Antibody variable domains targeting NKG2D receptor

Abstract

Antibody heavy chain variable domains are described, which can be paired with antibody light chain variable domains to form antigen binding sites that target the NKG2D receptor on natural killer cells. Also described are proteins comprising the NKG2D antigen binding sites, pharmaceutical compositions and methods of treatment thereof, including for the treatment of cancer.

Description

[0001] This application is a divisional application of a Chinese patent application with application number 201980012297.4, filing date February 8, 2019, and invention title "Antibody Variable Domains Targeting the NKG2D Receptor".

[0002] Cross - reference to related applications

[0003] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 628,161, filed February 8, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes; and U.S. Provisional Patent Application No. 62 / 716,259, filed August 8, 2018. Field of the invention

[0004] The present invention provides proteins having antibody heavy and light chain variable domains that can pair to form an antigen - binding site targeting the natural killer group 2D (NKG2D) receptor on natural killer cells, pharmaceutical compositions comprising such proteins, and methods of treatment using such proteins and pharmaceutical compositions, including for the treatment of cancer. Background art

[0005] Despite numerous research efforts and scientific advances reported in the literature for treating the disease, cancer remains a major health problem. Some of the most commonly diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Current treatment options for these cancers are not effective for all patients and / or may have severe adverse side effects. Treating other types of cancer with existing treatment options also remains challenging.

[0006] Cancer immunotherapies are desirable because they are highly specific and can harness the patient's own immune system to promote the destruction of cancer cells. Fusion proteins such as bispecific T - cell engagers are cancer immunotherapies described in the literature that bind to tumor cells and T cells to promote tumor cell destruction. 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.

[0007] Natural killer (NK) cells are components of the innate immune system and constitute approximately 15% of circulating lymphocytes. NK cells infiltrate almost all tissues and were initially characterized by their ability to efficiently kill tumor cells without the need for priming, which differentiates them from T cells. Activated NK cells kill target cells in a manner similar to cytotoxic T cells, i.e., through lytic granules containing perforin and granzymes and through the death receptor pathway. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other white blood cells to the target tissue.

[0008] NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by the activation of killer cell immunoglobulin-like receptors (KIR). Alternatively, when NK cells encounter cancer cells, they are activated through their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of some immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein that is expressed by essentially all natural killer cells, where NKG2D acts as an activating receptor. The ability to modulate NK cell function through NKG2D is useful in various therapeutic scenarios including malignancies. Summary of the Invention

[0009] Antibodies to NKG2D have been identified that have important advantages in the design of therapeutic agents. For example, some of these antibodies not only bind to the human NKG2D receptor but also have one or more other advantages such as the ability to agonize the receptor; the ability to compete with the natural ligand for binding to the receptor; and / or the ability to cross-react with NKG2D from other species (e.g., cynomolgus monkey). These advantages can be achieved within the affinity range for NKG2D.

[0010] Accordingly, in one aspect, the present invention relates to an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTS YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAPNYGDTTHDYYYMDVWGKGTTVTVSS (SEQ ID NO:1, ADI-29379). In some embodiments, the antibody heavy chain variable domain has at least 95% identity to SEQ ID NO:1. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTFTSYYMH (SEQ ID NO:11) of SEQ ID NO:1 as the first complementarity determining region 1 (“CDR1”), IINPSGGSTSYAQKFQG (SEQ ID NO:12) as the second CDR (“CDR2”), and ARGAPNYGDTTHDYYYMDV (SEQ ID NO:13) as the third CDR (“CDR3”). In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYYMH (SEQ IDNO:45) of SEQ ID NO:1 as CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO:12) as CDR2, and GAPNYGDTTHDYYYMDV (SEQ ID NO:68) as CDR3.

[0011] On the other hand, the present invention relates to an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDVWGQGTTVTVSS (SEQ ID NO:3, ADI-29463). In some embodiments, the antibody heavy chain variable domain has at least 95% identity to SEQ ID NO:3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTF TGYYMH (SEQ ID NO:17) of SEQ ID NO:3 as the first complementarity determining region (“CDR1”), WINPN SGGTNYAQKFQG (SEQ ID NO:18) as the second CDR (“CDR2”), and ARDTGEYYDTDDHGMDV (SEQ ID NO:19) as the third CDR (“CDR3”). In some embodiments, the heavy chain variable domain comprises the amino acid sequence GYYMH (SEQ ID NO:92) of SEQ ID NO:3 as CDR1, WINPNSGGTNYAQ KFQG (SEQ ID NO:18) as CDR2, and DTGEYYDTDDHGMDV (SEQ ID NO:69) as CDR3.

[0012] On the other hand, the present invention relates to an antibody heavy chain variable domain having at least 90% identity with the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSS (SEQ ID NO:5, ADI-27744). In some embodiments, the antibody heavy chain variable domain has at least 95% identity with SEQ ID NO:5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYAMS (SEQ ID NO:23) of SEQ ID NO:5 as the first complementarity determining region (“CDR1”), AISGSGGSTYYADSVKG (SEQ ID NO:24) as the second CDR (“CDR2”), and AKDGGYYDSGAGDY (SEQ ID NO:25) as the third CDR (“CDR3”). In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYAMS (SEQ ID NO:47) of SEQ ID NO:5 as CDR1, AISGSGGSTYYADSVKG (SEQ ID NO:24) as CDR2, and DGGYYDSGAGDY (SEQ ID NO:70) as CDR3.

[0013] On the other hand, the present invention relates to an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSS (SEQ ID NO:7, ADI-27749). In some embodiments, the antibody heavy chain variable domain has at least 95% identity to SEQ ID NO:7. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYSMN (SEQ ID NO:29) of SEQ ID NO:7 as the first complementarity determining region (“CDR1”), SISSSSSYIYYADSVKG (SEQ ID NO:30) as the second CDR (“CDR2”), and ARGAPMGA AAGWFDP (SEQ ID NO:31) as the third CDR (“CDR3”). In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYSMN (SEQ ID NO:48) of SEQ ID NO:7 as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO:30) as CDR2, and GAPMGAAAGWFDP (SEQ ID NO:71) as CDR3.

[0014] On the other hand, the present invention relates to an antibody heavy chain variable domain having at least 90% identity with the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGQGTLVTVSS (SEQ ID NO:85, A49MI). In some embodiments, the antibody heavy chain variable domain has at least 95% identity with SEQ ID NO:85. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYSMN (SEQ ID NO:29) of SEQ ID NO:85 as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO:30) as CDR2, and ARGAPIGAAAGWFDP (SEQ ID NO:77) as CDR3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYSMN (SEQ ID NO:48) of SEQ ID NO:85 as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO:30) as CDR2, and GAPIGAAAGWFDP (SEQ ID NO:78) as CDR3.

[0015] On the other hand, the present invention relates to an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAGFAYGMDYYYMDVWGKGTTVTVSS (SEQ ID NO:9, ADI-29378). In some embodiments, the antibody heavy chain variable domain has at least 95% identity to SEQ ID NO:9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTFTSYYMH (SEQ ID NO:35) of SEQ ID NO:9 as the first complementarity determining region (“CDR1”), IINPSGGSTSYAQKFQG (SEQ ID NO:36) as the second CDR (“CDR2”), and AREGAGFAYGMDYYYMDV (SEQ ID NO:37) as the third CDR (“CDR3”). In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYYMH (SEQ ID NO:45) of SEQ ID NO:9 as CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO:36) as CDR2, and EGAGFAYGMDYYYMDV (SEQ ID NO:72) as CDR3.

[0016] The variable domain of the heavy chain of the present invention can optionally be coupled to an amino acid sequence having at least 90% identity to an antibody constant region, such as an IgG constant region including a hinge, CH2, and CH3 domains and having or not having a CH1 domain. In some embodiments, the amino acid sequence of the constant region has at least 90% identity to 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 to an antibody constant region from another mammal (such as a rabbit, dog, cat, mouse, or horse). Compared to the human IgG1 constant region, the constant region can contain one or more mutations, such as those located at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0017] In certain embodiments, mutations that can be included in the CH1 of the human IgG1 constant region can be located at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be included in the Cκ of the human IgG1 constant region can be located at amino acids E123, F116, S176, V163, S174, and / or T164.

[0018] In some embodiments, one of the heavy chain variable domains described herein binds to a light chain variable domain to form an antigen-binding site capable of binding NKG2D. For example, a heavy chain variable domain of an antibody having at least 90% identity to the amino acid sequence of SEQ ID NO:1 can be paired with a light chain variable domain of an antibody having at least 90% identity to the amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQ SVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTL TISSLQSEDFAVYYCQQYDDWPFTFGGGTKVEIK (SEQ ID NO:2, ADI-29379). In some embodiments, the antibody light chain variable domain has at least 95% identity to SEQ ID NO:2. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQSVSSNL A (SEQ ID NO:14) as the first complementarity determining region (“CDR”), GASTRAT (SEQ ID NO:15) as the second CDR, and QQYDDWPFT (SEQ ID NO:16) as the third CDR.

[0019] For example, a heavy chain variable domain of an antibody having at least 90% identity to the amino acid sequence of SEQ ID NO:3 can be paired with a light chain variable domain of an antibody having at least 90% identity to the amino acid sequence EIVLTQSPGTLSLSPGERAT LSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGS GSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK (SE Q ID NO:4, ADI-29463). In some embodiments, the antibody light chain variable domain has at least 95% identity to SEQID NO:4. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQSVSSNLA (SEQ ID NO:20) as the first complementarity determining region (“CDR”), GASTRAT (SEQ ID NO:21) as the second CDR, and QQDDYWPPT (SEQ ID NO:22) as the third CDR.

