Antibody variable domains targeting NKG2d receptor

Abstract

To provide antibody variable domains targeting the NKG2D receptor.SOLUTION: Antibody heavy chain variable domains that can be paired with antibody light chain variable domains to form an antigen-binding site targeting the NKG2D receptor on natural killer cells are described. Proteins comprising an NKG2D antigen-binding site, pharmaceutical compositions thereof, and therapeutic methods thereof including the treatment of cancer are also described. Antibodies to NKG2D have been identified as providing important advantages in the design of therapeutic agents. For example, some of these antibodies do not merely bind to the human NKG2D receptor, but have one or more further advantages such as the ability to agonize the receptor, the ability to compete with a natural ligand for binding to the receptor, and / or the ability to cross-react with NKG2D from other species such as cynomolgus. These advantages can be achieved across a range of affinities for NKG2D.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 628,161, filed February 8, 2018, the disclosure of which is incorporated by reference in its entirety for all purposes, and U.S. Provisional Patent Application No. 62 / 716,259, filed August 8, 2018.

[0002] FIELD OF THEINVENTION The present invention provides proteins having an antibody heavy chain variable domain and an antibody light chain variable domain that can pair to form an antigen-binding site that targets the natural killer group 2D (NKG2D) receptor on natural killer cells, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer. [Background technology]

[0003] Cancer remains a serious health problem, despite substantial research efforts and scientific advances reported in the literature to treat this disease. The most frequently diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Current treatment options for these cancers are not effective for all patients and / or have substantial side effects. Other types of cancer also remain difficult to treat using existing treatment options.

[0004] Cancer immunotherapy is desirable because it is highly specific and can use the patient's own immune system to promote the destruction of cancer cells.Fusion proteins such as bispecific T cell derivatives are cancer immunotherapies described in the literature that bind to tumor cells and T cells to promote the destruction of tumor cells.Antibodies that bind to specific tumor-associated antigens and specific immune cells have been described in the literature.See, for example, WO2016 / 134371 and WO2015 / 095412.

[0005] Natural killer (NK) cells are components of the innate immune system and account for approximately 15% of circulating lymphocytes. NK cells were originally characterized by their ability to infiltrate virtually all tissues and effectively kill tumor cells without the need for priming to distinguish them from T cells. Activated NK cells kill target cells in a manner similar to cytotoxic T cells, i.e., via cytotoxic granules containing perforin and granzymes, as well as via death receptor pathways. Activated NK cells also secrete proinflammatory cytokines, such as IFN-gamma, and chemokines that promote the recruitment of other leukocytes to target tissues. NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIRs). 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 expressed by essentially all natural killer cells where NKG2D serves as an activating receptor. The ability to modulate NK cell function through NKG2D is useful in a variety of therapeutic situations, including malignancies. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention [Means for solving the problem]

[0007] Antibodies against NKG2D have been confirmed to provide important advantages in the design of therapeutic agents. For example, some of these antibodies not only bind to the human NKG2D receptor, but have one or more additional advantages, such as the ability to stimulate the receptor, compete with natural ligands for binding to the receptor, and / or cross-react with NKG2D from other species, such as cynomolgus monkeys. These advantages can be achieved over the entire range of NKG2D affinities.

[0008] Thus, one aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAPNYGDTTHDYYYMDVWGKGTTVTVSS (SEQ ID NO: 1, ADI-29379). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTFTSYYMH (SEQ ID NO: 11) 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") of SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYYMH (SEQ ID NO:45) as CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO:12) as CDR2, and GAPNYGDTTHDYYYMDV (SEQ ID NO:68) as CDR3 of SEQ ID NO:1.

[0009] Another aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDVWGQGTTVTVSS (SEQ ID NO: 3, ADI-29463). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTFTGYYMH (SEQ ID NO: 17) as the first complementarity determining region ("CDR1"), WINPNSGGTNYAQKFQG (SEQ ID NO: 18) as the second CDR ("CDR2"), and ARDTGEYYDTDDHGMDV (SEQ ID NO: 19) as the third CDR ("CDR3") of SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence GYYMH (SEQ ID NO: 92) as CDR1, WINPNSGGTNYAQKFQG (SEQ ID NO: 18) as CDR2, and DTGEYYDTDDHGMDV (SEQ ID NO: 69) as CDR3 of SEQ ID NO:3.

