heavy chain portion (VH-CH1) connected to the Fc domain (hinge-CH2-CH3), which is connected at the C-terminus of the Fc to a single-chain variable fragment (scFv) binding to NKG2D. The NKG2D-binding scFv is represented by the amino acid sequence of SEQ ID NO: 161 and includes a heavy chain portion (VH-CH1) connected to the heavy chain variable domain (VH-CH1) via a (G4S)4 linker. H The light chain variable structural domain (V) of (SEQ ID NO: 94) L (SEQ ID NO: 98). As shown in SEQ ID NO: 162, the C end of the Fc structural domain is connected to the V using a short SGSGGGGS connector (SEQ ID NO: 168). L (SEQ ID NO: 98) N-terminus of the structural domain.

[0123] NKG2D combined with scFv

[0124]

[0125] F4-BCMAFc-AJ chain B-NKG2D combined with scFv

[0126]

[0127] The scFv in NKG2D-binding-F4-TriNKET-BCMA includes a light chain variable domain (denoted as (V)) that is attached to the NKG2D binding site of the heavy chain variable domain using a (G4S)4 connector. L(G4S)4V H Connect the light and heavy chain variable domains of scFv (SEQ ID NO: 162) to form V. L -(G4S)4-V H V L and V H Includes 100V L -44V H SS bridges (generated by substitutions of G100C and G44C, respectively) (cysteine ​​residues are indicated by bold italic underlines). (G4S)4 is the italicized sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 164) in SEQ ID NO: 161 and SEQ ID NO: 162. The Fc domain in SEQ ID NO: 162 contains an S354C substitution, which forms a disulfide bond with the Y349C substitution in another Fc domain (SEQ ID NO: 163 described below). The Fc domain in SEQ ID NO: 162 includes Q347R, D399V, and F405T substitutions.

[0128] The second polypeptide (i.e., anti-BCMA VH-CH1-Fc) and the fourth polypeptide (i.e., anti-BCMA-full LC) form a second BCMA-binding Fab fragment. Anti-BCMA VH-CH1-Fc includes a heavy chain moiety containing a heavy chain variable domain (SEQ ID NO: 148) and a CH1 domain, wherein the heavy chain variable domain is linked to the CH1 domain, and the CH1 domain is linked to the Fc domain. Anti-BCMA-full LC includes a light chain moiety containing a light chain variable domain (SEQ ID NO: 152) and a light chain constant domain.

[0129] Anti-BCMA V H -CH1-Fc

[0130]

[0131] SEQ ID NO: 163 represents the heavy chain portion of the second anti-BCMA Fab fragment, comprising a heavy chain variable domain (SEQ ID NO: 148) at the BCMA binding site and a CH1 domain linked to the Fc domain (hinge-CH2-CH3). The Fc domain in SEQ ID NO: 163 includes a Y349C substitution, which forms a disulfide bond with the S354C substitution in the CH3 domain of the Fc domain linked to the NKG2D-bound scFv (SEQ ID NO: 162). The Fc domain in SEQ ID NO: 163 also includes K360E and K409W substitutions.

[0132] SEQ ID NO: 165 represents the light chain portion of the Fab fragment, which includes a light chain variable domain (SEQ ID NO: 152) and a light chain constant domain at the BCMA binding site.

[0133] Anti-BCMA-full LC

[0134]

[0135] In one exemplary embodiment, the Fc domain of the scFv fragment linked to NKG2D contains mutations of K360E and K409W, and the Fc domain linked to the BCMA Fab fragment contains matching mutations of Q347R, D399V, and F405T for forming a heterodimer.

[0136] In one exemplary embodiment, the Fc domain of the scFv linked to the NKG2D bond includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc of the Fab fragment linked to the BCMA bond.

[0137] F3 TriNKET is a heterodimeric multispecific antibody containing three peptides: the first immunoglobulin heavy chain, the second immunoglobulin heavy chain, and the immunoglobulin light chain. Figure 2 The first immunoglobulin heavy chain includes, from the N-terminus to the C-terminus, an NKG2D-binding scFv and a first Fc (CH2-CH3) domain, wherein the NKG2D-binding scFv is connected to the first Fc (CH2-CH3) domain via a linker or hinge. Figure 2 The second immunoglobulin heavy chain comprises a (VH-CH1) domain and a second Fc (CH2-CH3) domain from the N-terminus to the C-terminus, wherein the second Fc domain is connected to the (VH-CH1) domain at its N-terminus via a linker or hinge, and wherein the (VH-CH1) domain pairs with the light chain to form a Fab (a BCMA-binding chain). Figure 2 The TriNKET referred to as "NKG2D-Combination-F3-TriNKET-BCMA" can refer to... Figure 2 The TriNKET described herein. Another exemplary TriNKET of this disclosure is NKG2D-binding-F3-TriNKET-BCMA, the sequence of which is described below (CDR (Kabat number) is underlined).

[0138] The exemplary NKG2D-binding-F3-TriNKET-BCMA includes a BCMA-binding Fab fragment comprising a heavy chain portion containing a heavy chain variable domain (SEQ ID NO: 148) and a CH1 domain, and a light chain portion containing a light chain variable domain (SEQ ID NO: 152) and a light chain constant domain, wherein the heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the Fc domain. The NKG2D-binding-F3-TriNKET-BCMA further includes an NKG2D-binding scFv connected to the Fc domain (SEQ ID NO: 166).

[0139] SEQ ID NO: 163 indicates that... Figure 2 The exemplary second immunoglobulin heavy chain of NKG2D-binding-F3-TriNKET-BCMA shown includes a heavy chain portion of an anti-BCMA Fab fragment, the heavy chain portion comprising a heavy chain variable domain (SEQ ID NO: 148) of the BCMA binding site and a CH1 domain linked to the Fc domain. The Fc domain in SEQ ID NO: 163 includes a Y349C substitution, which forms a disulfide bond with an S354C substitution in the CH3 domain of the Fc of the NKG2D-binding scFv (SEQ ID NO: 166) to form NKG2D-binding-F3-TriNKET-BCMA. In SEQ ID NO: 163, the Fc domain also includes K360E and K409W substitutions.

[0140] In the exemplary first immunoglobulin heavy chain of NKG2D-binding-F3-TriNKET-BCMA, the scFv in NKG2D-binding-F3-TriNKET-BCMA includes a light chain variable domain (denoted as (V)) that is linked to the NKG2D binding site of the heavy chain variable domain using a (G4S)4 linker (SEQ ID NO: 164). L (G4S)4V H It is connected to the Fc domain. In NKG2D-binding-F3-TriNKET-BCMA, the light and heavy chain variable domains of scFv (SEQ ID NO: 161) are connected to form V. L -(G4S)4-V H V L and V H Includes 100V L -44V H SS bridges (generated by substitution of G100C and G44C respectively) (cysteine ​​residues are indicated by bold italic underline); and V H It is connected to the Fc domain via Ala-Ser.

[0141] SEQ ID NO: 166 represents the complete sequence (scFv-Fc) of scFv connected to the Fc domain via a hinge containing Ala-Ser. The Fc domain connected to the scFv includes Q347R, D399V and F405T substitutions.

[0142] F3-NKG2D combined with scFv, -Fc-AJ chain B[V L (G4S)4V H )]

[0143]

[0144] In an exemplary embodiment of NKG2D-binding-F3-TriNKET-BCMA, the Fc domain of the first immunoglobulin heavy chain linked to the NKG2D-binding scFv fragment contains mutations of K360E and K409W, and the Fc domain of the second immunoglobulin heavy chain linked to the BCMA Fab fragment contains matching mutations of Q347R, D399V, and F405T for forming heterodimers.

[0145] In an exemplary embodiment of NKG2D-binding-F3-TriNKET-BCMA, the Fc domain of the first immunoglobulin heavy chain linked to the NKG2D-binding scFv includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc domain of the second immunoglobulin heavy chain linked to the BCMA-binding Fab fragment.

[0146] Multispecific binding proteins can bind to cells expressing the NKG2D receptor, including but not limited to NK cells, γδT cells, and CD8 cells. + αβ T cells. After NKG2D binding, multispecific binding proteins can block the binding of natural ligands (such as ULBP6 and MICA) to NKG2D and activate the NKG2D receptor.

[0147] The multispecific binding protein binds to cells expressing CD16, which is an Fc receptor on the surface of leukocytes (including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells).

[0148] The proteins disclosed herein have a K+ of 10 nM or less. DThe affinity of [the material] combines with NKG2D, for example, approximately 10 nM, approximately 9 nM, approximately 8 nM, approximately 7 nM, approximately 6 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1.5 nM, approximately 1 nM, approximately 0.5 nM and approximately 1 nM, approximately 1 nM and approximately 2 nM, approximately 2 nM and approximately 3 nM, approximately 3 nM and 4 nM, approximately 4 nM and approximately 5 nM, approximately 5 nM and [other values]. Between approximately 6 nM, between approximately 6 nM and approximately 7 nM, between approximately 7 nM and approximately 8 nM, between approximately 8 nM and approximately 9 nM, between approximately 9 nM and approximately 10 nM, between approximately 1 nM and approximately 10 nM, between approximately 2 nM and approximately 10 nM, between approximately 3 nM and approximately 10 nM, between approximately 4 nM and approximately 10 nM, between approximately 5 nM and approximately 10 nM, between approximately 6 nM and approximately 10 nM, between approximately 7 nM and approximately 10 nM, or between approximately 8 nM and approximately 10 nM.

[0149] After binding to NKG2D and CD16 receptors on natural killer cells and tumor-associated antigens on cancer cells, the multispecific binding protein can bind to more than one NK activation receptor and can block the binding of natural ligands to NKG2D. In some embodiments, the protein can stimulate NK cells in humans. In some embodiments, the protein can stimulate NK cells in humans and other species such as rodents and cynomolgus monkeys.

[0150] Upon binding to NKG2D and CD16 receptors on cytotoxic T cells and tumor-associated antigens on cancer cells, the multispecific binding protein can bind to more than one activating receptor and can block the binding of natural ligands to NKG2D. In some embodiments, the protein can stimulate cytotoxic T cells in humans. In some embodiments, the protein can stimulate cytotoxic T cells in humans and other species such as rodents and cynomolgus monkeys.

[0151] NKG2D binding site

[0152] Table 1 lists the peptide sequences of the combined heavy chain variable domain and light chain variable domain that can bind to NKG2D. In some embodiments, the heavy chain variable domain and light chain variable domain are arranged in a Fab configuration. In some embodiments, the heavy chain variable domain and light chain variable domain are fused together to form scFv.

[0153] The NKG2D binding domain can vary in its binding affinity to NKG2D; however, it can activate cells expressing NKG2D, such as NK cells and cytotoxic T cells.

[0154] Unless otherwise noted, the CDR sequences provided in Table 1 were determined using Kabat.

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 110 may be paired with the light chain variable domain represented by SEQ ID NO: 111 to form an antigen-binding site that can bind to NKG2D, as described in US 9,273,136.

[0166] SEQ ID NO: 110

[0167]

[0168] SEQ ID NO: 111

[0169]

[0170] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 112 may be paired with the light chain variable domain represented by SEQ ID NO: 113 to form an antigen-binding site that can bind to NKG2D, as described in US 7,879,985.

[0171] SEQ ID NO: 112

[0172]

[0173] SEQ ID NO: 113

[0174]

[0175] Tumor-associated antigen binding sites

[0176] This disclosure provides a BCMA binding site comprising a heavy chain variable domain and a light chain variable domain. In some embodiments, the BCMA binding site is hinged to an antibody Fc domain or a portion thereof sufficient to bind CD16, or an antigen-binding site for binding CD16, of the disclosed protein. The disclosed protein provides monovalent or bivalent BCMA binding and has one or two BCMA binding sites. In some embodiments, the disclosed protein has two BCMA binding sites, each BCMA binding site being hinged to an antibody Fc domain or a portion thereof sufficient to bind CD16, or an antigen-binding site for binding CD16, of the disclosed protein.

[0177] Table 2 lists the peptide sequences of combined heavy chain variable domains and light chain variable domains that can bind to BCMA.

[0178]

[0179]

[0180]

[0181] Alternatively, the BCMA binding domain may include heavy chain variable domains and light chain variable domains as listed in 83A10 and MAB42 below.

[0182] 83A10 heavy chain variable structural domain (SEQ ID NO: 157):

[0183]

[0184] 83A10 light chain variable structural domain (SEQ ID NO: 158):

[0185]

[0186] Alternatively, novel antigen binding sites that can bind to BCMA can be identified by screening for binding to an amino acid sequence defined by SEQ ID NO: 156.