[0020] For example, an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence of SEQ ID NO:5 can be paired with an antibody light chain variable domain having at least 90% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK (SEQ ID NO:6, ADI-27744). In some embodiments, the antibody light chain variable domain has at least 95% identity to SEQ ID NO:6. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQGIDSWLA (SEQ ID NO:26) as the first complementarity determining region (“CDR”), AASSLQS (SEQ ID NO:27) as the second CDR, and QQGVSYPRT (SEQ ID NO:28) as the third CDR.

[0021] For example, an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence of SEQ ID NO:7 or 85 can be paired with an antibody light chain variable domain having at least 90% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK (SEQ ID NO:8, ADI-27749). In some embodiments, the antibody light chain variable domain has at least 95% identity to SEQ ID NO:8. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQGISSWLA (SEQ ID NO:32) as the first complementarity determining region (“CDR”), AASSLQS (SEQ ID NO:33) as the second CDR, and QQGVSFPRT (SEQ ID NO:34) as the third CDR.

[0022] For example, an antibody heavy chain variable domain having at least 90% identity to the amino acid sequence of SEQ ID NO:9 can be paired with an antibody light chain variable domain having at least 90% identity to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSDNWPFTFGGGTKVEIK (SEQ ID NO:10, ADI-29378). In some embodiments, the antibody light chain variable domain has at least 95% identity to SEQ ID NO:10. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO:38) as the first complementarity determining region (“CDR”), DASNRAT (SEQ ID NO:39) as the second CDR, and QQSDNWPFT (SEQ ID NO:40) as the third CDR.

[0023] When the heavy chain variable domain binds to the light chain variable domain to form an antigen binding site capable of binding NKG2D, the antigen binding site can be included in a variety of structures, such as a typical antibody structure having two identical heavy chains and two identical light chains to form a pair of antigen binding sites capable of binding NKG2D; bispecific, trispecific, tetra-specific or other multi-specific antibodies; or smaller structures, such as scFv (where the heavy chain variable domain is linked to the light chain variable domain).

[0024] In some embodiments, any NKG2D antigen-binding site disclosed in the present invention is included in a protein that further includes a separate antigen-binding site that binds to a tumor-associated antigen, which can allow the protein to interact with NK cells and tumor cells simultaneously. Tumor-associated antigens can be, for example, CD33, HER2, EpCAM, CD2, CD3, CD8, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD37, CD38, CD40, CD45RO, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CSAp, CA-125, TAG-72, EFGR / ERBB1, IGF1R, HER2, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA, and IL-6, MAGE-A3, B7.1, B7.2, CTLA4 or PD1.

[0025] In some embodiments, any NKG2D antigen-binding site disclosed in the present invention is included in a protein that further contains a tumor-associated antigen site and a CD16-binding site. The CD16-binding site can be an additional antigen-binding site or an antibody constant region or a portion thereof, such as the IgG1 constant region (which can optionally include one or more mutations that affect, for example, effector activity or CD16-binding affinity).

[0026] Another aspect of the present invention provides a method of enhancing tumor cell death and treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of the protein described herein to treat cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Represents a multispecific binding protein comprising an NKG2D-binding domain (right arm), a tumor-associated antigen-binding domain (left arm), and an Fc domain or a portion thereof that binds CD16.

[0028] Figure 2 Represents a multispecific binding protein comprising an NKG2D-binding structure or a tumor-associated antigen-binding domain (either of which can be in scFv form) and an Fc domain or a portion thereof that binds CD16.

[0029] Figures 3A - 3Eis a profile of the NKG2D binding affinity of the NKG2D binding domain measured by surface plasmon resonance. Figure 3A is the NKG2D binding affinity of the NKG2D binding domain ADI-27744 measured by surface plasmon resonance; Figure 3B is the NKG2D binding affinity of the NKG2D binding domain ADI-29379 measured by surface plasmon resonance; Figure 3C is the NKG2D binding affinity of the NKG2D binding domain ADI-27749 measured by surface plasmon resonance; Figure 3D is the NKG2D binding affinity of the NKG2D binding domain ADI-29463 measured by surface plasmon resonance; and Figure 3E is the NKG2D binding affinity of the NKG2D binding domain ADI-29378 measured by surface plasmon resonance.

[0030] Figure 4A -H is a profile of the competitive NKG2D binding of the NKG2D binding domain ADI-27744 (A44) and ULBP6 or other NKG2D antibodies measured by surface plasmon resonance. Figure 4A Shows the profile of injecting an NKG2D monoclonal antibody containing ADI-27744 onto immobilized NKG2D and then injecting ULBP6. Figure 4B Shows the profile of injecting ULBP6 onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody containing ADI-27744. Figure 4C Shows the profile of injecting the MS monoclonal antibody onto immobilized NKG2D and then injecting ULBP6. Figure 4D Shows the profile of injecting MS onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody containing ADI-27744. Figure 4E Shows the profile of injecting 1D11 onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody containing ADI-27744. Figure 4F Shows the profile of injecting MAB139 onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody containing ADI-27744. Figure 4G Shows the profile of injecting an NKG2D monoclonal antibody containing ADI-27744 onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody (A49) containing ADI-27749; and Figure 4H Shows the profile of injecting an NKG2D monoclonal antibody containing ADI-27744 onto immobilized NKG2D and then injecting an NKG2D monoclonal antibody containing F47.

[0031] Figure 5 is a line graph showing the binding profile of TriNKET targeting CD33 to NKG2D expressed on EL4 cells.

[0032] Figure 6 is a line graph showing the binding profile of TriNKET targeting HER2 to NKG2D expressed on EL4 cells.

[0033] Figure 7 is a bar graph showing the binding profile of TriNKET targeting CD33 to CD33 expressed on Mv4-11 cells.

[0034] Figure 8 is a bar graph showing the binding profile of TriNKET targeting HER2 to HER2 expressed on 786-O cells.

[0035] Figure 9 is a bar graph showing the binding profile of TriNKET targeting HER2 to HER2 expressed on NCI-H661 cells.

[0036] Figure 10 is a bar graph showing the activation of human NK cells co-cultured with NCI-H661 cells expressing HER2 mediated by TriNKET targeting HER2.

[0037] Figure 11 is a bar graph showing the activation of human NK cells co-cultured with SkBr-3 cells expressing HER2 mediated by TriNKET targeting HER2.

[0038] Figure 12 is a bar graph showing the activation of human NK cells co-cultured with human AML Mv4-11 cells expressing CD33 mediated by TriNKET targeting CD33.

[0039] Figure 13 is a line graph showing the cytotoxicity of TriNKET targeting CD33 on resting NK cells against Molm-13 cancer cells expressing CD33.

[0040] Figure 14 is a line graph showing the cytotoxicity of TriNKET targeting CD33 on activated NK cells against Molm-13 cancer cells expressing CD33.

[0041] Figure 15 is a bar graph showing the cytotoxicity of TriNKET targeting HER2 on resting NK cells against 786-O cancer cells expressing HER2.

[0042] Figure 16Bar graph showing that HER2-targeted TriNKET renders activated NK cells cytotoxic to HER2-expressing 786-O cancer cells.

[0043] Figure 17 Represents TriNKET in the Triomab format, which is a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. The Triomab format is a heterodimeric construct containing 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0044] Figure 18 Represents TriNKET in the KiH common light chain (LC) format, which involves the knot-in-hole (KIH) technology. KiH is a heterodimer containing 2 Fabs that bind to target 1 and 2 and an Fc stabilized by heterodimerization mutations. The TriNKET in the KiH format may be a heterodimeric construct with 2 fabs that bind to target 1 and target 2, which contains 2 different heavy chains and a common light chain paired with the two HCs.

[0045] Figure 19 Represents TriNKET in the format of dual variable domain immunoglobulin (DVD-Ig TM ) which combines the target-binding domains of two monoclonal antibodies via a flexible naturally occurring linker and generates a tetravalent IgG-like molecule. DVD-Ig TM is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of the Fab targeting antigen 1. The construct contains a normal Fc.

[0046] Figure 20 Represents TriNKET in the orthogonal Fab interface (Ortho-Fab) format, which is a heterodimeric construct containing 2 Fabs that bind to target 1 and target 2 and are fused to the Fc. The orthogonal interface ensures LC-HC pairing. Heterodimerization is ensured by mutations in the Fc.

[0047] Figure 21 Represents TriNKET in the two-in-one Ig format.

[0048] Figure 22 Represents TriNKET in the ES format, which is a heterodimeric construct containing 2 different Fabs that bind to target 1 and target 2 and are fused to the Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0049] Figure 23TriNKET in the form of Fab-arm exchange: An antibody that generates a bispecific antibody by exchanging Fab arms through the exchange of a heavy chain and the linked light chain (half-molecule) with a heavy-light chain pair from another molecule. The Fab-arm exchange form (cFae) is a heterodimer that contains two Fabs that bind to target 1 and 2, and an Fc that is stabilized by a heterodimerization mutation.

[0050] Figure 24 TriNKET in the form of SEED body, which is a heterodimer that contains two Fabs that bind to target 1 and 2, and an Fc that is stabilized by a heterodimerization mutation.