[0010] Another aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSS (SEQ ID NO:5, ADI-27744). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO:5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYAMS (SEQ ID NO:23) as the first complementarity determining region ("CDR1"), AISGSGGGSTYYADSVKG (SEQ ID NO:24) as the second CDR ("CDR2"), and AKDGGYYDSGAGDY (SEQ ID NO:25) as the third CDR ("CDR3") of SEQ ID NO:5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYAMS (SEQ ID NO:47) as CDR1, AISGSGGSTYYADSVKG (SEQ ID NO:24) as CDR2, and DGGYYDSGAGDY (SEQ ID NO:70) as CDR3 of SEQ ID NO:5.

[0011] Another aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSS (SEQ ID NO: 7, ADI-27749). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYSMN (SEQ ID NO: 29) as the first complementarity determining region ("CDR1"), SISSSSSYIYYADSVKG (SEQ ID NO: 30) as the second CDR ("CDR2"), and ARGAPMGAAAGWFDP (SEQ ID NO: 31) as the third CDR ("CDR3") of SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYSMN as CDR1 (SEQ ID NO:48), SISSSSSYIYYADSVKG as CDR2 (SEQ ID NO:30), and GAPMGAAAGWFDP as CDR3 (SEQ ID NO:71) of SEQ ID NO:7.

[0012] Another aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGQGTLVTVSS (SEQ ID NO: 85, A49MI). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO: 85. In some embodiments, the heavy chain variable domain comprises the amino acid sequence FTFSSYSMN (SEQ ID NO: 29) as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO: 30) as CDR2, and ARGAPIGAAAGWFDP (SEQ ID NO: 77) as CDR3 of SEQ ID NO: 85. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYSMN (SEQ ID NO:48) as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO:30) as CDR2, and GAPIGAAAGWFDP (SEQ ID NO:78) as CDR3 of SEQ ID NO:85.

[0013] Another aspect of the invention relates to an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAGFAYGMDYYYMDVWGKGTTVTVSS (SEQ ID NO: 9, ADI-29378). In some embodiments, the antibody heavy chain variable domain is at least 95% identical to SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence YTFTSYYMH (SEQ ID NO: 35) 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") of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequence SYYMH (SEQ ID NO:45) as CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO:36) as CDR2, and EGAGFAYGMDYYYMDV (SEQ ID NO:72) as CDR3 of SEQ ID NO:9.

[0014] The antibody heavy chain variable domain of the invention may optionally be coupled to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an antibody constant region, for example an IgG constant region comprising a hinge, CH2 domain, and CH3 domain, with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical 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 is at least 90% identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. The one or more mutations may be included in the constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439, relative to the human IgG1 constant region. Exemplary substituents 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.

[0015] In certain embodiments, variants that may be included in CH1 of the human IgG1 constant region may be amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, variants that may be included in Cκ of the human IgG1 constant region may be amino acids E123, F116, S176, V163, S174, and / or T164.

[0016] In some embodiments, one of the heavy chain variable domains described herein is combined with a light chain variable domain to form an antigen-binding site that can bind to NKG2D. For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 can be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDDWPFTFGGGTKVEIK (SEQ ID NO: 2, ADI-29379). In some embodiments, the antibody light chain variable domain is at least 95% identical to SEQ ID NO: 2. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQSVSSNLA (SEQ ID NO: 14) as a first complementarity determining region ("CDR"), GASTRAT (SEQ ID NO: 15) as a second CDR, and QQYDDWPFT (SEQ ID NO: 16) as a third CDR.

[0017] For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO:3 can be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK (SEQ ID NO:4, ADI-29463). In some embodiments, the antibody light chain variable domain is at least 95% identical to SEQ ID NO:4. In some embodiments, the light chain variable domain comprises the amino acid sequence RASQSVSSNLA (SEQ ID NO:20) as a first complementarity determining region ("CDR"), GASTRAT (SEQ ID NO:21) as a second CDR, and QQDDYWPPT (SEQ ID NO:22) as a third CDR.