[0187] SEQ ID NO: 156

[0188]

[0189] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is primarily concentrated in the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406(6793): 267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using phage display libraries or yeast surface-displayed cDNA libraries, or by designing mutations based on the known three-dimensional structure of the interaction.

[0190] Assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, resulting in both homodimeric and heterodimeric assembly of each antibody heavy chain. Preferential assembly of heterodimers can be facilitated by incorporating different mutations into the CH3 domain of the constant region of each antibody heavy chain, as illustrated in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1, and different amino acid substitution pairs can be incorporated into the first and second polypeptides, allowing the two chains to selectively heterodimerize. The amino acid substitution positions described below are based on EU index numbers as in Kabat.

[0191] In one case, the amino acid substitution in the first polypeptide replaces 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 the second polypeptide replaces the original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), so that the larger amino acid substitution (protrusion) fits onto the surface of the smaller amino acid substitution (cavity). For example, one polypeptide may be incorporated with a T366W substitution, and another polypeptide may be incorporated with three substitutions, including T366S, L368A, and Y407V.

[0192] The variable domain of the antibody heavy chain of the present invention may optionally be coupled to an amino acid sequence having at least 90% identity with an antibody constant region (such as an IgG constant region, including a hinge, CH2 and CH3 domains having or not having a CH1 domain). In some embodiments, the amino acid sequence of the constant region has at least 90% identity with a human antibody constant region (such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region). In some other embodiments, the amino acid sequence of the constant region has at least 90% identity with an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. 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 replacements 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, and T366. S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K , D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[0193] In some embodiments, mutations in CH1, which can be incorporated into the human IgG1 constant region, may be located at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In some embodiments, mutations in CK, which can be incorporated into the human IgG1 constant region, may be located at amino acids E123, F116, S176, V163, S174, and / or T164.

[0194] Amino acid substitutions may be selected from the following substitution groups shown in Table 3.

[0195]

[0196]

[0197] Alternatively, amino acid substitutions may be selected from the following groups of substitutions shown in Table 4.

[0198] Table 4 First polypeptide Second polypeptide Group 1 K409W D399V / F405T Group 2 Y349S E357W Group 3 K360E Q347R Group 4 K3 60E / K409W Q347R / D399V / F405T Group 5 Q347E / K360E / K409W Q347R / D399V / F405T Group 6 Y349S / K409W E357W / D399V / F405T

[0199] Alternatively, amino acid substitutions may be selected from the following groups of substitutions shown in Table 5.

[0200] Table 5 First polypeptide Second polypeptide Group 1 T366K / L351K L351D / L368E Group 2 T366K / L351K L351D / Y349E Group 3 T366K / L351K L351D / Y349D Group 4 T366K / L351K L351D / Y349E / L368E Group 5 T366K / L351K L351D / Y349D / L368E Group 6 E356K / D399K K392D / K409D

[0201] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6.

[0202]

[0203] Alternatively, at least one amino acid substitution may be selected from the following groups of substitutions in Table 7, wherein the position indicated in the first polypeptide column is replaced by any known negatively charged amino acid, and the position indicated in the second polypeptide column is replaced by any known positively charged amino acid.

[0204] Table 7 First polypeptide Second polypeptide K392, K370, K409 or K439 D399, E356 or E357

[0205] Alternatively, at least one amino acid substitution may be selected from the following group in Table 8, wherein the position indicated in the first polypeptide column is replaced by any known positively charged amino acid, and the position indicated in the second polypeptide column is replaced by any known negatively charged amino acid.

[0206] Table 8 First polypeptide Second polypeptide D399, E356 or E357 K409, K439, K370 or K392

[0207] Alternatively, amino acid substitutions may be selected from the following groups in Table 9.

[0208] Table 9 First polypeptide Second polypeptide T350V, L351Y, F405A and Y407V T350V, T366L, K392L and T394W

[0209] Alternatively or additionally, the structural stability of heteropolymeric proteins can be improved 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).

[0210] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0211] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more locations selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at location T366.

[0212] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0213] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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.

[0214] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411.

[0215] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407.

[0216] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405.

[0217] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, K360, Q347, and K409.

[0218] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399.

[0219] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399, and the amino acid sequence of another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0220] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the 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 another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409.

[0221] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more locations selected from the group consisting of Y349, L351, L368, K392, and K409, and the amino acid sequence of another polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more locations selected from the group consisting of L351, E356, T366, and D399.

[0222] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitution of S354C, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitution of Y349C.

[0223] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitution of Y349C, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitution of S354C.

[0224] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with K360E and K409W, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with O347R, D399V and F405T.

[0225] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with O347R, D399V, and F405T, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with K360E and K409W.

[0226] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitution of T366W, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region by the substitutions of T366S, T368A, and Y407V.

[0227] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with T366S, T368A, and Y407V, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that it is substituted with T366W.

[0228] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that T350V, L351Y, F405A, and Y407V are substituted, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that T350V, T366L, K392L, and T394W are substituted.

[0229] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that T350V, T366L, K392L, and T394W are substituted, and the amino acid sequence of another polypeptide chain in the antibody constant region differs from that of the IgG1 constant region in that T350V, L351Y, F405A, and Y407V are substituted.

[0230] The multispecific proteins described above can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding the second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding the 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 host cells to produce multimeric proteins.

[0231] To achieve the highest yield of multispecific proteins, different ratios of the first, second, and third expression vectors can be explored to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix, to generate a cell bank.

[0232] Clones can be cultured under conditions suitable for bioreactor scale-up and maintain the expression of multispecific proteins. Multispecific proteins can be separated and purified using methods known in the art, including centrifugation, deep filtration, cell lysis, homogenization, freeze-thaw cycles, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0233] II. Characteristics of multispecific proteins

[0234] The multispecific binding proteins of this disclosure, comprising an NKG2D-binding scFv and a BCMA-binding domain (e.g., NKG2D-binding-F4-TriNKET-BCMA or NKG2D-binding-F3-TriNKET-BCMA), are more effective in reducing tumor growth and killing cancer cells. For example, the multispecific binding proteins of this disclosure targeting BCMA-expressing tumors / cancer cells are more effective than the anti-BCMA monoclonal antibody MAB42. Compared to the anti-BCMA monoclonal antibody MAB42, the TriNKET of this disclosure's NKG2D-binding-F4-TriNKET-BCMA is more effective in promoting NK-mediated cell lysis in BCMA-expressing human cancer cell lines.

[0235] NKG2D-binding-F4-TriNKET-BCMA exhibits weak binding to cells expressing NKG2D. However, compared to MAB42 anti-BCMA mAbs, the multispecific binding proteins containing the NKG2D binding domain described herein (e.g., NKG2D-binding-F4-TriNKET-BCMA or NKG2D-binding-F3-TriNKET-BCMA) demonstrate significant advantages in target cell efficacy and maximal lysis.

[0236] Therefore, compared with monoclonal antibodies, the multispecific binding proteins described in this article (e.g., NKG2D-binding-F4-TriNKET-BCMA or NKG2D-binding-F3-TriNKET-BCMA) are advantageous in treating cancers expressing BCMA.

[0237] III. Therapeutic Applications

[0238] The proteins disclosed herein can be used to activate cytotoxic T cells or natural killer cells. In some embodiments, this document provides a method for activating cytotoxic T cells by exposing them to the proteins disclosed herein. In some embodiments, this document provides a method for activating natural killer cells by exposing them to the proteins disclosed herein.

[0239] Therefore, this document provides methods for enhancing tumor cell death by exposing tumor cells to the proteins disclosed herein in the presence of cytotoxic T cells or natural killer cells. In some embodiments, this document provides methods for enhancing tumor cell death by exposing tumor cells to the proteins disclosed herein in the presence of cytotoxic T cells. In some embodiments, this document provides methods for enhancing tumor cell death by exposing tumor cells to the proteins disclosed herein in the presence of natural killer cells.

[0240] This article also provides methods for enhancing the immune response against BCMA-expressing cancer cells in subjects by administering the proteins or formulations disclosed herein to the subjects.

[0241] This invention provides a method for treating cancer using the multispecific binding protein and / or pharmaceutical compositions described herein. The method can be used to treat a variety of cancers by administering a therapeutically effective amount of the multispecific binding protein described herein to a patient in need. In some embodiments, cancer can be treated using proteins that express BCMA as disclosed herein.

[0242] Treatment methods can be characterized based on the type of cancer to be treated. For example, in some implementations, the cancer is breast cancer, ovarian cancer, esophageal cancer, bladder cancer, or stomach cancer, salivary duct cancer, lung adenocarcinoma, or an invasive form of uterine cancer, such as serous endometrial cancer.

[0243] In some other embodiments, the cancer to be treated by the multispecific binding protein described herein and / or the pharmaceutical composition described herein is brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer.In other implementation schemes, the cancers include squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (such as angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, bile duct cancer, thyroid cancer, acral lentigines-like melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, and bartholin gland carcinoma. Carcinoma, basal cell carcinoma, gallbladder cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid tumor, bile duct cancer, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antral cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease. Diseases, including ileal cancer, insulinoma, intraepithelial neoplasia, mesothelial squamous cell carcinoma, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigines, malignant melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal carcinoma, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, plasmacytoma, pseudosarcoma, and pulmonary blastoma. Rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus carcinoma, skin cancer, small cell carcinoma, small bowel cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumors, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, subepithelial carcinoma, superficial diffuse melanoma, T-cell leukemia, tongue cancer, undifferentiated tumor, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vasoactive intestinal peptide tumor (VIPoma), vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0244] In some other embodiments, the cancer to be treated by the multispecific binding protein described herein and / or the pharmaceutical composition described herein is a non-Hodgkin lymphoma, such as B-cell lymphoma or T-cell lymphoma. In some embodiments, the non-Hodgkin lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In some other implementations, non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0245] In some embodiments, the cancer to be treated by the multispecific binding protein and / or the pharmaceutical composition described herein is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the DLBCL is germinal center B cell (GCB) DLBCL. In some embodiments, the DLBCL is activated B cell (ABC) DLBCL.

[0246] In some embodiments, the cancer to be treated by the multispecific binding protein and / or the pharmaceutical composition described herein is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, or acute myeloid leukemia. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is chronic lymphocytic leukemia. In some embodiments, the cancer is acute myeloid leukemia.

[0247] Cancers to be treated can be characterized by the presence of specific antigens expressed on the surface of cancer cells. In some implementations, in addition to BCMA, cancer cells may express one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD1.

[0248] IV. Combination Therapy

[0249] Another aspect of the invention provides combination therapies. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0250] Exemplary therapeutic agents that can be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, retinoic acid, ribomustin, gemcitabine, vincristine, etoposide, cladribine, dibromomannitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, and raltitrexed. (raltitrexed), daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptiniumAcetate, Ketanserin, Doxifluridine, Etretinate, Isotretinoin, Streptozocin, Nimustine, Vindesine, Flutamide, Drogenil, Butocin, Carmofur, Razoxane, Sizofilan, Carboplatin, Mitolactalol, Tegafur, Ifosfami The following drugs are listed: de, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ (IFN-γ), colony-stimulating factor 1, colony-stimulating factor 2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variants thereof, which may exhibit differential binding to their homologous receptors or increased or decreased serum half-life.

[0251] For certain cancers, such as multiple myeloma, additional therapy may be one or more of the following: lenalidomide, pomalidomide, thalidomide, bortezomib, dexamethasone, cyclophosphamide, doxorubicin, carfilzomib, iaxizomib, cisplatin, doxorubicin, etoposide, and anti-CD38 antibodies (such as daratumumab, panobinostat, and elotuzumab), which may be alone, one of the combinations listed above, or any other combination.

[0252] An additional class of agents that can be used as part of a combination therapy in cancer treatment is the immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of the following: (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 U.S. Food and Drug Administration for the treatment of melanoma.

[0253] Other agents that can be used as part of combination therapy in cancer treatment are monoclonal antibody agents that target non-checkpoint targets (such as herceptin) and non-cytotoxic agents (such as tyrosine kinase inhibitors).

[0254] Other classes of anticancer agents include, for example: (i) inhibitors selected from the following: ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors. The formulation, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositol 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25 or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF and G-CSF.

[0255] The protein of this invention can also be used as an adjunct to surgical removal of the primary lesion.