[0051] Figure 25 TriNKET in the form of LuZ-Y, where a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer that contains two different scFabs that bind to target 1 and 2 and are fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

[0052] Figure 26 TriNKET in the form of Cov-X body.

[0053] Figures 27A - 27B TriNKET in the form of Kλ body, which is a heterodimer construct with two different Fabs fused to an Fc stabilized by a heterodimerization mutation: Fab1 targeting antigen 1 contains κLC, while the second Fab targeting antibody 2 contains λLC. Figure 27A An exemplary representation of one form of Kλ body; Figure 27B An exemplary representation of another Kλ body.

[0054] Figure 28 An Oasc-Fab heterodimer construct that includes a Fab that binds to target 1 fused to an Fc and a scFab that binds to target 2. Heterodimerization is ensured by a mutation in the Fc.

[0055] Figure 29 DuetMab, which is a heterodimer construct containing two different Fabs that bind to antigens 1 and 2 and an Fc stabilized by a heterodimerization mutation. Fabs 1 and 2 contain differential S-S bridges that ensure correct LC and HC pairing.

[0056] Figure 30 CrossmAb, which is a heterodimer construct with two different Fabs that bind to target 1 and 2 and are fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are exchanged, for example, CH1 is fused in tandem with VL, while CL is fused in tandem with VH.

[0057] Figure 31 is Fit-Ig, which is a homodimer construct in which the Fab that binds antigen 2 is fused to the N-terminus of the HC of the Fab that binds antigen 1. The construct contains wild-type Fc.

[0058] Figure 32 is a series of line graphs showing the binding of TriNKET A* and TriNKET A to human NKG2D by SPR assay. The upper graph represents kinetic fitting, while the lower graph represents steady-state affinity fitting.

[0059] Figure 33 is a line graph showing the potency of TriNKET A and TriNKET A* in mediating NK cell cytotoxicity against target cells.

[0060] Figure 34 is a line graph showing the potency of TriNKET A and TriNKET A* in mediating NK cell cytotoxicity against target cells. Detailed Description

[0061] The present invention provides an antibody heavy chain variable domain that can pair with an antibody light chain variable domain to form an antigen-binding site that targets the NKG2D receptor on natural killer cells, a protein comprising the NKG2D antigen-binding site, a pharmaceutical composition comprising such a protein, and a method of treatment for treating cancer using such a protein and pharmaceutical composition. Aspects of the present invention are set forth in the following sections; however, aspects of the present invention described in a particular section are not limited to any particular section.

[0062] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0063] Unless the context is inappropriate, the terms "a" and "an" as used herein mean "one or more" and include the plural.

[0064] 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., mouse, ape, horse, cow, pig, dog, cat, etc.), and more preferably include humans.

[0065] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy chain ("H") and the light chain ("L"). Three highly variable segments within the V regions of the heavy and light chains are referred to as "hypervariable regions," which are inserted between more conserved flanking segments called "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences that naturally occur between and adjacent to the hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to one another in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementary determining regions" or "CDRs." In certain animals such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, thereby providing a "single-domain antibody." The antigen-binding site can be present in a whole antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, which uses a peptide linker to join the heavy-chain variable domain to the light-chain variable domain, resulting in a single polypeptide.

[0066] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to achieve a beneficial or desired result. The effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treatment" includes any effect that results in the improvement of a disorder, disease, condition, etc., or the alleviation of its symptoms, e.g., alleviation, reduction, modulation, improvement, or elimination.

[0067] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier such that the composition is particularly suitable for diagnostic or therapeutic applications in vivo or in vitro.

[0068] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th ed., Mack Publ. Co., Easton, PA

[1975] .

[0069] Throughout the specification, when a composition is described as having, including, or containing a particular component, or when a process and method are described as having, including, or containing a particular step, it is contemplated that there are also compositions of the invention consisting essentially of or consisting of the components, and processes and methods according to the invention consisting essentially of or consisting of the processing steps.

[0070] Generally, unless otherwise indicated, percentages of compositions are by weight. Additionally, if a variable is not defined, the previous definition of the variable applies.

[0071] NKG2D antigen-binding site

[0072] The present invention provides antigen-binding sites that bind NKG2D, and antigen heavy chain variable domains that can be used to generate such antigen-binding sites.

[0073] Antibody heavy chain variable domains and light chain variable domains that pair to form antigen-binding sites capable of binding and activating the NKG2D receptor have now been identified and are provided in Table 1 below. Unless otherwise indicated, the CDR sequences provided in Table 1 were determined according to Kabat.

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] An advantage of one or more of the above antibody heavy chain variable domain amino acid sequences is that they can bind to NKG2D from humans and cynomolgus monkeys to activate the receptor and compete with natural ligands for binding to the receptor. Other antigen-binding sites that bind NKG2D and share one or more of these properties are also particularly useful and can be identified by binding competition assays known in the art. For example, additional antigen-binding sites can be identified by competing with ADI-29379, ADI-29463, ADI-27744, ADI-27749, or ADI-29378 for binding to human and optionally cynomolgus monkey NKG2D.

[0081] Another advantage of the NKG2D binding sites comprising the above antibody heavy chain variable domain and light chain variable domain sequences is that they can bind NKG2D with high affinity. In some embodiments, the NKG2D binding site binds NKG2D with a K of 0.1 to 1000 nM. D The NKG2D binding site binds NKG2D with a K of 1 to 500 nM. D The NKG2D binding site binds NKG2D with a K of 5 to 100 nM. D The NKG2D binding site binds NKG2D with a K of 10 to 62 nM. D The NKG2D binding site binds NKG2D.

[0082] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:1, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:2. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:1 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:91, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:12, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:13 or 68. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:2 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:14, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:15, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:16.

[0083] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:3, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:4. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:3 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:92, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:18, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:69. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:4 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:20, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:21, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:22.

[0084] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:6. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:93, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:24, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:70. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:6 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:26, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:27, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:28.

[0085] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:7, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:7 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:71. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:34.

[0086] The amino acid residue M at position 102 of SEQ ID NO:7 in the CDR3 of the heavy chain variable domain may be mutated. In certain embodiments, M102 is replaced with an uncharged residue. In certain embodiments, M102 is replaced with a hydrophobic residue (Gly, Ala, Val, Leu, Ile, Pro, Phe or Trp). In certain embodiments, M102 is replaced with a polar residue (Ser, Thr, Cys, Asn, Gln or Tyr). In certain embodiments, M102 is replaced with Leu, Ile, Val, Gln or Phe.

[0087] Accordingly, in certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 83, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 83 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

[0088] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 84, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 84 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

[0089] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 85, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 85 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

[0090] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 86, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 86 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 79 or 80. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

[0091] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 41, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 41 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

[0092] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:9, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:10. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:9 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:91, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:36, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:72. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:10 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO:38, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:39, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO:40.

[0093] In certain embodiments, the present invention provides an antigen-binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the antibody heavy chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5, and the antibody light chain variable domain comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:6, and the antigen-binding site does not block the binding of anti-NKG2D antibodies MS, 1D11 and MAB139 to NKG2D.

[0094] In an embodiment, an antigen-binding site comprising an antibody heavy chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 7, 83, 84, 85, 86 or 87) and an antibody light chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 8) does not block the binding of an antigen-binding site comprising an antibody heavy chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 5) and an antibody light chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 6) to NKG2D.

[0095] In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 5) and an antibody light chain variable domain (which comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%)) identity to the amino acid sequence of SEQ ID NO: 6) binds to a unique epitope on NKG2D that is different from the MS, 1D11, MAB139, ADI-27749 and F47 binding epitopes.

[0096] Antibodies and multispecific binding proteins

[0097] In some embodiments of the invention, the NKG2D antigen-binding site formed by pairing the heavy chain variable domain of the antibody described herein with the light chain variable domain can be included in a larger protein, such as a full antibody, a multispecific binding protein, or a multispecific binding antibody. For example, the NKG2D binding site can be combined with a second component, such as a second antigen-binding site. In some embodiments, the second antigen-binding site binds one or more tumor-associated antigens, such as CD33, HER2, EpCAM, CD2, CD3, CD8, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD45RO, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CSAp, CA-125, TAG-72, EFGR / ERBB1, IGF1R, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA, and IL-6, MAGE-A3, B7.1, B7.2, CTLA4, or PD1. The binding of the multispecific protein to NKG2D and to the tumor-associated antigens on cancer cells brings the cancer cells close to natural killer cells, which promotes the direct or indirect destruction of cancer cells by natural killer cells.

[0098] In some embodiments, in addition to the NKG2D binding site and the tumor-associated antigen binding site, the multispecific binding protein may further include a domain that binds to CD16, which is an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. In some embodiments, the CD16 binding domain may include an antibody Fc region or a portion thereof. In some embodiments, the domain that binds to CD16 comprises the hinge, CH2, and CH3 domains of the antibody Fc region with or without the CH1 domain. In some embodiments, the antibody Fc region is derived from the Fc region of human and / or other mammalian immunoglobulins. It is known that within the Fc region, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mediated by the amino acid residues Asp265 - Glu 269, Asn297 - 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). In some embodiments, based on the known domains and amino acid residues, mutations can be selected within the CD16 binding domain to enhance or reduce its binding affinity to CD16. The selection methods are methods well known in the art, such as phage display libraries or yeast surface display cDNA libraries. Appropriate selection methods can also be designed by those skilled in the art based on the known three-dimensional structure of the interaction.