[0018] For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 can pair with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK (SEQ ID NO:6, ADI-27744). In some embodiments, the antibody light chain variable domain is at least 95% identical 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.

[0019] For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:85 can be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK (SEQ ID NO:8, ADI-27749). In some embodiments, the antibody light chain variable domain is at least 95% identical 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.

[0020] For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO:9 can be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSDNWPFTFGGGTKVEIK (SEQ ID NO:10, ADI-29378). In some embodiments, the antibody light chain variable domain is at least 95% identical to SEQ ID NO:10. In some embodiments, the antibody light chain variable domain comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO:38) as a first complementarity determining region ("CDR"), DASNRAT (SEQ ID NO:39) as a second CDR, and QQSDNWPFT (SEQ ID NO:40) as a third CDR.

[0021] When a heavy chain variable domain is combined with a light chain variable domain to form an antigen-binding site capable of binding to NKG2D, the antigen-binding site can be included in a variety of structures, e.g., 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 to NKG2D, a bispecific, trispecific, tetraspecific, or other multispecific antibody, or a smaller structure such as an scFv (in which a heavy chain variable domain is linked to a light chain variable domain).

[0022] In some embodiments, any of the NKG2D antigen-binding sites disclosed in the present invention may be comprised in a protein that also comprises a separate antigen-binding site that binds to a tumor-associated antigen, allowing the protein to simultaneously interact with NK cells and tumor cells. Tumor-associated antigens include, 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.

[0023] In some embodiments, any of the NKG2D antigen-binding sites disclosed in the present invention is comprised in a protein that also comprises a tumor-associated antigen site and a CD16-binding site, which may be an additional antigen-binding site or an antibody constant region or part thereof, such as an IgG1 constant region (which may, for example, optionally contain one or more mutations that affect effector activity or CD16-binding affinity).