[0256] The amount of multispecific binding protein and additional therapeutic agents, as well as the relative timing of administration, can be selected to achieve the desired combined therapeutic effect. For example, when administering combination therapy to a patient requiring such administration, the therapeutic agents in the combination, or one or more pharmaceutical compositions containing therapeutic agents, can be administered in any order (e.g., sequentially, in parallel, together, simultaneously, etc.). Furthermore, the multispecific binding protein can be administered, for example, while the additional therapeutic agent is exerting its preventive or therapeutic effect, and vice versa.

[0257] V. Pharmaceutical Composition

[0258] This disclosure is further characterized by pharmaceutical compositions comprising a therapeutically effective amount of the protein described herein. The compositions can be formulated for use in a variety of drug delivery systems. The compositions may also contain one or more physiologically acceptable excipients or carriers for appropriate formulation. Suitable formulations used in this disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief overview of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990).

[0259] The intravenous drug delivery formulations disclosed herein may be contained in a bag, pen, or syringe. In some embodiments, the bag may be connected to a channel containing a tube and / or needle. In some embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In some embodiments, the formulation may be lyophilized (freeze-dried) and contained in about 12-60 vials. In some embodiments, the formulation may be lyophilized and 45 mg of the lyophilized formulation may be contained in one vial. In some embodiments, about 40 mg to about 100 mg of the lyophilized formulation may be contained in one vial. In some embodiments, lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of protein in the intravenous drug formulation. In some embodiments, the formulation may be a liquid formulation and stored at about 250 mg / vial to about 1000 mg / vial. In some embodiments, the formulation may be a liquid formulation and stored at about 600 mg / vial. In some embodiments, the formulation may be a liquid formulation and stored at about 250 mg / vial.

[0260] The protein may be present in a liquid aqueous pharmaceutical preparation, wherein the liquid aqueous pharmaceutical preparation comprises a therapeutically effective amount of the protein in a buffer solution forming the preparation.

[0261] These compositions can be sterilized using conventional sterilization techniques or by aseptic filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, with the lyophilized formulation combined with a sterile aqueous carrier prior to administration. The pH of the formulation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form can be packaged in multiple single-dose units, each containing a fixed amount of one or more of the aforementioned agents. The compositions in solid form can also be flexibly packaged in containers.

[0262] In some embodiments, this disclosure provides a formulation having an extended shelf life, comprising the protein of this disclosure in combination with the following substances: mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0263] In some embodiments, an aqueous formulation containing the proteins of this disclosure is prepared in a pH buffer solution. The pH range of the buffer solution of the present invention can be from about 4 to about 8, for example from about 4.5 to about 6.0 or from about 4.8 to about 5.5, or the pH can be from about 5.0 to about 5.2. The intermediate pH ranges described above are also intended to be part of this disclosure. For example, it is intended to include ranges using any combination of the above values ​​as upper and / or lower limits. Examples of buffer solutions 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 buffer solutions.

[0264] In some embodiments, the formulation includes a buffer system comprising citrate and phosphate to maintain a pH in the range of about 4 to about 8. In some embodiments, the pH range may be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or a pH range of about 5.0 to about 5.2. In some embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In some 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 hydrogen 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 some embodiments, the buffer system comprises 1-1.5 mg / mL citric acid, 0.25 to 0.5 mg / mL sodium citrate, 1.25 to 1.75 mg / mL disodium hydrogen phosphate dihydrate, 0.7 to 1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / mL sodium chloride. In some embodiments, sodium hydroxide is used to adjust the pH of the formulation.

[0265] The formulation may also contain a polyol, which acts as a tension modifier and stabilizes the antibody. The polyol is added to the formulation in an amount that can be varied relative to the required isotonicity. In some embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be varied relative to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In some embodiments, the polyol that may be used as a tension modifier in the formulation is mannitol. In some embodiments, the concentration of mannitol may be from about 5 to about 20 mg / mL. In some embodiments, the concentration of mannitol may be from about 7.5 to 15 mg / mL. In some embodiments, the concentration of mannitol may be from about 10-14 mg / mL. In some embodiments, the concentration of mannitol may be from about 12 mg / mL. In some embodiments, the formulation may contain the polyol sorbitol.

[0266] Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamer (e.g., poloxamer 188). The amount of detergent added reduces the aggregation of the formulated antibody and / or minimizes the formation of particles in the formulation and / or reduces adsorption. In some embodiments, the formulation may contain a surfactant that is polysorbate. In some embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) dehydrated sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996). In some embodiments, the formulation may contain polysorbate 80 at a concentration between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL. In some implementations, about 0.1% polysorbate 80 may be added to the formulation.

[0267] In some embodiments, the protein product of this disclosure is formulated as a liquid preparation. The liquid preparation may be presented at a concentration of 10 mg / mL in a USP / Ph Eur Type I 50R vial, said vial being sealed with a rubber stopper and an aluminum crimp-sealed cap. The stopper may be made of an elastomer conforming to USP and Ph Eur standards. In some embodiments, the vial may be filled with 61.2 mL of the protein product solution to allow for an extractable volume of 60 mL. In some embodiments, the liquid preparation may be diluted with a 0.9% saline solution.

[0268] In some embodiments, the liquid formulation of this disclosure can be prepared in combination with a stable level of sugar to form a solution with a concentration of 10 mg / mL. In some embodiments, the liquid formulation can be prepared in an aqueous carrier. In some embodiments, a stabilizer may be added in an amount not exceeding that which may result in undesirable or unsuitable viscosity for intravenous administration. In some embodiments, the sugar may be a disaccharide, such as sucrose. In some embodiments, the liquid formulation may also contain one or more of a buffer, surfactant, and preservative.

[0269] In some embodiments, the pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[0270] Besides aggregation, deamidation is a common product variation of peptides and proteins, which can occur during fermentation, harvest / cell clarification, purification, drug substance / product storage, and sample analysis. Deamidation involves the loss of NH3 from proteins to form a hydrolyzable succinimide intermediate. The succinimide intermediate results in a 17 Dalton mass reduction in the parent peptide. Subsequent hydrolysis leads to an 18 Dalton mass increase. The succinimide intermediate is difficult to separate due to its instability under aqueous conditions. Therefore, deamidation is typically detectable as a 1 Dalton mass increase. Deamidation of asparagine yields either aspartic acid or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local peptide conformation, and tertiary structure. The amino acid residues adjacent to Asn in the peptide chain influence the rate of deamidation. Gly and Ser residues following Asn in the protein sequence result in higher sensitivity to deamidation.

[0271] In some embodiments, the liquid formulations of this disclosure can be stored under pH and humidity conditions to prevent deamination of the protein products.

[0272] The aqueous carriers of interest in this article are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for the preparation of liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0273] Preservatives may be optionally added to the formulations described herein to reduce bacterial activity. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0274] Intravenous (IV) preparations may be a preferred route of administration in certain circumstances, such as when a patient receives all medications via IV in a hospital post-transplantation. In some embodiments, the liquid preparation is diluted with a 0.9% sodium chloride solution prior to administration. In some embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0275] In some embodiments, salt or buffer components may be added in amounts from 10 mM to 200 mM. The salt and / or buffer are pharmaceutically acceptable and derived from various known acids (inorganic and organic) that "form a base" with a metal or amine. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycine salt, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may be used as counterions.

[0276] Preservatives may be optionally added to the formulations described herein to reduce bacterial activity. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0277] The aqueous carriers of interest in this article are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for the preparation of liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextran solution.

[0278] The protein disclosed herein may be present in a lyophilized formulation comprising the protein and a lyophilization protectant. The lyophilization protectant may be a sugar, such as a disaccharide. In some embodiments, the lyophilization protectant may be sucrose or maltose. The lyophilized formulation may also contain one or more of a buffer, surfactant, thickener, and / or preservative.

[0279] The amount of sucrose or maltose used to stabilize the lyophilized pharmaceutical product may be at least 1:2 in weight ratio of protein to sucrose or maltose. In some embodiments, the weight ratio of protein to sucrose or maltose may be from 1:2 to 1:5.

[0280] In some embodiments, the pH of the formulation can be set by adding a pharmaceutically acceptable acid and / or base prior to lyophilization. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[0281] Before lyophilization, the pH of the solution containing the protein of this disclosure can be adjusted to between 6 and 8. In some embodiments, the pH range of the lyophilized pharmaceutical product can be between 7 and 8.

[0282] In some embodiments, salt or buffer components may be added in amounts from 10 mM to 200 mM. The salt and / or buffer are pharmaceutically acceptable and derived from various known acid (inorganic and organic) "base-forming" metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycine salt, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may be used as counterions.

[0283] In some embodiments, a "polymerizer" may be added. A "polymerizer" is a compound that increases the mass of the lyophilized mixture and contributes to the physical structure of the lyophilized filter cake (e.g., promoting the production of a substantially uniform lyophilized filter cake with an open pore structure). Illustrative polymerizers include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such polymerizers.

[0284] Preservatives may be optionally added to the formulations described herein to reduce bacterial activity. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0285] In some embodiments, the lyophilized drug product can be reconstituted with an aqueous carrier. The aqueous carriers of interest herein are pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing liquid formulations after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffers (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextran solution.

[0286] In some embodiments, the lyophilized pharmaceutical product of this disclosure is reconstituted using sterile water for injection, USP (SWFI), or 0.9% sodium chloride injection (USP). During reconstitution, the lyophilized powder is dissolved into a solution.

[0287] In some embodiments, the lyophilized protein product of this disclosure is reconstituted with about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0288] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied in order to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition and administration mode without being toxic to the patient.

[0289] For each patient, a specific dose may be a uniform dose, such as 50–5000 mg of protein. Alternatively, the dose may be adjusted based on the patient’s approximate weight or surface area. Other factors for determining an appropriate dose may include the disease or symptom to be treated or prevented, the severity of the disease, the route of administration, and the patient’s age, sex, and medical condition. Those skilled in the art may further refine the calculations necessary to determine an appropriate therapeutic dose in a conventional manner, particularly based on the dose information and assays disclosed herein. The dose may also be determined by using known assays that determine the dose, in conjunction with appropriate dose-response data. The dose for an individual patient may be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex may be measured in the patient to determine whether dose adjustment is necessary to achieve or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and / or complexes and their doses are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43–53, 2001; Steimer et al., Clinica Chimica Acta 308:33–41, 2001).

[0290] Generally, the body weight-based dosage is from about 0.01 μg to about 100 mg per kilogram of body weight, such as about 0.01 μg to about 100 mg per kilogram of body weight, about 0.01 μg to about 50 mg per kilogram of body weight, about 0.01 μg to about 10 mg per kilogram of body weight, about 0.01 μg to about 1 mg per kilogram of body weight, about 0.01 μg to about 100 μg per kilogram of body weight, about 0.01 μg to about 50 μg per kilogram of body weight, about 0.01 μg to about 10 μg per kilogram of body weight, about 0.01 μg to about 1 μg per kilogram of body weight, about 0.1 μg to about 100 mg per kilogram of body weight, about 0.1 μg to about 50 mg per kilogram of body weight, about 0.1 μg to about 10 mg per kilogram of body weight, and about 0.1 μg to about 10 mg per kilogram of body weight. mg, approximately 0.1 μg to approximately 100 μg per kilogram of body weight, approximately 0.1 μg to approximately 10 μg per kilogram of body weight, approximately 0.1 μg to approximately 1 mg per kilogram of body weight. μg, about 1 μg to about 100 mg per kilogram of body weight, about 1 μg to about 50 mg per kilogram of body weight, about 1 μg to about 10 mg per kilogram of body weight, about 1 μg to about 1 mg per kilogram of body weight, about 1 μg to about 100 μg per kilogram of body weight, about 1 μg to about 50 μg per kilogram of body weight, about 1 μg to about 10 μg per kilogram of body weight, about 10 μg to about 100 mg per kilogram of body weight, about 10 μg to about 50 μg per kilogram of body weight, about 50 μg to about 100 mg per kilogram of body weight, about 10 μg to about 100 mg per kilogram of body weight, about 10 μg to about 50 μg per kilogram of body weight, about 50 μg to about 100 mg per kilogram of body weight, about 10 μg to about 100 mg per kilogram of body weight, about 10 μg to about 50 μg per kilogram of body weight, about 50 μg to about 100 mg per kilogram of body weight, about 100 μg to about 100 mg per kilogram of body weight, about 10 μg to about 50 μg per kilogram of body weight, about 100 μg to about 100 mg per kilogram of body weight, about 1 ... Approximately 50 μg to about 50 mg per gram of body weight, approximately 50 μg to about 10 mg per kilogram of body weight, approximately 50 μg to about 1 mg per kilogram of body weight, approximately 50 μg to about 100 μg per kilogram of body weight, approximately 100 μg to about 100 mg per kilogram of body weight, approximately 100 μg to about 50 mg per kilogram of body weight, approximately 100 μg to about 10 mg per kilogram of body weight, approximately 100 μg to about 1 mg per kilogram of body weight, approximately 1 mg to about 100 mg per kilogram of body weight, approximately 1 mg to about 50 mg per kilogram of body weight, approximately 1 mg to about 10 mg per kilogram of body weight, approximately 10 mg to about 50 mg per kilogram of body weight, approximately 50 mg to about 100 mg per kilogram of body weight.