[0099] The multispecific binding proteins described herein can take several forms. For example, one form is a heterodimeric, multispecific antibody that includes a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain and the first immunoglobulin heavy chain together form an antigen binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain includes a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain and the second immunoglobulin heavy chain together form an antigen binding site that binds to a tumor antigen. The first Fc domain and the second Fc domain together are capable of binding CD16( Figure 1 ).

[0100] Another exemplary form relates to heterodimeric, multispecific antibodies that include a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain fused via a linker or antibody hinge to a single-chain variable fragment (scFv) composed of a heavy-chain variable domain and a light-chain variable domain that pair and bind to NKG2D. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy-chain variable domain, and an optional CH1 heavy-chain domain. The immunoglobulin light chain includes a light-chain variable domain and a constant light-chain domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to a tumor-associated antigen. The first Fc domain and the second Fc domain together are capable of binding CD16( Figure 2 ). By combining the various forms of the NKG2D-binding fragment described herein, other forms of multispecific binding proteins can be designed.

[0101] One or more additional binding motifs can optionally be fused via a linker sequence to the C-terminus of the constant region CH3 domain. In certain embodiments, the antigen-binding site can be a single-chain or disulfide-stabilized variable region (scFv) or can form a tetravalent or trivalent molecule.

[0102] In some embodiments, the multispecific binding protein is in the Triomab form, which is a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. The Triomab form is a heterodimeric construct that contains 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0103] In some embodiments, the multispecific binding protein is in the KiH common light chain (LC) form, which involves the knot-in-hole (KIH) technology. KIH involves engineering the C H 3 domain to create a "knot" or "hole" in each heavy chain to facilitate heterodimerization. The concept behind the "knot-in-hole (KiH)" Fc technology is to introduce a "knot" in one CH3 domain (CH3A) by replacing a small residue with a large residue (i.e., T366W in EU numbering CH3A ). To accommodate the "knot", a smaller residue (i.e., T366S / L368A / Y407V CH3B)Replace the residues closest to the knot to create a complementary "hole" surface on another CH3 domain (CH3B). Optimize the "hole" mutations by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol (1997) 270(1):26–35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) 426(9):1947–57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol Immunol (2014) 58(1):132–8) show that hydrophobic interactions driven by spatial complementarity at the interface between CH3 domain cores thermodynamically favor heterodimerization, while knob-knob and hole-hole interfaces disfavor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0104] In some embodiments, the multispecific binding protein is a dual variable domain immunoglobulin (DVD-Ig TM ) form that combines the target-binding domains of two monoclonal antibodies via a flexible naturally occurring linker and generates a tetravalent IgG-like molecule. DVD-Ig TMIt is a homodimer construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of the Fab targeting antigen 1. The construct contains a normal Fc.

[0105] In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab), which is a heterodimer construct containing two Fabs that bind to target 1 and target 2 and are fused to the Fc. LC-HC pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc. In the ortho-Fab IgG method (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191–8), structure-based regional design introduces complementary mutations only at the LC and HC VH-CH1 interface in one Fab, without making any changes to the other Fab.

[0106] In some embodiments, the multispecific binding protein is in the form of a tandem Ig. In some embodiments, the multispecific binding protein is in the ES form, which is a heterodimer construct containing two Fabs that bind to target 1 and target 2 and are fused to the Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0107] In some embodiments, the multispecific binding protein is in the form of a Kappa body, which is a heterodimer construct having two different Fabs fused to the Fc stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains kappa LC, and the second Fab targeting antigen 2 contains lambda LC.

[0108] In some embodiments, the multispecific binding protein is in the form of Fab arm exchange (antibodies that generate bispecific antibodies by exchanging the heavy chain and the linked light chain (half molecule) with a heavy-light chain pair from another molecule). The Fab arm exchange form (cFae) is a heterodimer that contains two Fabs that bind to targets 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0109] In some embodiments, the multispecific binding protein is in the form of a SEED body, which is a heterodimer comprising two Fabs that bind to targets 1 and 2, and an Fc stabilized by heterodimerization mutations. The Strand Exchange Engineered Domain (SEED) platform is designed to generate asymmetric and bispecific antibody-like molecules, and this ability extends the therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins within the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while disfavoring the homodimerization of the AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

[0110] In some embodiments, the multispecific binding protein is in the LuZ-Y form, where a leucine zipper is used to induce the heterodimerization of two different HCs. (Wranik, B.J. et al., J. Biol. Chem. (2012), 287:43331-9). The LuZ-Y form is a heterodimer that comprises two different scFabs that bind to targets 1 and 2 and are fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

[0111] In some embodiments, the multispecific binding protein is in the Cov-X body form (in a bispecific CovX body, two different peptides are linked together using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. Although the pharmacophore is responsible for the functional activity, the antibody scaffold confers a long half-life and an Ig-like distribution. The pharmacophore can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. (Doppalapudi V.R. et al., PNAS (2010), 107(52); 22611-22616).

[0112] In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer, which comprises a Fab that binds to target 1 and an scFab that binds to target 2, both fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0113] In some embodiments, the multispecific binding protein is in the DuetMab form, which is a heterodimer construct comprising two different Fabs that bind to antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure proper LC and HC pairing.

[0114] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a heterodimer construct having two different Fabs that bind to targets 1 and 2 and are fused to the Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are exchanged, for example, CH1 is fused in tandem with VL, and CL is fused in tandem with VH.

[0115] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a homodimer construct in which the Fab that binds antigen 2 is fused to the N-terminus of the HC of the Fab that binds antigen 1. The construct comprises wild type.

[0116] Heterodimeric antibody heavy chain

[0117] Assembly of the heterodimeric antibody heavy chain can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. Preferential assembly of the heterodimeric heavy chain within the multispecific binding proteins described herein can be facilitated by incorporating different pairs of amino acid substitutions into the first CH3 domain within the first heavy chain polypeptide and the second CH3 domain within the second heavy chain polypeptide such that the two chains selectively heterodimerize with each other, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, US14 / 830336. In some embodiments, the multispecific binding protein comprises the Fc domain of human IgG1. The following lists various examples of amino acid substitutions within the human IgG1 Fc domain pair to facilitate heterodimerization of the two heavy chains. Each position of the amino acid substitution is numbered according to the EU index in Kabat.

[0118] 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), such that the larger amino acid substitution (protrusion) fits into the surface (cavity) of the smaller amino acid substitution. For example, one polypeptide can include the T366W substitution, while the other can include three substitutions, including T366S, L368A, and Y407V.

[0119] Alternatively, the amino acid substitutions can be selected from the following groups of substitutions shown in Table 2.

[0120] Table 2 First polypeptide Second polypeptide Group 1 S364E / F405A Y349K / T394F Group 2 S364H / D401K Y349T / T411E Group 3 S364H / T394F Y349T / F405A Group 4 S364E / T394F Y349K / F405A Group 5 S364E / T411E Y349K / D401K Group 6 S364D / T394F Y349K / F405A Group 7 S364H / F405A Y349T / T394F Group 8 S364K / E357Q L368D / K370S Group 9 L368D / K370S S364K Group 10 L368E / K370S S364K Group 11 K360E / Q362E D401K Group 12 L368D / K370S S364K / E357L Group 13 K370S S364K / E357Q Group 14 F405L K409R Group 15 K409R F405L

[0121] Alternatively, the amino acid substitution can be selected from the following substitution groups shown in Table 3.

[0122] Table 3 First polypeptide Second polypeptide Group 1 K409W D399V / F405T Group 2 Y349S E357W Group 3 K360E Q347R Group 4 K360E / K409W Q347R / D399V / F405T Group 5 Q347E / K360E / K409W Q347R / D399V / F405T Group 6 Y349S / K409W E357W / D399V / F405T

[0123] Alternatively, the amino acid substitution can be selected from the following substitution groups shown in Table 4.

[0124] Table 4 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

[0125] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 5.

[0126]

[0127] Alternatively, at least one amino acid substitution can be selected from the following substitution groups in Table 6, wherein the positions shown in the first polypeptide column are replaced by any known negatively charged amino acid, and the positions shown in the second polypeptide column are replaced by any known positively charged amino acid.

[0128]

[0129] Alternatively, at least one amino acid substitution can be selected from the following groups in Table 7, wherein the positions shown in the first polypeptide column are replaced by any known positively charged amino acid, and the positions shown in the second polypeptide column are replaced by any known negatively charged amino acid.

[0130] Table 7 First polypeptide Second polypeptide D399, E356 or E357 K409, K439, K370 or K392

[0131] Alternatively, the amino acid substitution can be selected from the following groups in Table 8.

[0132]

[0133] Alternatively, or additionally, the structural stability of the heterodimeric heavy chain within the multispecific binding protein can be increased by forming an artificial disulfide bond within the interface of the two polypeptides by introducing S354C on either the first or second polypeptide chain and Y349C on the opposite polypeptide chain.

[0134] The above-mentioned multispecific binding proteins can be prepared using recombinant DNA techniques well-known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding a first immunoglobulin light chain can be cloned into a third expression vector; a fourth nucleic acid sequence encoding a second immunoglobulin light chain can be cloned into a fourth expression vector; and the first, second, third, and fourth expression vectors can be stably co-transfected into a host cell to produce a multimeric protein.

[0135] To obtain the highest yield of the multispecific binding protein, different ratios of the first, second, third, and fourth expression vectors can be explored to determine the optimal ratio for transfection into the host cell. After transfection, individual clones can be isolated using methods known in the art (such as limiting dilution, ELISA, FACS, microscopy, or Clonepix) for generating a cell bank.

[0136] The clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of the expression of the multispecific protein. The multispecific binding protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0137] A protein comprising an antigen-binding site that competes with the NKG2D-binding site described herein

[0138] In certain embodiments, the present invention provides a protein that includes an antigen-binding site that competes with the NKG2D-binding site described herein for binding to NKG2D. The NKG2D-binding sites described herein include the amino acid sequences of SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 5 and 6; SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10; SEQ ID NO: 83 and 8; SEQ ID NO: 84 and 8; SEQ ID NO: 85 and 8; SEQ ID NO: 86 and 8; or SEQ ID NO: 87 and 8. These NKG2D-binding sites can bind to different epitopes on NKG2D that are calibrated by surface plasmon resonance. For example, ADI-27744 binds to an epitope on NKG2D that is different from ADI-27749 and other existing NKG2D antibodies, as shown in Example 2.

[0139] In some embodiments, the antigen-binding site of a protein that competes with the NKG2D-binding site comprises a heavy-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:1 and a light-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antigen-binding site of a protein that competes with the NKG2D-binding site comprises a heavy-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:3 and a light-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the antigen-binding site of a protein that competes with the NKG2D-binding site comprises a heavy-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5 and a light-chain variable domain having an amino acid sequence with at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:6.In some embodiments, the antigen-binding site of a protein that competes with the NKG2D-binding site comprises a heavy chain variable domain having an amino acid sequence with at least 50% (such as 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:7 and a light chain variable domain having an amino acid sequence with at least 50% (such as 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the antigen-binding site of a protein that competes with the NKG2D-binding site comprises a heavy chain variable domain having an amino acid sequence with at least 50% (such as 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:9 and a light chain variable domain having an amino acid sequence with at least 50% (such as 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:10.

[0140] In some embodiments, a protein comprising an antigen-binding site that competes with the NKG2D-binding site described herein further comprises a second antigen-binding site that binds a tumor-associated antigen and / or a CD16-binding site. In some embodiments, the CD16-binding site is an antibody constant region or a portion thereof that is capable of binding CD16. In some embodiments, the CD16-binding site comprises a human IgG1 Fc domain.

[0141] Cells expressing the protein

[0142] In one aspect, the present disclosure provides a cell comprising one or more nucleic acids encoding a protein, the protein comprising: an NKG2D binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 1, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 2; an NKG2D binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 3, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 4; an NKG2D binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 5, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 6; an NKG2D binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 7, 83, 84, 85, 86 or 87, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 8;or an NKG2D binding site having a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 9, and the light chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 10.;

[0143] Therapeutic applications

[0144] The present invention provides methods of using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein to enhance tumor cell death and / or treat cancer. The methods can be used to treat a variety of cancers. Desirably, the type of cancer to be treated matches the type of cancer cell to which the protein binds. Other aspects and embodiments of the treatment methods are described below.

[0145] Pharmaceutical composition

[0146] In one aspect, the present disclosure also relates to a pharmaceutical composition comprising an effective amount of a protein and a pharmaceutically acceptable carrier, wherein the protein comprises an NKG2D binding site described herein or an NKG2D binding site that competes with the NKG2D binding site described herein.

[0147] In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:1, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:2. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:3, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:4. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:6. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86 or 87, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:8.In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:9, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:10.

[0148] The compositions can be formulated for a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can be included in the compositions for proper formulation. Suitable formulations for the present disclosure are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th Edition, 1985. For a brief review of drug delivery methods, see, e.g., Langer (Science 249:1527-1533, 1990).

[0149] For example, the present disclosure may exist as an aqueous pharmaceutical formulation that includes a therapeutically effective amount of a protein in a buffer solution that forms the formulation. The aqueous carrier may include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffer solution (such as phosphate buffered saline), a sterile saline solution, Ringer's solution, or a dextrose solution. In certain embodiments, an aqueous formulation is prepared that contains the proteins disclosed herein in a pH buffer solution. The pH of the formulation is generally from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 7 to 8, such as from 7 to 7.5. Intermediate ranges of the above pH values are also intended to be part of the present disclosure. For example, ranges of values using combinations of any of the above values as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetates (such as sodium acetate), succinates (such as sodium succinate), gluconates, histidine, citrates, and other organic acid buffers. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system contains about 1.3 mg / ml of citric acid (such as 1.305 mg / ml), about 0.3 mg / ml of sodium citrate (such as 0.305 mg / ml), about 1.5 mg / ml of disodium phosphate dihydrate (such as 1.53 mg / ml), about 0.9 mg / ml of sodium dihydrogen phosphate dihydrate (such as 0.86), and about 6.2 mg / ml of sodium chloride (such as 6.165 mg / ml). In certain embodiments, the buffer system includes 1 - 1.5 mg / ml of citric acid, 0.25 to 0.5 mg / ml of sodium citrate, 1.25 to 1.75 mg / ml of disodium phosphate dihydrate, 0.7 to 1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / ml of sodium chloride. The pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0150] In some embodiments, the formulation includes an aqueous carrier that is pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare a liquid formulation. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffer solution (such as phosphate buffered saline), a sterile saline solution, Ringer's solution, or a dextrose solution.

[0151] The formulation may also include polyols, which can be used as tonicity agents and can stabilize the antibody. The polyol is added to the formulation in an amount that can vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added can also vary depending on the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) can be added compared to a disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the mannitol concentration can be from about 5 to about 20 mg / ml. In certain embodiments, the concentration of mannitol can be from about 7.5 to 15 mg / ml. In certain embodiments, the concentration of mannitol can be from about 10 - 14 mg / ml. In certain embodiments, the concentration of mannitol can be about 12 mg / ml. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0152] A detergent or surfactant can also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added should be such that it reduces aggregation of the formulated antibody and / or minimizes particle formation in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant, which is a polysorbate. In certain embodiments, the formulation may include the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996). In certain embodiments, the formulation may include from about 0.1 mg / mL to about 10 mg / mL of polysorbate 80, or from about 0.5 mg / mL to about 5 mg / mL. In certain embodiments, about 0.1% of polysorbate 80 can be added to the formulation.

[0153] In certain embodiments, the liquid formulation of the present disclosure can be combined with a stabilizing level of sugar to make a solution at a concentration of 10 mg / mL. In certain embodiments, the liquid formulation can be prepared in an aqueous carrier. In certain embodiments, the stabilizer can be added in an amount that does not result in a viscosity that is undesirable or not suitable for intravenous administration. In certain embodiments, the sugar can be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may further include one or more of a buffer, a surfactant, and a preservative, which are added to the formulations herein to reduce bacterial action. Adding a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0154] In some embodiments, the present disclosure provides a formulation having an extended shelf life, which comprises a protein of the present disclosure, as well as mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0155] Deamidation is a common product variant of peptides and proteins, which may occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from a protein, forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17 Da decrease in the mass of the parent peptide. Subsequent hydrolysis results in a 18 u increase in mass. The succinimide intermediate is difficult to isolate due to its instability in aqueous conditions. Therefore, deamidation is typically detectable as a 1 u increase in mass. Deamidation of asparagine yields aspartic acid or isoaspartic acid. Parameters affecting the deamidation rate include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser following Asn in the protein sequence result in higher sensitivity to deamidation. In certain embodiments, the liquid formulations of the present disclosure can be stored under conditions of pH and humidity that prevent deamination of the protein product.

[0156] In some embodiments, the formulation is a lyophilized formulation. In certain embodiments, the formulation is lyophilized (freeze-dried) and contained in about 12 - 60 vials. In certain embodiments, the formulation is lyophilized, and one vial can contain 45 mg of the lyophilized formulation. In certain embodiments, about 40 mg to about 100 mg of the lyophilized formulation is contained in one vial. In certain embodiments, the lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the protein in an intravenous drug formulation. The formulation can be a liquid formulation. In some embodiments, the liquid formulation is stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the liquid formulation is stored at about 600 mg / vial. In certain embodiments, the liquid formulation is stored at about 250 mg / vial.

[0157] In some embodiments, the lyophilized formulation comprises the protein described herein and a lyoprotectant. The lyoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant can be sucrose or maltose. The lyophilized formulation can also comprise one or more of a buffer, a surfactant, a filler, and / or a preservative. The amount of sucrose or maltose used to stabilize the lyophilized drug product can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

[0158] In certain embodiments, the pH of the formulation can be set by adding a pharmaceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present disclosure can be adjusted between 6 and 8. In certain embodiments, the pH range of the lyophilized drug product can be from 7 to 8.

[0159] In certain embodiments, a "filler" can be added. A "filler" is a compound that increases the mass of the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of a substantially uniform lyophilized cake that maintains an open pore structure). Exemplary fillers include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention can contain such fillers.

[0160] In certain embodiments, the lyophilized protein product can be formulated with an aqueous carrier. The aqueous carriers of interest herein are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and can be used to prepare liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solutions. In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with sterile water for injection USP (SWFI) or 0.9% sodium chloride injection USP. During reconstitution, the lyophilized powder dissolves into a solution. In certain embodiments, the lyophilized protein product of the present disclosure is formulated into approximately 4.5 mL of water for injection and diluted with a 0.9% saline solution (sodium chloride solution).

[0161] The protein composition can be sterilized by conventional sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized formulation can be combined with a sterile aqueous carrier prior to administration. The resulting composition in solid form can be packaged in multiple single-dose units, each unit containing a fixed amount of one or more of the above-mentioned agents. The composition in solid form can also be packaged in a container for flexible dosing.