[0291] Doses can be administered once or more daily, weekly, monthly, or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the repetition rate of administration based on the measured residence time and concentration of the targetable construct or complex in body fluids or tissues. Administration of the present invention can be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, via catheter infusion, or direct intralesional injection. This can be administered once or more daily, once or more weekly, once or more monthly, or once or more annually.

[0292] The foregoing description has described various aspects and embodiments of the invention. This patent application specifically considers all combinations and permutations of the aspects and embodiments.

[0293] Example

[0294] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are for illustrative purposes only and are not intended to limit the invention.

[0295] Example 1 - Primary human NK cell cytotoxicity assay:

[0296] Peripheral blood mononuclear cells (PBMCs) were isolated from the ochre layer of human peripheral blood sedimentation using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using negative selection with magnetic beads; the purity of the isolated NK cells was typically >90% CD3-CD56. + The isolated NK cells were left to stand overnight, and the rested NK cells were used in a cytotoxicity assay the next day.

[0297] DELFIA cytotoxicity assay:

[0298] Human cancer cell lines expressing BCMA were harvested from the culture, and the cells were washed with HBS and then... 6 Resuspend cells in growth medium at 0.5-1.0 × 10⁶ mL for labeling with BATDA reagent (Perkin Elmer AD0116). Label target cells according to the manufacturer's instructions. After labeling, wash cells three times with HBS and then incubate at 0.5-1.0 × 10⁶ mL. 5 / mL resuspended in culture medium. To prepare background wells, set aside aliquots of labeled cells and rotate the cells out of the medium. Carefully add 100 μl of culture medium in triplicate to the wells to avoid interfering with the precipitated cells. Add 100 μl of BATDA-labeled cells to each well of a 96-well plate. Reserve wells for spontaneous release from target cells and prepare wells to maximize the lysis of target cells by adding 1% Triton-X. Dilute the monoclonal antibody or TriNKET against BCMA (NKG2D-binding-F4-TriNKET-BCMA) in culture medium and add 50 μl of diluted mAb or TriNKET to each well. Harvest the quiescent NK cells from the culture, wash and thaw them according to the desired E:T ratio at a 10:10 ratio. 5 -2.0×10 6 / mL resuspended in culture medium. Add 50 μl of NK cells to each well of the plate to bring the total culture volume to 200 μl. Incubate the plates at 37°C with 5% CO2 for 2–3 hours before assay.

[0299] After culturing for 2-3 hours, remove the plates from the incubator and pellet the cells by centrifugation at 200g for 5 minutes. Transfer 20 μl of culture supernatant to a clean microplate provided by the manufacturer, and add 200 μl of room temperature europium solution to each well. Incubate the plates in the dark on a plate shaker at 250 rpm for 15 minutes. Read the plates using a Victor 3 or SpectraMaxi3X instrument. Calculate the specific lysis percentage as follows: Specific lysis percentage = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) * 100%.

[0300] FACS-based long-term cytotoxicity assays:

[0301] Following puromycin selection, human cancer cell lines expressing BCMA were harvested from stationary cultures and transduced to stably express NucLight Green (Essen BioScience 4475), and then... 5 / mL resuspended in culture medium. 100 μl of target cells were added to each well of a 96-well plate. NKG2D-binding-F4-TriNKET-BCMA TriNKET was diluted in culture medium, and 50 μl of each was added to the replicate wells. The purified human NK cells were harvested from the culture after standing overnight, washed, and rinsed with 4 × 10⁻⁶ cells / mL. 5 / mL resuspended in culture medium. For an effector cell:target cell (E:T) ratio of 1:1, 50 μl of NK cells were added to all wells except for the target control (which received 100 μl of culture medium). To use freshly treated PBMCs as effectors, an E:T ratio of 10:1 was used by comparison. Plates were incubated at 37°C with 5% CO2 for 30 hours.

[0302] After co-culture, cells were stained, fixed, and analyzed by flow cytometry. Residual target cells were detected with strong translocation in the FITC channel, where dead cells were excluded using viability staining. The number of green events was output and the kill percentage was calculated by comparing with a control sample containing only the target. Counting beads were included to ensure comparable recording volumes.

[0303] Example 2 - Evaluation of the binding of TriNKET to NKG2D positive cells

[0304] TriNKET binding in human whole blood

[0305] Add 100 μl of heparinized human whole blood to each tube / well. Add directly labeled TriNKET (NKG2D-binding-F4-TriNKET-BCMA) or mAb directly to the whole blood, along with a mixture of directly conjugated mAbs for immunophenotypic analysis, and incubate the samples at room temperature for 20 min. For directly labeled NKG2D-binding-F4-TriNKET-BCMA or mAb, add 2 mL of 1xRBC lysis / fixation buffer to each sample after incubation. Incubate the samples at room temperature for 15 min. Wash the samples once after lysis and then prepare for analysis.

[0306] Figure 3 and Figure 4 This demonstrates the lysis of human NK cells from BCMA-positive target cell lines within 2 hours in the presence of either anti-BCMA TriNKET (NKG2D-binding-F4-TriNKET-BCMA) or anti-BCMA monoclonal antibody. KMS12-PE cells with low and high BCMA expression were separately... Figure 3 ) and MM.1R cells ( Figure 4 NKG2D-binding-F4-TriNKET-BCMA was used as a target cell. It showed sub-nanomolar EC50 activity against both KMS12-PE and MM.1R cells. 50 Value. Compared to the anti-BCMA monoclonal antibody (MAB42), NKG2D-binding-F4-TriNKET-BCMA provides protection against both cell lines (KMS12-PE cells). Figure 3 ) and MM.1R cells ( Figure 4 Greater maximum specificity and efficiency.

[0307] BCMA surface stabilization via TriNKET

[0308] KMS12-PE or MM.1R cells were incubated with anti-BCMA monoclonal antibody (MAB42), bivalent TriNKET (NKG2D-binding-F4-TriNKET-BCMA), or monovalent TriNKET (A49-DB-TriNKET-BCMA). A49-DB-TriNKET-BCMA is a TriNKET in which the first antigen-binding site contains a Fab that binds NKG2D and the second antigen-binding site contains a Fab that binds BCMA, each linked to an Fc domain, thereby forming a bivalent antibody (WO2018 / 148566).

[0309] To assess total surface area BCMA, a saturation concentration of 100 μg / mL was used, while a saturation concentration of 100 ng / mL was chosen to study the stability of subsaturated surfaces. Each sample was aliquoted into three portions, with each aliquot placed on ice for 20 minutes and incubated at 37°C for 2 hours or 24 hours. After the incubation period, cells were washed and TriNKET binding was detected using anti-human IgG secondary antibody. After staining, cells were fixed and stored at 4°C, and all samples were analyzed at the end of the study.

[0310] TriNKET stabilized surface BCMA

[0311] Figure 5 The image shows staining of surface BCMA on KMS12-PE cells using either A49-DB-TriNKET-BCMA or BCMA monoclonal antibody (MAB42) after a specified incubation time. Both BCMAmAb and TriNKET were able to rapidly stabilize surface BCMA after incubation and maintain increased expression for up to 24 hours. Figure 6 The same effect was observed in the MM.1R cell line, which has a naturally high expression of BCMA.

[0312] At subsaturated concentrations of A49-DB-TriNKET-BCMA and anti-BCMA mAb (MAB42), a significant improvement in BCMA target cell binding was also observed with longer incubation times. Figure 7 The study demonstrated that the frenzied binding provided by anti-BCMA mAb (MAB42) and bivalent TriNKET (NKG2D-binding-F4-TriNKET-BCMA) promoted a rapid and sustained increase in binding to BCMA on KMS12-PE cells, while only a limited improvement in binding was observed when using monovalent TriNKET (A49-DB-TriNKET-BCMA). Figure 8A similar pattern was shown on MM.1R cells.

[0313] Example 3 - Divalent TriNKET mediates excellent long-term cytotoxicity

[0314] The ability of purified human NK depleted KMS12-PE cells expressing BCMA to be compared with that of the anti-BCMA monoclonal antibody MAB42 in the presence of bivalent TriNKET (NKG2D-binding-F4-TriNKET-BCMA). Figure 9 The depletion of KMS12-PE cells mediated by statically incubated NK cells (E:T ratio of 1:1) was demonstrated by flow cytometry after 20 hours. The bivalent BCMA TriNKET (NKG2D-binding-F4-TriNKET-BCMA) produced more potent killing than either the monoclonal antibody or the monovalent TriNKET (A49-DB-TriNKET-BCMA). Similar results were obtained using PBMCs at an E:T ratio of 10:1 instead of purified NK cells. Figure 10 Compared to either TriNKET form, anti-BCMA mAb reduced maximum killing power and efficacy in both effector cell types.

[0315] BCMA TriNKET has a very weak binding interaction with NKG2D on cells.

[0316] The KHYG-1 human NK cell line was used to evaluate NKG2D binding of TriNKET NKG2D-binding-F4-TriNKET-BCMA. The contribution of FcCD16 binding was investigated using KHYG-1 cells transduced to express CD16-F158V. TriNKET was diluted and incubated with KHYG-1 cells. TriNKET binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and median fluorescence intensity (“MFI”) is reported.

[0317] The ability of TriNKET to bind to NKG2D-expressing cells was investigated. Figure 11 As shown, almost no binding of TriNKET (NKG2D-binding-F4-TriNKET-BCMA and NKG2D-binding-F3-TriNKET-BCMA) was observed with KHYG-1 cells expressing NKG2D but not CD16. Conversely, when KHYG-1 cells were transduced to express a high-affinity variant of CD16, NKG2D-binding-F4-TriNKET-BCMA was able to bind to cells at an MFI level only slightly higher than that of the anti-BCMA monoclonal antibody MAB42. Figure 12However, NKG2D-binding-F3-TriNKET-BCMA was able to bind to CD16-expressing KHYG-1 cells with a higher MFI ( ). Figure 12 TriNKET does not bind to cells expressing NKG2D because it cannot bind to NKG2D-positive NK cells in whole blood. Figure 13A ) or CD8+ T cells ( Figure 13B Further evidence suggests that TriNKET can bind to B cells in whole blood at levels comparable to the IgG1 control. Figure 13D ), mononuclear cells ( Figure 13E ) and granulocytes ( Figure 13F (Combined)

[0318] Example 4 - TriNKET triggers CD8+ T cell lysis in BCMA+ tumor cells

[0319] Original substitution of CD8+ T cell cytotoxicity assay:

[0320] Primary human CD8+ effector T cell generation: PBMCs were isolated from the brown-yellow layer of human peripheral blood sedimentation using density gradient centrifugation. The isolated PBMCs were stimulated with 1 μg / ml concanavalin A (ConA) at 37°C for 18 h. ConA was then removed, and the cells were cultured at 37°C with 25 units / ml IL-2 for 4 days. CD8+ T cells were purified using negative selection with magnetic beads and then cultured at 37°C in medium containing 10 ng / ml IL-15 for 6–13 days.

[0321] Characterization of primary human CD8+ effector T cells: The purity of CD8+ T cells and the expression of NKG2D and CD16 in the human CD8+ effector T cells generated above were analyzed by flow cytometry. Cells were stained with fluorescently conjugated antibodies against CD3, CD8, NKG2D, and CD16, and then analyzed by flow cytometry.