[0162] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient.

[0163] For each patient, a specific dose can be a uniform dose, such as 50 - 5000 mg of protein. Alternatively, the dose for a patient can be adjusted according to the patient's approximate body weight or surface area. Other factors in determining a suitable dose can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine a suitable therapeutic dose is routine for those skilled in the art, particularly in light of the dose information and assays disclosed herein. The dose can also be determined by using known assays for determining dose in combination with appropriate dose - response data. The dose of an individual patient can be adjusted while monitoring disease progression. The blood level of the targetable construct or complex in the patient can be measured to see if the dose needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable construct and / or complex and its dose 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).

[0164] Typically, the dose based on body weight is from about 0.01 μg to about 100 mg per kg body weight, such as about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0.1 μg / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 100 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight. The dose can be administered once or multiple times per day, per week, per month, or per year, or even once every 2 to 20 years. One of ordinary skill 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, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion, or by direct injection into the lesion. It can be administered once or multiple times per day, once or multiple times per week, once or multiple times per month, or once or multiple times per year.

[0165] Enhanced Tumor Cell Death and Cancer Therapy

[0166] The present invention provides methods for enhancing patient tumor cell death and / or treating cancer. In some embodiments, the methods include exposing tumors and natural killer cells to the multispecific binding proteins disclosed herein. In some embodiments, the methods include administering to a patient in need thereof a therapeutically effective amount of the proteins described herein and / or their required formulations. In those embodiments, the multispecific binding protein may comprise: an antigen-binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:1, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:2; an antigen-binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:3, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:4; an antigen-binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:5, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:6; an antigen-binding site having a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86 or 87, and the light chain variable domain having an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:8;or an antigen-binding site having a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:9, and the light-chain variable domain has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO:10;

[0167] Ideally, the type of cancer to be treated matches the type of cancer cells bound by the multispecific binding proteins disclosed herein. For example, ideally, a multispecific binding protein that binds to EpCAM and NKG2D as described herein is used to treat cancers that express epithelial cell adhesion molecule (EpCAM), such as colon cancer that expresses EpCAM.

[0168] In some embodiments, the patient to be treated has cancer cells expressing one or more of the following: CD33, HER2, CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, CEA, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD1. In some embodiments, the patient to be treated has solid cancer, such as brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer.In still other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (such as angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytoma, Bartholin gland adenocarcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, 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, orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell carcinoma, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningiocarcinoma, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglioma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyoid cancer, submesothelial cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine body cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulvar cancer, well-differentiated carcinoma or Wilms' tumor.

[0169] In some embodiments, the patient to be treated has non-Hodgkin lymphoma, such as B-cell lymphoma or T-cell lymphoma. In certain embodiments, the non-Hodgkin lymphoma is 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, nodal 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 certain other embodiments, the non-Hodgkin lymphoma is 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.

[0170] In some embodiments, the proteins described herein are used in combination with other therapeutic agents to treat cancer patients. Exemplary therapeutic agents that can be used as part of a combination therapy for treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine,Lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ, colony stimulating factor-1, colony stimulating factor-2, denileukindiftitox, interleukin-2, luteinizing hormone releasing factor, and various variants of the above agents that may exhibit different binding to their cognate receptors and increased or decreased serum half-lives.

[0171] Another class of agents that can be used as part of a combination therapy for treating cancer are immune checkpoint inhibitors. 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 US Food and Drug Administration for the treatment of melanoma.

[0172] Other agents that can be used as part of a combination therapy for treating cancer are monoclonal antibody agents that target non-checkpoint targets (such as herceptin) and non-cytotoxic agents (such as tyrosine kinase inhibitors).

[0173] Other classes of anti-cancer agents include, for example: (i) inhibitors selected from the group consisting of 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, DNA-PK and mTOR inhibitors, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of 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 the group consisting of IL-12, IL-15, GM-CSF, and G-CSF.

[0174] The protein of the present invention can also be used as an adjunct to surgical resection of the primary lesion.

[0175] The amounts of the protein and the additional therapeutic agent, as well as the relative timing of administration, can be selected to achieve the desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need thereof, the therapeutic agents in the combination or the one or more pharmaceutical compositions comprising the therapeutic agents can be administered in any order, such as sequentially, jointly, together, simultaneously, etc. In addition, for example, the protein described herein can be administered during the period when the additional therapeutic agent exerts its prophylactic or therapeutic effect, and vice versa.

[0176] The foregoing description has described various aspects and embodiments of the present invention. The patent application specifically encompasses all combinations and permutations of the aspects and embodiments.

[0177] Examples

[0178] The present invention will now be more readily understood by reference to the following examples, which are for the purpose of illustrating certain aspects and embodiments of the present invention only and are not intended to limit the present invention.

[0179] Example 1 - Binding Affinities of Various NKG2D Binding Domains

[0180] The kinetics and affinity of various NKG2D binding domains were evaluated by surface plasmon resonance using a Biacore 8K instrument (GE Healthcare). Anti-human Fc antibody was immobilized on a CM5 chip using standard amine coupling chemistry. Human monoclonal antibodies containing various NKG2D binding domains were captured on the anti-human Fc chip at a density of approximately 100 RU. Solutions containing 0.411 - 100 nM soluble mouse Fc-human NKG2D dimer were injected over the captured NKG2D antibodies and control surfaces at 37 °C at 30 μl / min. The surface was regenerated between cycles by rapid injection of 10 mM glycine pH 1.8. To obtain the kinetic rate constants, the dual-reference data were fitted to a 1:1 interaction model using Biacore 8K evaluation software (GE Healthcare). The equilibrium binding constant K D is determined from the dissociation constant k d and the association constant k a by the ratio (k d / k a ). As shown in Table 9 and Figure 3, the binding affinity of the NKG2D binding domains for NKG2D ranges from 10 - 62 nM.

[0181]

[0182] Example 2 - Binding epitope clustering of ADI-27744 clone

[0183] Clustering of the ADI-27744 (A44) NKG2D binding domain was performed for a series of antibodies and ULBP6 (a natural ligand of NKG2D) by surface plasmon resonance using a Biacore 8K instrument. Briefly, mouse Fc-human NKG2D was captured at a density of approximately 100 RU using anti-mouse Fc antibody immobilized on a CM5 chip. Subsequently, antibodies were continuously injected at 25 °C at 30 μl / min, including NKG2D monoclonal antibodies containing ADI-27744, ADI-27749, F47 (sequences listed below), or 1D11 (a commercial monoclonal NKG2D antibody), ULBP6 (sequences listed below), MS (an NKG2D antibody from Novo Nordisk, sequences listed below), and MAB139 (an NKG2D antibody from R&D system, clone 149810). Biacore 8K evaluation software was used for all data analysis.

[0184] Table 10

[0185]

[0186]

[0187] ULBP amino acid sequence SEQ ID NO:67:

[0188]

[0189] Figure 4A Shows the profile of injecting an NKG2D monoclonal antibody containing ADI - 27744 onto immobilized NKG2D, and then injecting ULBP6. Figure 4B Shows the profile of injecting ULBP6 onto immobilized NKG2D, and then injecting an NKG2D monoclonal antibody containing ADI - 27744. These results indicate that the NKG2D monoclonal antibody including the antigen - binding site of ADI - 27744 does not block the binding of ULBP6 to NKG2D, that is, ADI - 27744 binds to an epitope on NBP2D different from ULBP6.

[0190] Figure 4C Shows the profile of injecting the MS monoclonal antibody onto immobilized NKG2D, and then injecting ULBP6. The MS monoclonal antibody blocks the binding of ULBP6 to NKG2D. Figure 4D -F shows the profile of injecting MS, 1D11 or MAB139 onto immobilized NKG2D, and then injecting an NKG2D monoclonal antibody containing ADI - 27744. Figure 4G -H shows the profile of injecting an NKG2D monoclonal antibody containing ADI - 27744 onto immobilized NKG2D, and then injecting an NKG2D monoclonal antibody containing ADI - 27749 or F47. ADI - 27744 does not block the binding of MS, 1D11 and MAB139 to NKG2D. ADI - 27749 and F47 do not block the binding of ADI - 27744 to NKG2D. These results indicate that ADI - 27744 binds to a unique epitope on NKG2D different from the binding epitopes of MS, 1D11, MAB139, ADI - 27749 and F47.

[0191] Example 3 - Trispecific binding protein binds to NKG2D

[0192] The EL4 mouse lymphoma cell line was engineered to express human NKG2D. Detection was performed as Figure 1The affinity of each trispecific binding protein (TriNKET) containing an NKG2D binding domain, a tumor-associated antigen binding domain (such as a CD33 or HER2 binding domain), and an Fc domain that binds CD16 to extracellular NKG2D expressed on EL4 cells was determined. Binding of the multispecific binding protein to NKG2D was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the background fold (FOB) was calculated using the mean fluorescence intensity (MFI) of cells expressing NKG2D compared to parental EL4 cells.

[0193] The TriNKETs tested included CD33-TriNKET-A44 (ADI-27744 and CD33 binding domain), CD33-TriNKET-A49 (ADI-27749 and CD33 binding domain), CD33-TriNKET-F63 (ADI-29463 and CD33 binding domain), HER2-TriNKET-A44 (ADI-27744 and CD33 binding domain), HER2-TriNKET-A49 (ADI-27749 and HER2 binding domain), HER2-TriNKET-F63 (ADI-29463 and HER binding domain), and HER2-TriNKET-E79 (ADI-29379 and HER2 binding domain). The HER2 binding domain consists of the heavy chain variable domain and the light chain variable domain of trastuzumab. The CD33 binding domain consists of the heavy chain variable domain and the light chain variable domain listed below.