[0322] Short-term CD8+ effector T cell DELFIA cytotoxicity assay: Human multiple myeloma KMS12-PE cells expressing the target of interest BCMA were harvested from culture. Cells were washed and resuspended in growth medium at 10⁶ / mL for labeling using BATDA reagent (Perkin Elmer AD0116). Target cells were labeled according to the manufacturer's instructions. After labeling, cells were washed three times with HBS and then inoculated with 0.5 × 10⁶ cells / mL. 5Resuspend cells in culture medium at 50 μL / mL. Add 100 μL of BATDA-labeled cells to each well of a 96-well plate. Reserve wells for spontaneous release from target cells and prepare wells to maximize target cell lysis by adding 1% Triton-X. Dilute TriNKET and mAb in culture medium and add 50 μL / well to the plate. Harvest CD8+ effector T cells from the culture, wash, and resuspend at 5 × 10⁻⁶ mL / well. 6 / mL resuspended in culture medium (E:T ratio = 50:1). Then, 50 μl of CD8+ T cells were added to each well of the plate to bring the total culture volume to 200 μl. Before assay, the plates were incubated at 37°C with 5% CO2 for 3.5 hours. After incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 500g for 5 minutes. Then, 20 μl of culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μl of europium solution at room temperature was added to each well. The plates were protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plates were read using a SpectraMax i3X instrument.

[0323] The specific lysis percentage is calculated as follows: Specific lysis percentage = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) * 100%.

[0324] Characterization of CD8+ effector T cells for cytotoxicity assays

[0325] like Figure 14 As shown, CD8+ effector T cells generated by ConA stimulation and cultured with IL-15 had high purity (CD3+CD8+ cells > 99%) and all expressed NKG2D but not CD16.

[0326] When co-cultured with activated CD8+ T cells, NKG2D-binding-F4-TriNKET-BCMA enhanced the lysis of KMS12-PE cells.

[0327] Cell lysis of KMS12-PE cells as determined by DELFIA assay: in cells derived from donor 1 ( Figure 15A ) and donor 2 ( Figure 15B In the presence or absence of IL-15-stimulated CD8+ T cells, 60 nM of NKG2D-binding-F4-TriNKET-BCMA, anti-BCMA mAb, or unrelated TriNKET was added to the culture of KMS12-PE target cells. In the absence of TriNKET / mAb, activated CD8+ T cells co-cultured with KMS12-PE cells were used as background T cells for killing.

[0328] Figures 15A-15BThe results of the DELFIA cytotoxicity assay using primary human CD8+ effector T cells from two healthy donors and KMS12-PE target cells are shown. As shown in the figure, NKG2D-binding-F4-TriNKET-BCMA enhanced the lysis of KMS12-PE cells when co-cultured with activated CD8+ T cells, but not in the absence of effector cells. Parental anti-BCMA mAb or unrelated TriNKET did not enhance the lysis of CD8+ T cells from either donor.

[0329] Example 5 - TriNKET-stimulated NK cell activation

[0330] Co-culture activation of purified human NK cells: Human cancer cell lines expressing BCMA were harvested from the culture and the cells were regulated to 1×10⁶ cells / year. 6 Cells / mL. Dilute TriNKET / mAb in culture medium. Harvest statically held NK cells from the culture and wash. Introduce purified NK cells at 1×10⁻⁶ cells / mL. 6 Cells were resuspended at 100 cells / mL to achieve an E:T ratio of 1:1. All co-cultures were supplemented with hIL-2, Brefeldin-A, monensin, and fluorophore-conjugated anti-CD107a and incubated for 4 hours. Intracellular staining of live NK cells was achieved after fixation with permeabilization / wash buffer and fluorophore-conjugated anti-IFNγ.

[0331] Figures 16A-16B This demonstrates the activation of human NK cells within 4 hours in the presence of BCMA-positive target cell lines and in the presence of anti-BCMA TriNKET or monoclonal antibodies. Figure 16A In this study, KMS12-PE cells (with low BCMA expression) were used as target cells. As shown in the figure, compared with anti-BCMA mAb, TriNKET, which targets BCMA, mediated more significant activation of human NK cells in co-culture with BCMA-positive KMS12-PE myeloma cells. Figure 16B In this study, H929 cells (high BCMA expression) were used as target cells. As shown in the figure, compared with anti-BCMA mAbs, BCMA-targeting TriNKET mediated more significant activation of human NK cells in co-culture with BCMA-positive H929 myeloma cells. Therefore, F4-TriNKET triggered increased degranulation and IFNγ production with sub-nanomolar EC50 values ​​against both high and low BCMA-expressing cells. Compared with BCMA monoclonal antibodies, F4TriNKET stimulated a larger proportion of NK cells to the greatest extent and enhanced efficacy against both cell lines.

[0332] Exemplary Implementation

[0333] Implementation Scheme 1: A protein comprising: (a) a first antigen-binding site comprising a single-chain variable fragment (scFv) that binds NKG2D; the scFv that binds NKG2D comprising a heavy chain variable domain and a light chain variable domain; (b) a second antigen-binding site that binds B cell maturation antigen (BCMA); and (c) an antibody Fc domain sufficient to bind CD16 or a portion thereof or a third antigen-binding site that binds CD16.

[0334] Implementation Scheme 2: The protein according to Implementation Scheme 1 further includes an additional antigen-binding site for binding BCMA.

[0335] Implementation Scheme 3: The protein according to Implementation Scheme 1 or 2, wherein the second antigen binding site that binds to BCMA is the Fab fragment.

[0336] Implementation Scheme 4: The protein according to any one of Implementation Schemes 1-3, wherein the second antigen binding site binding BCMA and the additional antigen binding site are Fab fragments.

[0337] Implementation Scheme 5: The protein according to Implementation Scheme 1 or 2, wherein the second antigen binding site binding BCMA and the additional antigen binding site are scFv, each of the scFv comprising a heavy chain variable domain and a light chain variable domain.

[0338] Implementation Scheme 6: The protein according to any one of Implementation Schemes 1-5, wherein the heavy chain variable domain of the scFv that binds NKG2D is located at the N-terminus or C-terminus of the light chain variable domain of the scFv that binds NKG2D.

[0339] Implementation Scheme 7: The protein according to Implementation Scheme 6, wherein the light chain variable domain of the scFv that binds NKG2D is located at the N-terminus of the heavy chain variable domain of the scFv that binds NKG2D.

[0340] Implementation Scheme 8: The protein according to any one of Implementation Schemes 1-7, wherein the scFv that binds to NKG2D is linked to an antibody Fc domain or a portion thereof sufficient to bind CD16 or a third antigen binding site that binds CD16.

[0341] Implementation Scheme 9: The protein according to Implementation Scheme 8, wherein the scFv that binds to NKG2D is connected via a hinge containing Ala-Ser to an antibody Fc domain or a portion thereof sufficient to bind CD16 or a third antigen binding site that binds CD16.

[0342] Implementation Scheme 10: The protein according to Implementation Scheme 8, wherein the scFv that binds to NKG2D is connected via a flexible linker comprising an amino acid sequence comprising SEQ ID NO: 168 to the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind CD16 or to a third antigen-binding site that binds CD16.

[0343] Implementation Scheme 11: The protein according to Implementation Scheme 10, wherein the C-terminus of the antibody Fc domain is linked to the N-terminus of the light chain variable domain of the scFv that binds NKG2D.

[0344] Implementation Scheme 12: The protein according to any one of Implementation Schemes 1-11, wherein a disulfide bridge is formed between the heavy chain variable domain and the light chain variable domain of the scFv within the NKG2D-binding scFv.

[0345] Implementation Scheme 13: The protein according to Implementation Scheme 12, wherein a disulfide bridge is formed between C44 from the heavy chain variable structural domain and C100 from the light chain variable structural domain.

[0346] Implementation Scheme 14: The protein according to any one of Implementation Schemes 1-13, wherein the heavy chain variable domain is connected to the light chain variable domain via a flexible linker within the scFv that binds NKG2D.

[0347] Implementation Scheme 15: The protein according to Implementation Scheme 14, wherein the flexible linker comprises (GlyGlyGlyGlySer)n (SEQ ID NO: 198), where n is an integer between 1 and 10.

[0348] Implementation Scheme 16: The protein according to any one of Implementation Schemes 5 to 15, wherein the second antigen binding site and the additional antigen binding site scFv are each connected via a hinge containing Ala-Ser to an antibody Fc domain or a portion thereof sufficient to bind CD16 or a third antigen binding site binding CD16.

[0349] Implementation Scheme 17: The protein according to any one of Implementation Schemes 5 to 16, wherein the second antigen binding site and the additional antigen binding site scFv are connected to the antibody Fc domain via a hinge containing Ala-Ser.

[0350] Implementation Scheme 18: The protein according to Implementation Scheme 16 or 17, wherein a disulfide bridge is formed between the heavy chain variable domain and the light chain variable domain of the second antigen binding site, the additional antigen binding site, or both.

[0351] Implementation Scheme 19: The protein according to Implementation Scheme 18, wherein a disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain at the second antigen binding site, the additional antigen binding site, or both.

[0352] Implementation Scheme 20: The protein according to any one of Implementation Schemes 1 to 19, wherein the light chain variable domain of the NKG2D-binding scFv is located at the N-terminus of the heavy chain variable domain of the NKG2D-binding scFv, wherein the light chain variable domain of the NKG2D-binding scFv is connected to the heavy chain variable domain of the NKG2D-binding scFv via a flexible linker consisting of the amino acid sequence of SEQ ID NO: 167, and the NKG2D-binding scFv is connected to the antibody Fc domain via a hinge containing Ala-Ser.

[0353] Implementation Scheme 21: The protein according to any one of Implementation Schemes 1-20, wherein the scFv binding NKG2D comprises: (a) a heavy chain variable domain comprising complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 191, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; (b) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 193, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; and (c) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; (d) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 95, 96, and 97; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; and (e) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 188, 88, and 189, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 91, 92, and 93, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 185, 104, and 192, respectively. (f) Light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 107, 108, and 109; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 185, 72, and 159; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 75, 76, and 77; (g) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 186, 80, and 187; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 83, 84, and 85; and (h) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 107, 108, and 109; and heavy ...8, 109; and heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 107, 108, and 109; and The heavy chain variable domains of the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences NO: 190, 96, and 194;and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; (i) heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 195, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; (i) heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 196, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; (k) heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; Heavy chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 197; and light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively; or (l) heavy chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 190, 96, and 160, respectively; and light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 99, 100, and 101, respectively.

[0354] Implementation Scheme 22: Implementation scheme, the protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D contains a heavy chain variable domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 94.

[0355] Implementation Scheme 23: The protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D comprises a heavy chain variable domain having at least 90% identity with SEQ ID NO: 94 and a light chain variable domain having at least 90% identity with SEQ ID NO: 98.

[0356] Implementation Scheme 24: The protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D comprises a heavy chain variable domain having at least 95% identity with SEQ ID NO: 94 and a light chain variable domain having at least 95% identity with SEQ ID NO: 98.

[0357] Implementation Scheme 25: The protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D comprises a heavy chain variable domain having the same identity as SEQ ID NO: 94 and a light chain variable domain having the same identity as SEQ ID NO: 98.

[0358] Implementation Scheme 26: The protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D comprises a heavy chain variable domain identical to SEQ ID NO: 169 and a light chain variable domain identical to SEQ ID NO: 98.

[0359] Implementation Scheme 27: The protein according to any one of Implementation Schemes 1-21, wherein the scFv that binds NKG2D comprises a heavy chain variable domain having the same identity as SEQ ID NO: 171 and a light chain variable domain having the same identity as SEQ ID NO: 98.

[0360] Implementation Scheme 28: The protein according to any one of Implementation Schemes 1-21, wherein the scFv that binds NKG2D comprises a heavy chain variable domain having the same identity as SEQ ID NO: 173 and a light chain variable domain having the same identity as SEQ ID NO: 98.

[0361] Implementation Scheme 29: The protein according to any one of Implementation Schemes 1-21, wherein the scFv that binds NKG2D comprises a heavy chain variable domain having the same identity as SEQ ID NO: 175 and a light chain variable domain having the same identity as SEQ ID NO: 98.

[0362] Implementation Scheme 30: The protein according to any one of Implementation Schemes 1-21, wherein the scFv that binds NKG2D includes a heavy chain variable domain that is identical to SEQ ID NO: 177 and a light chain variable domain that is identical to SEQ ID NO: 98.

[0363] Implementation Scheme 31: The protein according to any one of Implementation Schemes 1-21, wherein the scFv binding NKG2D comprises a heavy chain variable domain identical to SEQ ID NO: 179 and a light chain variable domain identical to SEQ ID NO: 98.

[0364] Implementation Scheme 32: The protein according to any one of Implementation Schemes 1-31, wherein the second antigen-binding site binding to BCMA comprises: (a) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 149, 150, and 151, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 153, 154, and 155, respectively; (b) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 115, 116, and 117, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 120, 121, and 123, respectively; (c) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 120, 121, and 123, respectively; (d) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 125, 126, and 127; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively; and (e) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 137, 138, and 139, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 141, 142, and 143, respectively. Light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 145, 146, and 147; or (f) heavy chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 115, 116, and 117, respectively; and light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NO: 120, 121, and 122, respectively.

[0365] Implementation Scheme 33: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 148 and a light chain variable domain having at least 90% identity with SEQ ID NO: 152; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 148 and a light chain variable domain having at least 95% identity with SEQ ID NO: 152; or (c) a heavy chain variable domain having identity with SEQ ID NO: 148 and a light chain variable domain having identity with SEQ ID NO: 152.

[0366] Implementation Scheme 34: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 and a light chain variable domain having at least 90% identity with SEQ ID NO: 119; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 119; or (c) a heavy chain variable domain having identity with SEQ ID NO: 114 and a light chain variable domain having identity with SEQ ID NO: 119.

[0367] Implementation Scheme 35: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 124 and a light chain variable domain having at least 90% identity with SEQ ID NO: 128; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 124 and a light chain variable domain having at least 95% identity with SEQ ID NO: 128; or (c) a heavy chain variable domain having identity with SEQ ID NO: 124 and a light chain variable domain having identity with SEQ ID NO: 128.

[0368] Implementation Scheme 36: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 132 and a light chain variable domain having at least 90% identity with SEQ ID NO: 136; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 132 and a light chain variable domain having at least 95% identity with SEQ ID NO: 136; or (c) a heavy chain variable domain having identity with SEQ ID NO: 132 and a light chain variable domain having identity with SEQ ID NO: 136.

[0369] Implementation Scheme 37: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 140 and a light chain variable domain having at least 90% identity with SEQ ID NO: 144; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 140 and a light chain variable domain having at least 95% identity with SEQ ID NO: 144; or (c) a heavy chain variable domain having identity with SEQ ID NO: 140 and a light chain variable domain having identity with SEQ ID NO: 144.

[0370] Implementation Scheme 38: The protein according to any one of Implementation Schemes 1-32, wherein the second antigen binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 and a light chain variable domain having at least 90% identity with SEQ ID NO: 118; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 118; or (c) a heavy chain variable domain having identity with SEQ ID NO: 114 and a light chain variable domain having identity with SEQ ID NO: 118.

[0371] Implementation Scheme 39: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding BCMA comprises: (a) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 149, 150, and 151, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 153, 154, and 155, respectively; (b) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 115, 116, and 117, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 120, 121, and 123, respectively; and (c) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 120, 121, and 123, respectively; (d) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 125, 126, and 127; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively; (e) Heavy chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 135, 126, and 127; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 125, 126, and 127, respectively; and light chain variable domains comprising CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 125, 126, and 127, respectively. Light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 145, 146, and 147; or (f) heavy chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 115, 116, and 117, respectively; and light chain variable domains comprising the CDR1, CDR2, and CDR3 sequences represented by amino acid sequences of SEQ ID NO: 120, 121, and 122, respectively.

[0372] Implementation Scheme 40: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 148 and a light chain variable domain having at least 90% identity with SEQ ID NO: 152; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 148 and a light chain variable domain having at least 95% identity with SEQ ID NO: 152; or (c) a heavy chain variable domain having identity with SEQ ID NO: 148 and a light chain variable domain having identity with SEQ ID NO: 152.

[0373] Implementation Scheme 41: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 and a light chain variable domain having at least 90% identity with SEQ ID NO: 119; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 119; or (c) a heavy chain variable domain having identity with SEQ ID NO: 114 and a light chain variable domain having identity with SEQ ID NO: 119.

[0374] Implementation Scheme 42: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 124 and a light chain variable domain having at least 90% identity with SEQ ID NO: 128; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 124 and a light chain variable domain having at least 95% identity with SEQ ID NO: 128; or (c) a heavy chain variable domain having identity with SEQ ID NO: 124 and a light chain variable domain having identity with SEQ ID NO: 128.

[0375] Implementation Scheme 43: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 132 and a light chain variable domain having at least 90% identity with SEQ ID NO: 136; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 132 and a light chain variable domain having at least 95% identity with SEQ ID NO: 136; or (c) a heavy chain variable domain having identity with SEQ ID NO: 132 and a light chain variable domain having identity with SEQ ID NO: 136.

[0376] Implementation Scheme 44: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 140 and a light chain variable domain having at least 90% identity with SEQ ID NO: 144; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 140 and a light chain variable domain having at least 95% identity with SEQ ID NO: 144; or (c) a heavy chain variable domain having identity with SEQ ID NO: 140 and a light chain variable domain having identity with SEQ ID NO: 144.

[0377] Implementation Scheme 45: The protein according to any one of Implementation Schemes 2-38, wherein the additional antigen-binding site binding to BCMA comprises: (a) a heavy chain variable domain having at least 90% identity with SEQ ID NO: 114 and a light chain variable domain having at least 90% identity with SEQ ID NO: 118; (b) a heavy chain variable domain having at least 95% identity with SEQ ID NO: 114 and a light chain variable domain having at least 95% identity with SEQ ID NO: 118; or (c) a heavy chain variable domain having identity with SEQ ID NO: 114 and a light chain variable domain having identity with SEQ ID NO: 118.

[0378] Implementation Scheme 46: The protein according to any one of Implementation Schemes 1-45, wherein the antibody Fc domain or a portion thereof sufficient to bind CD16 or the third antigen binding site for binding CD16 is an antibody Fc domain comprising the hinge and CH2 domain of a human IgG1 antibody.

[0379] Implementation Scheme 47: The protein according to any one of Implementation Schemes 1-45, wherein the antibody Fc domain or a portion thereof sufficient to bind CD16 or the third antigen binding site for binding CD16 is an antibody Fc domain comprising an amino acid sequence having at least 90% identity with amino acids 234-332 of the human IgG1 antibody.

[0380] Implementation Scheme 48: The protein according to Implementation Scheme 46 or 47, wherein the antibody Fc domain comprises an amino acid sequence having at least 90% identity with the Fc domain of human IgG1 and differing at one or more positions selected from the group consisting of: Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0381] Implementation Scheme 49: The protein according to Implementation Scheme 48, wherein the antibody Fc domain is an Fc domain of human IgG1 containing Q347R, D399V and F405T substitutions.

[0382] Implementation Scheme 50: The protein according to Implementation Scheme 49, wherein the antibody Fc domain is linked to scFv that binds NKG2D.

[0383] Implementation Scheme 51: The protein according to Implementation Scheme 48, wherein the Fc domain is the Fc domain of human IgG1 containing K360E and K409W substitutions.

[0384] Implementation Scheme 52: The protein according to Implementation Scheme 51, wherein the Fc domain is linked to the second antigen binding site.

[0385] Implementation Scheme 53: The protein according to any one of Implementation Schemes 1-33 and 46-52, comprising the amino acid sequence of SEQ ID NO: 162.

[0386] Implementation Scheme 54: The protein according to any one of Implementation Schemes 1-33 and 46-52, comprising an amino acid sequence containing SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 165.

[0387] Implementation Scheme 55: The protein according to any one of Implementation Schemes 1-33 and 46-52, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 162.

[0388] Implementation Scheme 56: The protein according to any one of Implementation Schemes 1-33 and 46-52, comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 162.

[0389] Implementation Scheme 57: The protein according to any one of Implementation Schemes 1-33 and 46-52, comprising an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 162.

[0390] Implementation Scheme 58: A protein according to any one of Implementation Schemes 1-33 and 46-52, comprising an amino acid sequence having at least 90% or at least 95% or at least 99% identity with the amino acid sequence of SEQ ID NO: 162, and further comprising SEQ ID NO: 163 and SEQ ID NO: 165.

[0391] Implementation Scheme 59: The protein according to any one of Implementation Schemes 1-58, wherein the protein binds to NKG2D with KD at 2 nM to 120 nM, as measured by surface plasmon resonance.

[0392] Implementation Scheme 60: The protein according to any one of Implementation Schemes 1-59, wherein the protein activates natural killer cells or cytotoxic T cells upon binding.

[0393] Implementation Scheme 61: A formulation comprising a protein according to any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0394] Implementation Scheme 62: A cell comprising one or more nucleic acids encoding proteins according to any one of Implementation Schemes 1-60.

[0395] Implementation Scheme 63: A method for enhancing tumor cell death, comprising exposing the tumor to a protein according to any one of Implementation Schemes 1-60 in the presence of natural killer cells or cytotoxic T cells.

[0396] Implementation Scheme 64: A method of treating cancer in a subject, comprising administering to the subject a protein according to any one of Implementation Schemes 1-60 or a preparation according to Implementation Scheme 61.

[0397] Implementation Scheme 65: The method as described in Implementation Scheme 64, wherein the cancer is selected from the group consisting of: multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, and acute myeloid leukemia.

[0398] Implementation Scheme 66: The method as described in Implementation Scheme 64 or 65, wherein the cancer expresses BCMA.

[0399] Implementation Scheme 67: A method for stimulating cytotoxic T cells, comprising exposing the cytotoxic T cells to a protein according to any one of Implementation Schemes 1-60.

[0400] Implementation Scheme 68: A method for stimulating natural killer cells, comprising exposing the natural killer cells to a protein according to any one of Implementation Schemes 1-60.

[0401] Incorporate by reference

[0402] The full disclosure of each of the patent documents and scientific articles mentioned in this article is incorporated herein by reference for all purposes.