[0194] SEQ ID NO:49

[0195]

[0196] SEQ ID NO:50

[0197]

[0198] All TriNKETs bound to NKG2D on EL4 cells, but with different affinities. CD33-TriNKET-A44 showed the same binding profile as HER2-TriNKET-A44, CD33-TriNKET-A49 did so for HER2-TriNKET-A49, and CD33-TriNKET-F63 did the same for HER2-TriNKET-F63. The NKG2D binding affinity of each clone was similar between cells expressing human and mouse NKG2D ( Figures 5 - 6 ).

[0199] Example 4 - Binding of Trispecific Binding Proteins to Human Tumor Antigens

[0200] The trispecific binding protein binds to CD33

[0201] The human AML cell line MV4-11 expressing CD33 was used to determine the binding of TriNKET to tumor-associated antigens. TriNKET and the parental CD33 monoclonal antibody were incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and the background fold (FOB) was calculated using the mean fluorescence intensity (MFI) from TriNKET and the parental monoclonal CD33 antibody normalized to the secondary antibody control.

[0202] Compared to the parental CD33 antibody, CD33-TriNKET-A44, CD33-TriNKET-A49, and CD33-TriNKET-F63 showed comparable levels of binding to CD33 ( Figure 7 ).

[0203] The trispecific binding protein binds to HER2

[0204] Human cancer cell lines expressing HER2 were used to determine the binding of TriNKET to tumor-associated antigens. The renal cell carcinoma cell line 786-O expresses low levels of HER2, and the human lung cancer cell line NCI-H661 expresses moderate levels of HER2. TriNKET and an optional parental HER2 monoclonal antibody (trastuzumab) were incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and the background fold (FOB) was calculated using the mean fluorescence intensity (MFI) from TriNKET and trastuzumab normalized to the secondary antibody control.

[0205] Compared to trastuzumab, HER2-TriNKET-A44, HER2-TriNKET-A49, and HER2-TriNKET-F63 showed comparable levels of binding to HER2 expressed on 786-O cells ( Figure 8 ). Binding of HER2-TriNKET-E79 to HER2 expressed on NCI-H661 cells was shown ( Figure 9 ).

[0206] Example 5 - Activation of NK cells by trispecific binding proteins

[0207] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 +), and the purity of the isolated NK cells is usually >90%. The isolated NK cells are cultured in a medium containing 100 ng / mL IL-2 for activation or rested overnight in the absence of cytokines. The IL-2-activated NK cells are used within 24 - 48 hours after activation.

[0208] Human cancer cells expressing tumor antigens are harvested and resuspended in the medium at 2x10 6 cells / mL. Monoclonal antibodies or TriNKETs targeting the tumor antigen are diluted in the medium. The activated NK cells are harvested, washed, and resuspended in the medium at 2x10 6 cells / mL. Then, in the presence of IL-2, the cancer cells are mixed with the monoclonal antibody / TriNKET and the activated NK cells. Brefeldin A and monensin are also added to the mixed culture to block the transport of proteins out of the cells for intracellular cytokine staining. A fluorophore-conjugated anti-CD107a is added to the mixed culture, and the culture is incubated for 4 hours. Then, samples are prepared using fluorophore-conjugated antibodies against CD3, CD56, and IFN-γ for FACS analysis. CD107a and IFN-γ staining are analyzed in CD3-CD56 + cells to evaluate NK cell activation. An increase in CD107a / IFN-γ double-positive cells indicates better activation of NK cells through the engagement of two activating receptors rather than one receptor.

[0209] TriNKET mediates the activation of human NK cells co-cultured with HER2-expressing NCI-H661 cells ( Figure 10 ) and SkBr-3 cells ( Figure 11 ), as shown by the increased CD107a degranulation and IFN-γ production. Compared with the monoclonal antibody trastuzumab, TriNKET shows excellent activation of human NK cells in the presence of human cancer cells.

[0210] TriNKET mediates the activation of human NK cells co-cultured with human AML Mv4-11 cells expressing CD33, as shown by the increased CD107a degranulation and IFN-γ production ( Figure 12 ). Compared with the monoclonal anti-CD33 antibody, TriNKET shows excellent activation of human NK cells in the presence of human cancer cells.

[0211] Example 6 - Trispecific binding protein achieves cytotoxicity against target cancer cells

[0212] Peripheral blood mononuclear cells (PBMCs) are isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56+ ), and the purity of the isolated NK cells is usually >90%. The isolated NK cells are cultured in a medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. The next day, the IL-2-activated or rested NK cells are used for cytotoxicity assays.

[0213] To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, the Cyto Tox 96 non-radioactive cytotoxicity assay from Promega (G1780) was used according to the manufacturer's instructions. Briefly, human cancer cells expressing tumor antigens were harvested, washed, and resuspended in medium at 1 - 2x10 5 cells / mL. Rested and / or activated NK cells were harvested, washed, and resuspended in the same medium as the cancer cells at 10 5 -2.0×10 6 cells / mL. In each well of a 96-well plate, 50 μl of the cancer cell suspension was mixed with 50 μl of the NK cell suspension in the presence or absence of TriNKET targeting the tumor antigen expressed on the cancer cells. After incubation at 37 °C with 5% CO 2 for 3 hours and 15 minutes, 10x lysis buffer was added to the wells containing only cancer cells and the wells containing only medium, respectively, to obtain maximum lysis and negative reagent controls. The plate was then returned to the incubator for an additional 45 minutes to achieve a total incubation of 4 hours. The cells were then pelleted, and the culture supernatant was transferred to a new 96-well plate and mixed with the substrate for development. The new plate was incubated at room temperature for 30 minutes, and the absorbance was read at 492 nm on a SpectraMax i3x. The percentage of specific lysis of the cancer cells was calculated as follows: % specific lysis = ((experimental lysis - spontaneous lysis from NK cells alone - spontaneous lysis from cancer cells alone) / (maximum lysis - negative reagent control)) * 100%.

[0214] TriNKET mediates the cytotoxicity of human NK cells against CD33-positive Molm-13 human AML cell line. As Figure 13 shown, rested human NK cells were mixed with Molm-13 cancer cells, and TriNKET was able to enhance the cytotoxic activity of rested human NK cells against cancer cells in a dose-responsive manner. The dashed line represents the cytotoxic activity of rested NK cells without TriNKET. As Figure 14 shown, activated human NK cells were mixed with Molm-13 cancer cells, and TriNKET even further enhanced the cytotoxic activity of activated human NK cells against cancer cells in a dose-responsive manner.

[0215] TriNKET mediates the cytotoxicity of human NK cells against the HER2-positive 786-O human renal cell carcinoma cell line. As Figure 15 shown, resting human NK cells were mixed with 786-O cancer cells, and TriNKET was able to enhance the cytotoxic activity of resting human NK cells against cancer cells in a dose-responsive manner (each TriNKET was added at 5, 1, 0.2 μg / ml in the assay, and the results are shown in three columns from left to right for each TriNKET in Figures 15 - 16 . The dashed line represents the cytotoxic activity of resting NK cells against 786-O cells in the absence of TriNKET. As Figure 16 shown, activated human NK cells were mixed with 786-O cells, and TriNKET even further enhanced the cytotoxic activity of activated human NK cells against cancer cells in a dose-responsive manner. The dashed line represents the cytotoxic activity of activated NK cells against 786-O cells in the absence of TriNKET.

[0216] Example 7 - Variants of ADI-27749 and TriNKETs Containing the Variants

[0217] As described above, ADI-27749 (A49) particularly contains a heavy chain CDR3 having the amino acid sequence GAPMGAAAGWFDP (SEQ ID NO:71). The Met at position 102 of SEQ ID NO:7 (i.e., position 4 of this CDR3 sequence) can be replaced with Gln, Leu, Ile, Phe, or Val, thereby generating the NKG2D antibodies A49MQ, A49ML, A49MI, A49MF, and A49MV having the corresponding heavy chain variable regions, light chain variable regions, and CDR sequences provided in Table 1, respectively.

[0218] Using the parameter settings of avg_pro_patch_cdr_hyd, the effects of these mutations on hydrophobicity were analyzed using the MOE2018.01 program. The residues were mutated using the protein builder module, and the entire Fab was minimized after connecting all the residues. Dynamic property sampling was performed using the lowMD scheme in BIOMOE. As shown in Table 11, these mutations had no substantial negative impact on the predicted hydrophobicity of A49Fab.

[0219]

[0220] The hydrophobicity of TriNKET containing A49 (“TriNKET A”) and mutant forms of TriNKET A in which Met is replaced by Ile, Leu, Val, Gln, or Phe (“TriNKET A*”) was tested by analytical hydrophobic interaction chromatography (HIC). Each TriNKET also binds to a first tumor antigen. As shown in Table 12, the retention times of TriNKET A* are similar to those of TriNKET A.