[0403] equivalent

[0404] The invention may be implemented in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments should be considered illustrative in all respects and not limiting of the invention as described herein. Consequently, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all variations falling within the equivalent meaning and scope of the claims are intended to be encompassed therein. sequence list <110> Dragonfly Therapeutics, Inc. <120> Multispecific binding proteins that combine BCMA, NKG2D, and CD16, and their usage methods <130> 14247-425-228 <140> TBA <141> 2019-08-08 <150> 62 / 716,207 <151> 2018-08-08 <160> 198 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning the heavy chain variable region of ADI-27705 <400> 1 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-27705 light chain variable region <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloning ADI-27705–VH <400> 3 Gly Ser Phe Ser Gly Tyr Tyr Trp Ser 1 5 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-27705–VH <400> 4 Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 of ADI-27705–VH <400> 5 Ala Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro 1 5 10 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning the heavy chain variable region of ADI-27724 <400> 6 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-27724 light chain variable region <400> 7 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone ADI-27740 heavy chain variable region <400> 8 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone ADI-27740 light chain variable region <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone the variable region of the heavy chain of ADI-27741 <400> 10 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-27741 light chain variable region <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Tyr Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning the heavy chain variable region of ADI-27743 <400> 12 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 13 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-27743 light chain variable region <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-28153 heavy chain variable region <400> 14 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-28153 light chain variable region <400> 15 Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-28226 (C26) heavy chain variable region <400> 16 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-28226(C26) light chain variable region <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone heavy chain variable region of ADI-28154 <400> 18 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Clone of the variable region of ADI-28154 light chain <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone the variable region of the ADI-29399 heavy chain <400> 20 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 21 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-29399 light chain variable region <400> twenty one Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> twenty two <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning the heavy chain variable region of ADI-29401 <400> twenty two Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 23 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of the variable region of the ADI-29401 light chain <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ile Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI - 29403 heavy chain variable region <400> 24 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 25 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29403 light chain variable region <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 26 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29405 heavy chain variable region <400> 26 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 27 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-29405 light chain variable region <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of the heavy chain variable region of ADI-29407 <400> 28 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 29 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29407 light chain variable region <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone the heavy chain variable region of ADI-29419 <400> 30 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 31 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-29419 light chain variable region <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Ser Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning the heavy chain variable region of ADI-29421 <400> 32 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 33 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29421 light chain variable region <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Ser Tyr Ser Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 34 ​​​​​​​​​​​​​​​​​​​​Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 35 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29424 light chain variable region <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Phe Ile Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 36 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29425 heavy chain variable region <400> 36 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 37 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-29425 light chain variable region <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone heavy chain variable region of ADI-29426 <400> 38 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 39 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone ADI-29426 light chain variable region <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 40 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Clone the heavy chain variable region of ADI-29429 <400> 40 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-29429 light chain variable region <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Leu Tyr Ser Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29447(F47) heavy chain variable region <400> 42 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 43 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29447(F47) light chain variable region <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Phe Ile Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-27727 heavy chain variable region <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 三十 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27727-VH's CDR1 (non-Kabat) <400> 45 Gly Thr Phe Ser Ser Tyr Ala Ile Ser 1 5 <210> 46 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 cloned from ADI-27727-VH <400> 46 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 47 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Cloning the CDR3 (non-Kabat) of ADI-27727-VH <400> 47 Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met 1 5 10 15 Asp Val <210> 48 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-27727 light chain variable region <400> 48 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 49 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR1 cloned from ADI-27727-VL <400> 49 Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Ala <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 cloned from ADI-27727-VL <400> 50 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 of ADI-27727-VL <400> 51 Gln Gln Tyr Tyr Ser Thr Pro Ile Thr 1 5 <210> 52 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29443(F43) heavy chain variable region <400> 52 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29443(F43)-VH's CDR1 (non-Kabat) <400> 53 Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly 1 5 10 <210> 54 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29443(F43)-VH <400> 54 Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 55 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29443(F43)-VH's CDR3 (non-Kabat) <400> 55 Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr 1 5 10 <210> 56 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29443(F43) light chain variable region <400> 56 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Thr Trp Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloning ADI-29443(F43)-VL <400> 57 Arg Ala Ser Gln Ser Val Ser Arg Tyr Leu Ala 1 5 10 <210> 58 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29443(F43)-VL <400> 58 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 cloned from ADI-29443(F43)-VL <400> 59 Gln Gln Phe Asp Thr Trp Pro Pro Thr 1 5 <210> 60 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29404(F04) heavy chain variable region <400> 60 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 61 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29404(F04) light chain variable region <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Tyr Asp Ser Tyr Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 62 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-28200 heavy chain variable region <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning the CDR1 (non-Kabat) of the ADI-28200-VH <400> 63 Gly Thr Phe Ser Ser Tyr Ala Ile Ser 1 5 <210> 64 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-28200-VH <400> 64 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 65 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 for ADI-28200-VH <400> 65 Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val <210> 66 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Cloning the ADI-28200 light chain variable region <400> 66 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR1 cloned from ADI-28200-VL <400> 67 Glu Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-28200-VL <400> 68 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 69 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-28200-VL's CDR3 <400> 69 Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr 1 5 <210> 70 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Clone the heavy chain variable region of ADI-29379 (E79) <400> 70 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr 100 105 110 Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29379(E79)-VH's CDR1 (non-Kabat) <400> 71 Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29379(E79)-VH <400> 72 Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 73 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29379(E79)-VH's CDR3 (non-Kabat) <400> 73 Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr 1 5 10 15 Met Asp Val <210> 74 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloned ADI-29379(E79) light chain variable region <400> 74 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asp Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 75 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Clone of CDR1 of ADI-29379 (E79)–VL <400> 75 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 76 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29379(E79)–VL <400> 76 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 77 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 for cloned ADI-29379(E79)–VL <400> 77 Gln Gln Tyr Asp Asp Trp Pro Phe Thr 1 5 <210> 78 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29463(F63) heavy chain variable region <400> 78 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29463(F63)-VH's CDR1 (non-Kabat) <400> 79 Tyr Thr Phe Thr Gly Tyr Tyr Met His 1 5 <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29463(F63)-VH <400> 80 Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29463(F63)-VH <400> 81 Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp 1 5 10 15 Val <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloned ADI-29463(F63) light chain variable region <400> 82 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Asp Tyr Trp Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 83 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 cloned from ADI-29463(F63)-VL <400> 83 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 84 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29463(F63)-VL <400> 84 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 85 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29463(F63)-VL <400> 85 Gln Gln Asp Asp Tyr Trp Pro Pro Thr 1 5 <210> 86 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Clone the heavy chain variable region of ADI-27744 (A44) <400> 86 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27744(A44)-VH's CDR1 (non-Kabat) <400> 87 Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 <210> 88 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-27744(A44)-VH <400> 88 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 89 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27744(A44)-VH's CDR3 (non-Kabat) <400> 89 Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr 1 5 10 <210> 90 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27744(A44) light chain variable region <400> 90 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloning ADI-27744(A44)–VL <400> 91 Arg Ala Ser Gln Gly Ile Asp Ser Trp Leu Ala 1 5 10 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-27744(A44)–VL <400> 92 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-27744(A44)–VL <400> 93 Gln Gln Gly Val Ser Tyr Pro Arg Thr 1 5 <210> 94 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27749(A49) heavy chain variable region <400> 94 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR1 (non-Kabat) for ADI-27744(A44), A49MI, A49MQ, A49ML, A49MF, and A49MV-VH. <400> 95 Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5 <210> 96 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clones of ADI-27744 (A44), A49MI, A49MQ, A49ML, A49MF, and A49MV-VH. <400> 96 Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 97 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27744(A44)-VH's CDR3 (non-Kabat) <400> 97 Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 98 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-27749(A49), A49MQ, A49MF, A49MV, and A49-common sequences Light chain variable region <400> 98 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 99 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR1 of ADI-27744(A44), A49MI, A49MQ, A49ML, A49MF, A49MV, and A49-common sequence – VL <400> 99 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 100 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clones of ADI-27744(A44), A49MI, A49MQ, A49ML, A49MF, A49MV, and A49-common sequence-VL. <400> 100 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of cloned ADI-27744(A44), A49MI, A49MQ, A49ML, A49MF, A49MV, and A49-common sequence-VL. <400> 101 Gln Gln Gly Val Ser Phe Pro Arg Thr 1 5 <210> 102 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Clone of ADI-29378 (E78) heavy chain variable region <400> 102 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Gly Phe Ala Tyr Gly Met Asp Tyr Tyr Tyr Met 100 105 110 Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29378(E78)-VH's CDR1 (non-Kabat) <400> 103 Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5 <210> 104 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29378(E78)-VH <400> 104 Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 105 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Cloning ADI-29378(E78)-VH's CDR3 (non-Kabat) <400> 105 Ala Arg Glu Gly Ala Gly Phe Ala Tyr Gly Met Asp Tyr Tyr Tyr Met 1 5 10 15 Asp Val <210> 106 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Cloned ADI-29378(E78) light chain variable region <400> 106 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Asn Trp Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 107 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloned ADI-29378(E78)–VL <400> 107 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 108 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 clone of ADI-29378(E78)–VL <400> 108 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 109 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29378(E78)–VL <400> 109 Gln Gln Ser Asp Asn Trp Pro Phe Thr 1 5 <210> 110 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable structural domain <400> 110 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 111 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable domain <400> 111 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 112 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable domain <400> 112 Gln Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Asp Asp Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly His Ile Ser Tyr Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Asn Trp Asp Asp Ala Phe Asn Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 113 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable structural domain <400> 113 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 114 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Clone 1 in US14 / 776649 Heavy Chain Variable Domain <400> 114 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Pro Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Phe Ala Ser Gly Asn Ser Glu Tyr Asn Gln Lys Phe 50 55 60 Thr Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Leu Tyr Asp Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 115 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1, the first clone in US14 / 776649–VH <400> 115 Asp Tyr Tyr Ile Asn 1 5 <210> 116 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 1 in US14 / 776649–VH <400> 116 Trp Ile Tyr Phe Ala Ser Gly Asn Ser Glu Tyr Asn Gln Lys Phe Thr 1 5 10 15 Gly <210> 117 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 1 in US14 / 776649–VH <400> 117 Leu Tyr Asp Tyr Asp Trp Tyr Phe Asp Val 1 5 10 <210> 118 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Clone 1 in US14 / 776649 Light Chain Variable Domain <400> 118 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Glu Thr 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 119 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Clone 1 in US14 / 776649 Light Chain Variable Domain <400> 119 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Ser Gln Ser 85 90 95 Ser Ile Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 120 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR1, the first clone in US14 / 776649–VL <400> 120 Lys Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 121 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 1 in US14 / 776649–VL <400> 121 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 1 in US14 / 776649–VL <400> 122 Ala Glu Thr Ser His Val Pro Trp Thr 1 5 <210> 123 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 1 in US14 / 776649–VL <400> 123 Ser Gln Ser Ser Ile Tyr Pro Trp Thr 1 5 <210> 124 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Cloning 2 in heavy-chain variable domains (PCT / US15 / 64269) <400> 124 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe 50 55 60 Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 125 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of clone 2 in PCT / US15 / 64269–VH <400> 125 Asp Tyr Ser Ile Asn 1 5 <210> 126 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 2 in PCT / US15 / 64269–VH <400> 126 Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe Arg 1 5 10 15 <210> 127 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 2 in PCT / US15 / 64269–VH <400> 127 Asp Tyr Ser Tyr Ala Met Asp Tyr 1 5 <210> 128 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Cloning 2 in Light Chain Variable Domains (PCT / US15 / 64269) <400> 128 Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly 1 5 10 15 Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu 20 25 30 Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg 85 90 95 Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 129 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of clone 2 in PCT / US15 / 64269–VL <400> 129 Arg Ala Ser Glu Ser Val Thr Ile Leu Gly Ser His Leu Ile His 1 5 10 15 <210> 130 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2, clone 2 in PCT / US15 / 64269–VL <400> 130 Leu Ala Ser Asn Val Gln Thr 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 2 in PCT / US15 / 64269–VL <400> 131 Leu Gln Ser Arg Thr Ile Pro Arg Thr 1 5 <210> 132 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Clones 3 in US14 / 122,391 Heavy Chain Variable Domain <400> 132 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Ile Tyr Asn Gly Tyr Asp Val Leu Asp Asn Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 133 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of the third clone in US14 / 122,391–VH <400> 133 Asn Tyr Trp Met His 1 5 <210> 134 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 3 in US14 / 122,391–VH <400> 134 Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 135 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3, the third clone in US14 / 122,391–VH <400> 135 Gly Ala Ile Tyr Asn Gly Tyr Asp Val Leu Asp Asn 1 5 10 <210> 136 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Clone 3 in US14 / 122,391 Light Chain Variable Structural Domain <400> 136 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 137 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1, the third clone in US14 / 122,391–VL <400> 137 Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 138 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 3 in US14 / 122,391–VL <400> 138 Tyr Thr Ser Asn Leu His Ser 1 5 <210> 139 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3, the third clone in US14 / 122,391–VL <400> 139 Gln Gln Tyr Arg Lys Leu Pro Trp Thr 1 5 <210> 140 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Clones 4 in heavy-chain variable domains (US20170051068) <400> 140 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ile Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg His Asp Gly Ala Thr Ala Gly Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 141 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of Clone 4 in US20170051068–VH <400> 141 Ser Ser Ser Tyr Phe Trp Gly 1 5 <210> 142 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 4 in US20170051068–VH <400> 142 Ser Ile Tyr Tyr Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 143 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 4 in US20170051068–VH <400> 143 His Asp Gly Ala Thr Ala Gly Leu Phe Asp Tyr 1 5 10 <210> 144 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Clone 4 in the Light Chain Variable Domain (US20170051068) <400> 144 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Pro Pro Gly Gln Ala Pro Val Val Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 145 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of clone 4 in US20170051068–VL <400> 145 Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His 1 5 10 <210> 146 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 4 in US20170051068–VL <400> 146 Asp Asp Ser Asp Arg Pro Ser 1 5 <210> 147 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 4 in US20170051068–VL <400> 147 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10 <210> 148 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Clone 5 (Mab42) in heavy-chain variable structural domains (WO2017021450) <400> 148 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 149 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of clone 5 (Mab42) in WO2017021450–VH <400> 149 Asp Asn Ala Met Gly 1 5 <210> 150 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 5 (Mab42) in WO2017021450–VH <400> 150 Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 151 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 5 (Mab42) in WO2017021450–VH <400> 151 Val Leu Gly Trp Phe Asp Tyr 1 5 <210> 152 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Clone 5 (Mab42) in the variable structural domain of light chains (WO2017021450) <400> 152 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Glu 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile His Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 153 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of clone 5 (Mab42) in WO2017021450–VL <400> 153 Arg Ala Ser Gln Ser Val Ser Asp Glu Tyr Leu Ser Trp 1 5 10 <210> 154 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of clone 5 (Mab42) in WO2017021450–VL <400> 154 His Ser Ala Ser Thr Arg Ala Thr 1 5 <210> 155 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 5 (Mab42) in WO2017021450–VL <400> 155 Gln Gln Tyr Gly Tyr Pro Pro Asp Phe Thr 1 5 10 <210> 156 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> BCMA combined domain <400> 156 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu 50 55 60 Ile Ile Ser Leu Ala Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile 65 70 75 80 Asn Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu 85 90 95 Leu Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu 100 105 110 Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys 115 120 125 Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His Cys Phe 130 135 140 Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val Thr Thr Lys 145 150 155 160 Thr Asn Asp Tyr Cys Lys Ser Leu Pro Ala Ala Leu Ser Ala Thr Glu 165 170 175 Ile Glu Lys Ser Ile Ser Ala Arg 180 <210> 157 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 83A10 Heavy Chain Variable Domain <400> 157 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 158 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 83A10 light chain variable structural domain <400> 158 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 159 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29379(E79)–VH <400> 159 Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr Met Asp 1 5 10 15 Val <210> 160 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone A49-common sequence – VH <220> <221> misc_feature <222> (4)..(4) <223> Xaa=Met, Leu, Ile, Val, Gln or Phe <400> 160 Gly Ala Pro Xaa Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 161 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> NKG2D combined with scFv <400> 161 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg 85 90 95 Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser 145 150 155 160 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ser 165 170 175 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 <210> 162 <211> 702 <212> PRT <213> Artificial Sequence <220> <223> C-terminus of Fc domain (F4-BCMAFc-AJ chain B-NKG2D binding scFv) <400> 162 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val Tyr Thr Leu Pro Pro 340 345 350 Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Val Ser Asp 385 390 395 400 Gly Ser Phe Thr Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Ser 435 440 445 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val 450 455 460 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 465 470 475 480 Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 485 490 495 Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro 500 505 510 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 515 520 525 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly 530 535 540 Val Ser Phe Pro Arg Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 545 550 555 560 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 565 570 575 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 580 585 590 Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 595 600 605 Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys 610 615 620 Leu Glu Trp Val Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr 625 630 635 640 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 645 650 655 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 660 665 670 Val Tyr Tyr Cys Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp 675 680 685 Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 690 695 700 <210> 163 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> Anti-BCMA VH-CH1-Fc <400> 163 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Glu Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Trp Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 164 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 164 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 165 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-full LC <400> 165 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Glu 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile His Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 166 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> F3-NKG2D binding scFv-Fc-AJ chain B [VL(G4S)4VH)] <400> 166 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg 85 90 95 Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser 145 150 155 160 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val Ser 165 170 175 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Asp Lys Thr His Thr 245 250 255 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gln Pro Arg Glu Pro Arg Val Tyr Thr Leu Pro Pro 370 375 380 Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Val Ser Asp 420 425 430 Gly Ser Phe Thr Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 <210> 167 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Connector sequence <400> 167 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 168 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Connector sequence <400> 168 Ser Gly Ser Gly Gly Gly Gly Ser 1 5 <210> 169 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning the A49MI heavy chain variable region <400> 169 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Ile Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 170 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49MI-VH's CDR3 (non-Kabat) <400> 170 Ala Arg Gly Ala Pro Ile Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 171 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49MQ heavy chain variable region <400> 171 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Gln Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 172 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of Cloned A49MQ-VH (non-Kabat) <400> 172 Ala Arg Gly Ala Pro Gln Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 173 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Clone A49ML heavy chain variable region <400> 173 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Leu Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 174 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49ML-VH's CDR3 (non-Kabat) <400> 174 Ala Arg Gly Ala Pro Leu Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 175 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning the A49MF heavy chain variable region <400> 175 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Phe Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 176 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49MF-VH's CDR3 (non-Kabat) <400> 176 Ala Arg Gly Ala Pro Phe Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 177 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning the A49MV heavy chain variable region <400> 177 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Val Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 178 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49MV-VH's CDR3 (non-Kabat) <400> 178 Ala Arg Gly Ala Pro Val Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 179 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49 - Common Sequence Heavy Chain Variable Region <220> <221> misc_feature <222> (102)..(102) <223> Xaa can be any naturally occurring amino acid. <400> 179 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Pro Xaa Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 180 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Cloning A49-shared sequence-VH CDR3 (non-Kabat) <220> <221> misc_feature <222> (6)..(6) <223> Xaa=Met, Leu, Ile, Val, Gln or Phe <400> 180 Ala Arg Gly Ala Pro Xaa Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 15 <210> 181 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 cloned from ADI-27727, 28200–VH <400> 181 Ser Tyr Ala Ile Ser 1 5 <210> 182 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 of ADI-27727-VH <400> 182 Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 183 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloning ADI-29443(F43)-VH <400> 183 Ser Ser Ser Tyr Tyr Trp Gly 1 5 <210> 184 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29443(F43)-VH <400> 184 Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr 1 5 10 <210> 185 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 clones of ADI-29379(E79) and 29378(E78)-VH <400> 185 Ser Tyr Tyr Met His 1 5 <210> 186 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloned ADI-29463(F63)-VH <400> 186 Gly Tyr Tyr Met His 1 5 <210> 187 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of ADI-29463(F63)-VH <400> 187 Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp Val 1 5 10 15 <210> 188 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 for cloning ADI-27744(A44)-VH <400> 188 Ser Tyr Ala Met Ser 1 5 <210> 189 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 of ADI-27744(A44)-VH <400> 189 Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr 1 5 10 <210> 190 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 clones of ADI-27744 (A44), A49MI, A49ML, A49MV, and A49-common sequence-VH <400> 190 Ser Tyr Ser Met Asn 1 5 <210> 191 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Cloning CDR3 of ADI-27744(A44)–VH <400> 191 Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 192 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of clone 29378(E78)–VH <400> 192 Glu Gly Ala Gly Phe Ala Tyr Gly Met Asp Tyr Tyr Tyr Met Asp Val 1 5 10 15 <210> 193 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of A49MI–VH <400> 193 Gly Ala Pro Ile Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 194 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of A49MQ-VH <400> 194 Gly Ala Pro Gln Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 195 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 cloned from A49ML-VH <400> 195 Gly Ala Pro Leu Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 196 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of A49MF-VH <400> 196 Gly Ala Pro Phe Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 197 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 clone of A49MV-VH <400> 197 Gly Ala Pro Val Gly Ala Ala Ala Gly Trp Phe Asp Pro 1 5 10 <210> 198 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Connector sequence <220> <221> repeat <222> (1)..(5) <223> Amino acids can be repeated 1-10 times. <400> 198 Gly Gly Gly Gly Ser 1 5