[0221]

[0222] The thermal stabilities of TriNKET A and TriNKET A* were examined by differential scanning calorimetry (DSC) at pH 6.0 in 20 mM histidine, 260 mM sucrose, and 0.005% PS-80. T m values are shown in Table 13, where T m is the midpoint transition temperature of a single domain. Compared to TriNKET A, the M102 mutation decreased the T m3 and T m4 values of the two most stable transitions by 0.6 and 0.7 °C, with little effect on the earlier transitions (T m and T m1 and T m2 ). Thus, the M102 mutation has only a marginal effect on the overall thermal stability of TriNKET A.

[0223]

[0224] The binding of TriNKET A and TriNKET A* to the fusion protein of human NKG2D and murine Fc (“mFc-hNKG2D”) was characterized by surface plasmon resonance (SPR) at 37 °C. Two different fits, namely steady-state affinity fitting and kinetic fitting, were used to obtain equilibrium affinity data ( Figure 32 ). Kinetic constants and equilibrium affinity constants were calculated, and data from two independent experiments of TriNKET A* and three independent experiments of TriNKET A were averaged.

[0225]

[0226]

[0227] As shown in Table 14, the equilibrium affinity constants (K D) They are very similar between repeated experiments, indicating that the measurement parameters have high confidence. Compared with TriNKET A, the affinity of the M102 variant for human NKG2D is reduced by less than 2-fold. The K D of TriNKET A* is (6.87 ± 0.16) x 10 -7 M, while the K D of TriNKET A is (4.87 ± 0.83) x 10 -7 M (calculated by affinity fitting). When calculating K D from kinetic fitting, a similar difference in affinity was observed. The stoichiometry of NKG2D bound to TriNKET A* is 0.85 ± 0.12, similar to 1.01 ± 0.11 for TriNKET A, which confirms that each NKG2D dimer binds to one molecule of TriNKET A*. This indicates that the M102 mutation has only a small effect on the binding of A49-containing TriNKET to human NKG2D.

[0228] Finally, the effect of the M102 mutation on the potency of TriNKET was evaluated in a cytotoxicity assay. Briefly, KHYG-1 cells expressing the high-affinity variant (158V) of CD16a were generated by retroviral transduction. After transduction, the cells were selected in growth medium containing puromycin to generate a selected population of KHYG-1-CD16V cells. The selected population was maintained in medium containing 10 ng / mL human IL-2. To prepare KHYG-1-CD16V cells for use as effectors in the cytotoxicity assay, the cells were harvested from the culture, pelleted, washed 3 times in medium without IL-2, and resuspended in medium without IL-2, and allowed to stand for 24 hours.

[0229] Human cancer cell lines expressing the target of interest were harvested from the culture. The cells were washed with HBS and resuspended at 10 6 cells / mL in growth medium for labeling with BATDA reagent (Perkin Elmer C136-100). The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed 3 times with HBS and resuspended at 0.5 x 10 5 cells / mL in medium. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate.

[0230] TriNKET was serially diluted in medium, and 50 μl of the diluted TriNKET was added to each well. Resting NK cells were harvested from the culture, washed, and resuspended at 1.0 x 10 6Cells / mL were resuspended in the medium. 50 μl of NK cells were added to each well of the plate to achieve the desired E:T ratio of 10:1, and the total volume of the culture in each well was made 200 μl. The plate was incubated at 37 °C and 5% CO 2 for 2 - 3 hours.

[0231] After incubation, the plate was removed from the incubator and centrifuged at 200×g for 5 minutes to pellet the cells. 20 μl of the culture supernatant was transferred to a clean microplate provided by the manufacturer. The supernatant from the labeled cells incubated alone without NK cells was used to measure the spontaneous release of TDA. The supernatant from the labeled cells incubated with 1% Triton-X was used to measure the maximum lysis of the target cells. The supernatant from the labeled cells before incubation for 2 - 3 hours was used to measure the background and for quality control purposes.

[0232] 200 μl of room temperature europium solution (Perkin Elmer C135-100) was added to each well containing the culture supernatant. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. Fluorescence was measured using a SpectraMax i3X instrument. The fluorescence level represents the lysis of the target cells. The value of % specific lysis was calculated as: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) × 100%.

[0233] To measure the activities of TriNKET A and TriNKET A*, a cell line expressing the first tumor antigen was selected as the target cell. Two different batches of TriNKET A were used for comparison. The Figure 33 % specific lysis values were plotted, and the EC50 and maximum % specific lysis values were summarized in Table 15. The EC50 and maximum % specific lysis values of TriNKET A* were similar to those of TriNKET A, indicating that the M102 mutation did not affect the biological activity of TriNKET A.

[0234]

[0235] To confirm that the lack of effect of the M102 mutation on TriNKET activity was not tumor antigen-specific, TriNKET A and TriNKET A* that bind to a second different tumor antigen were constructed. Using a cell line expressing the second tumor antigen as the target cell and KHYG-1-CD16V cells as effector cells, the activities of the two TriNKETs were compared in a cytotoxicity assay. As Figure 34 shown, TriNKET A* exhibited activity equivalent to that of TriNKET A.

[0236] Incorporated by reference

[0237] The entire disclosure of every patent document and scientific and technical article mentioned herein is incorporated by reference for all purposes.

[0238] equivalent solutions

[0239] Without departing from the spirit or essential characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, the foregoing embodiments should be considered illustrative in all respects and not restrictive of the present invention described herein. Accordingly, the scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and range of equivalence of the claims are intended to be included herein.

Claims

1. A multispecific binding protein, comprising: (a) A Fab that binds to NKG2D, comprising: (i) An antibody heavy chain variable domain comprising the complementarity determining region 1 (CDR1) amino acid sequence of SEQ ID NO:48, the complementarity determining region 2 (CDR2) amino acid sequence of SEQ ID NO:30, and the complementarity determining region 3 (CDR3) amino acid sequence of SEQ ID NO:71, 78, 74, 76, 80, or 82; and (ii) An antibody light chain variable domain comprising the CDR1 amino acid sequence of SEQ ID NO:32, the CDR2 amino acid sequence of SEQ ID NO:33, and the CDR3 amino acid sequence of SEQ ID NO:34; (b) A single-chain variable fragment (scFv) that binds to a tumor-associated antigen; and (c) A first antibody constant domain and a second antibody constant domain that together bind to CD16, wherein the Fab comprises (i) a heavy chain portion comprising an antibody heavy chain variable domain and a CH1 domain; and (ii) a light chain portion comprising an antibody light chain variable domain and a light chain constant domain; and wherein the first antibody constant domain is linked to the C-terminus of the scFv, and the second antibody constant domain is linked to the C-terminus of the CH1 domain.

2. The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of the Fab comprises the CDR1 amino acid sequence of SEQ ID NO:29, the CDR2 amino acid sequence of SEQ ID NO:30, and the CDR3 amino acid sequence of SEQ ID NO:

31.

3. The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of the Fab comprises the CDR1 amino acid sequence of SEQ ID NO:48, the CDR2 amino acid sequence of SEQ ID NO:30, and the CDR3 amino acid sequence of SEQ ID NO:

71.

4. The multispecific binding protein according to claim 2, wherein the antibody heavy chain variable domain of the Fab is composed of an amino acid sequence having at least 90% identity to SEQ ID NO:7, and the antibody light chain variable domain of the Fab is composed of an amino acid sequence having at least 90% identity to SEQ ID NO:

8.

5. The multispecific binding protein according to claim 2, wherein the antibody heavy chain variable domain of the Fab is composed of the amino acid sequence of SEQ ID NO:7, and the antibody light chain variable domain of the Fab is composed of the amino acid sequence of SEQ ID NO:

8.

6. The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of the Fab comprises the CDR1 amino acid sequence of SEQ ID NO:29, the CDR2 amino acid sequence of SEQ ID NO:30, and the CDR3 amino acid sequence of SEQ ID NO:

77.

7. The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of the Fab comprises the CDR1 amino acid sequence of SEQ ID NO: 48, the CDR2 amino acid sequence of SEQ ID NO: 30, and the CDR3 amino acid sequence of SEQ ID NO:

78.

8. The multispecific binding protein according to claim 6, wherein the antibody heavy chain variable domain of the Fab consists of an amino acid sequence having at least 90% identity with SEQ ID NO: 85, and the antibody light chain variable domain of the Fab consists of an amino acid sequence having at least 90% identity with SEQ ID NO:

8.

9. The multispecific binding protein according to claim 6, wherein the antibody heavy chain variable domain of the Fab consists of the amino acid sequence of SEQ ID NO: 85, and the antibody light chain variable domain of the Fab consists of the amino acid sequence of SEQ ID NO:

8.

10. The multispecific binding protein according to any one of claims 1-9, wherein the amino acid sequences of the first antibody constant domain and the second antibody constant domain each have at least 90% identity with the human IgG1 constant region.

11. A formulation comprising the multispecific binding protein according to any one of claims 1-10 and a pharmaceutically acceptable carrier.

12. A cell comprising one or more nucleic acids encoding the multispecific binding protein according to any one of claims 1-10.

13. Use of the multispecific binding protein according to any one of claims 1-10 or the formulation according to claim 11 in the preparation of a medicament for enhancing tumor cell death.

14. The use according to claim 13, wherein, the tumor and natural killer cells are exposed to the multispecific binding protein.

15. Use of the multispecific binding protein according to any one of claims 1-10 or the formulation according to claim 11 in the preparation of a medicament for treating cancer.

16. The use according to claim 15, wherein the cancer is selected from the group consisting of: acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancers infiltrated by myeloid-derived suppressor cells, cancers with extracellular matrix deposition, cancers with high levels of reactive stroma, and cancers with neovascularization.

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