Claims

1. A protein comprising: (a) a first antigen binding site, comprising a single chain variable fragment (scFv), which binds to NKG2D and comprises a heavy chain complementary determining region 1 (CDR1) sequence consisting of the amino acid sequence of SEQ ID NO: 190, a heavy chain complementary determining region 2 (CDR2) sequence consisting of the amino acid sequence of SEQ ID NO: 96, a heavy chain complementary determining region 3 (CDR3) sequence consisting of the amino acid sequence of SEQ ID NO: 191, a light chain CDR1 sequence consisting of the amino acid sequence of SEQ ID NO: 99, a light chain CDR2 sequence consisting of the amino acid sequence of SEQ ID NO: 100, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 101; (b) a second antigen binding site that binds to B cell maturation antigen (BCMA) and comprises a heavy chain CDR1 sequence consisting of the amino acid sequence of SEQ ID NO: 149, a heavy chain CDR2 sequence consisting of the amino acid sequence of SEQ ID NO: 150, a heavy chain CDR3 sequence consisting of the amino acid sequence of SEQ ID NO: 151, a light chain CDR1 sequence consisting of the amino acid sequence of SEQ ID NO: 153, a light chain CDR2 sequence consisting of the amino acid sequence of SEQ ID NO: 154, and a light chain CDR3 sequence consisting of the amino acid sequence of SEQ ID NO: 155; (c) an additional antigen binding site that binds to BCMA and comprises a heavy chain CDR1 sequence consisting of the amino acid sequence of SEQ ID NO: 149, a heavy chain CDR2 sequence consisting of the amino acid sequence of SEQ ID NO: 150, a heavy chain CDR3 sequence consisting of the amino acid sequence of SEQ ID NO: 151, a light chain CDR1 sequence consisting of the amino acid sequence of SEQ ID NO: 153, a light chain CDR2 sequence consisting of the amino acid sequence of SEQ ID NO: 154, and a light chain CDR3 sequence consisting of the amino acid sequence of SEQ ID NO: 155; and (d) an antibody Fc domain or a portion thereof sufficient to bind to CD16; Wherein the protein comprises the amino acid sequence of SEQ ID NO:

162.

2. The protein according to claim 1, wherein: The second antigen binding site and the additional antigen binding site that binds BCMA are Fab fragments.

3. The protein of claim 1, wherein the heavy chain variable domain of the scFv that binds NKG2D is located at the C-terminus of the light chain variable domain of the scFv.

4. The protein of claim 3, wherein the light chain variable domain is located N-terminal to the heavy chain variable domain of the scFv that binds NKG2D.

5. The protein of claim 1, wherein the scFv that binds to NKG2D is linked to the antibody Fc domain or a portion thereof sufficient to bind CD16.

6. The protein of claim 5, wherein the scFv that binds to NKG2D: (a) linked via a hinge comprising Ala-Ser to the antibody Fc domain or a portion thereof sufficient to bind CD16; or (b) linked to the C-terminus of the antibody Fc domain or a portion thereof sufficient to bind CD16 via a flexible linker comprising the amino acid sequence of SEQ ID NO:

168.

7. The protein of claim 6, wherein the C-terminus of the antibody Fc domain is linked to the N-terminus of the light chain variable domain of the scFv that binds NKG2D.

8. The protein of claim 1, wherein within the scFv that binds NKG2D, a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv.

9. The protein of claim 8, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

10. The protein of claim 1, wherein within the scFv that binds NKG2D, the heavy chain variable domain is connected to the light chain variable domain via a flexible linker.

11. The protein of claim 10, wherein the flexible linker comprises (GlyGlyGlyGlySer) n , where n is an integer between 1 and 10.

12. The protein of claim 2, wherein the second antigen binding site and the additional antigen binding site scFv are linked to the antibody Fc domain or portion thereof sufficient to bind CD16 via a hinge comprising Ala-Ser.

13. The protein of claim 2, wherein the second antigen binding site and the additional antigen binding site scFv are linked to the antibody Fc domain via a hinge comprising Ala-Ser.

14. The protein of claim 12, wherein a disulfide bridge is formed between the heavy chain variable domain and the light chain variable domain of the second antigen binding site and / or the additional antigen binding site.

15. The protein of claim 14, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

16. The protein of claim 1, wherein the scFv that binds to NKG2D comprises a light chain variable domain located at the N-terminus of the heavy chain variable domain, wherein the light chain variable domain is connected to the heavy chain variable domain of the scFv via a flexible linker, the flexible linker consisting of the amino acid sequence of SEQ ID NO: 167, and the scFv that binds to NKG2D is connected to the antibody Fc domain via a hinge comprising Ala-Ser.

17. The protein according to claim 1, wherein: The first antigen binding site that binds to NKG2D is composed of a heavy chain variable domain identical to SEQ ID NO: 94 and a light chain variable domain identical to SEQ ID NO:

98.

18. The protein of claim 1, wherein the second antigen binding site and / or the additional antigen binding site that binds BCMA consists of a heavy chain variable domain identical to SEQ ID NO: 148 and a light chain variable domain identical to SEQ ID NO:

152.

19. The protein of claim 1, wherein the antibody Fc domain comprises the hinge and CH2 domains of a human IgG1 antibody.

20. The protein of claim 19, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody.

21. The protein of claim 19, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of: Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

22. The protein of claim 1, wherein the Fc domain is an Fc domain of human IgG1 comprising Q347R, D399V, and F405T substitutions.

23. The protein of claim 22, wherein the Fc domain comprising the substitution is linked to the scFv that binds NKG2D.

24. The protein of claim 1, wherein the Fc domain is an Fc domain of human IgG1 comprising K360E and K409W substitutions.

25. The protein of claim 24, wherein the Fc domain comprising the substitution is linked to the second antigen binding site.

26. The protein of claim 1, comprising: (a) the amino acid sequence of SEQ ID NO: 162; or (b) The amino acid sequences of SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO:

165.

27. A formulation comprising the protein according to any one of claims 1 to 26 and a pharmaceutically acceptable carrier.

28. A cell comprising one or more nucleic acids encoding a protein according to any one of claims 1-26.

29. Use of the protein according to any one of claims 1 to 26 in the preparation of a medicament for directly and / or indirectly enhancing tumor cell death, by exposing tumors and natural killer cells to the protein, wherein the tumor cells are multiple myeloma tumor cells.

30. Use of the protein according to any one of claims 1 to 26 or the preparation according to claim 27 in the preparation of a medicament for treating multiple myeloma.

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