[0024] Another aspect of the invention provides a method of enhancing tumor cell death and treating cancer in a patient, comprising administering to a patient in need of treatment a therapeutically effective amount of a protein described herein to treat the cancer. The present invention provides, for example, the following: (Item 1) An antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:7. (Item 2) The amino acid sequence A complementarity determining region 1 (CDR1) sequence represented by the amino acid sequence of SEQ ID NO: 48; A complementarity determining region 2 (CDR2) sequence represented by the amino acid sequence of SEQ ID NO: 30; and a complementarity determining region 3 (CDR3) sequence represented by the amino acid sequence of SEQ ID NO: 44. (Item 3) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29; 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. The antibody heavy chain variable domain of. (Item 4) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 48; 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: 71. The antibody heavy chain variable domain of item 1. (Item 5) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29; 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. (Item 6) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 48; 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: 78. (Item 7) An antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1. (Item 8) The amino acid sequence a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 11; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 12; 8. The antibody heavy chain variable domain according to item 7, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 13. (Item 9) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 45; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 12; 8. The antibody heavy chain variable domain according to item 7, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 68. (Item 10) An antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3. (Item 11) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 17; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 18; 11. The antibody heavy chain variable domain according to item 10, comprising: a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 19. (Item 12) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 46; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 18; 11. The antibody heavy chain variable domain according to item 10, comprising: a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 69. (Item 13) An antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5. (Item 14) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 23; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 24; 14. The antibody heavy chain variable domain according to claim 13, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 25. (Item 15) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 47; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 24; 14. The antibody heavy chain variable domain according to claim 13, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 70. (Item 16) An antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9. (Item 17) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 35; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 36; 17. The antibody heavy chain variable domain according to claim 16, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 37. (Item 18) The amino acid sequence A CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 45; A CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 36; 17. The antibody heavy chain variable domain of claim 16, comprising a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 72. (Item 19) 19. An antibody heavy chain comprising the antibody heavy chain variable domain according to any one of items 1 to 18 and an amino acid sequence that is at least 90% identical to an antibody constant region. (Item 20) 20. The antibody heavy chain of claim 19, wherein the antibody constant region is a human IgG constant region comprising a hinge, CH2, and CH3 domain. (Item 21) 21. The antibody heavy chain of claim 20, wherein the antibody constant region is a human IgG constant region further comprising a CH1 domain. (Item 22) 22. The antibody heavy chain according to any one of items 19 to 21, wherein the antibody constant region is 90% identical to an IgG1 constant region. (Item 23) 23. The antibody heavy chain of item 22, wherein the amino acid sequence at least 90% identical to the antibody constant region is different from the amino acid sequence of the IgG1 constant region at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, or K439, or any combination thereof. (Item 24) The amino acid sequence having at least 90% identity to the antibody constant region is selected from the group consisting of Q347E, Q34 7R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S 364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, 24. The antibody heavy chain of item 23, which differs from the amino acid sequence of the IgG1 constant region by a substitution selected from the group consisting of: K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E, or any combination thereof. (Item 25) 7. An antigen-binding site comprising the antibody heavy chain variable domain according to any one of items 1 to 6 and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:8. (Item 26) 26. The antigen-binding site of claim 25, wherein the light chain variable domain comprises a CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 32, a CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 33, and a CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 34. (Item 27) K of 2–120 nM as measured by surface plasmon resonance D 27. The antigen-binding site according to item 25 or 26, having the following structure: (Item 28) 10. An antigen-binding site comprising the antibody heavy chain variable domain according to any one of items 7 to 9, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2. (Item 29) 29. The antigen-binding site of item 28, wherein the light chain variable domain comprises a CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 14, a CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 15, and a CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 16. (Item 30) K between 5 and 500 nM as measured by surface plasmon resonance D 30. The antigen-binding site according to item 28 or 29, having the following structure: (Item 31) 13. An antigen-binding site comprising the antibody heavy chain variable domain according to any one of items 10 to 12 and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:4. (Item 32) 32. The antigen-binding site of claim 31, wherein the light chain variable domain comprises a CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 20, a CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 21, and a CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 22. (Item 33) K of 6–600 nM as measured by surface plasmon resonance D 33. The antigen-binding site according to item 31 or 32, having the following structure: (Item 34) 16. An antigen-binding site comprising the antibody heavy chain variable domain according to any one of items 13 to 15, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:6. (Item 35) 35. The antigen-binding site of claim 34, wherein the light chain variable domain comprises a CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 26, a CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 27, and a CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 28. (Item 36) K between 1 and 100 nM as measured by surface plasmon resonance D 36. The antigen-binding site according to item 34 or 35, having the following structure: (Item 37) 19. An antigen-binding site comprising the antibody heavy chain variable domain according to any one of items 16 to 18, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. (Item 38) 38. The antigen-binding site of claim 37, wherein the light chain variable domain comprises a CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 38, a CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 39, and a CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 40. (Item 39) K of 6–600 nM as measured by surface plasmon resonance D 39. The antigen-binding site according to item 37 or 38, having the following structure: (Item 40) A protein comprising the antigen-binding site according to any one of items 25 to 39, which binds to NKG2D, and a further antigen-binding site. (Item 41) 41. The protein according to item 40, wherein the further antigen-binding site binds to a tumor-associated antigen. (Item 42) 42. The protein according to item 41, wherein the tumor associated antigen is selected from the group consisting of CD33, HER2, EpCAM, CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD1. (Item 43) the antigen-binding site that binds to NKG2D comprises a first antibody heavy chain variable domain, and the further antigen-binding site comprises a second antibody heavy chain variable domain; 43. The protein according to any one of Items 40 to 42, wherein the first antibody heavy chain variable domain is present in a first polypeptide further comprising a first antibody constant region, and the second antibody heavy chain variable domain is present in a second polypeptide further comprising a second antibody constant region. (Item 44) 44. The protein of item 43, wherein the first antibody constant region and the second antibody constant region form a complex capable of binding to CD16. (Item 45) 45. The protein according to item 43 or 44, wherein the first antibody constant region and the second antibody constant region each comprise a hinge, a CH2, and a CH3 domain. (Item 46) 46. ​​The protein according to any one of items 43 to 45, wherein the first antibody constant region and the second antibody constant region each further comprise a CH1 domain. (Item 47) 47. The protein according to any one of Items 43 to 46, wherein the amino acid sequences of the first antibody constant region and the second antibody constant region are at least 90% identical to a human IgG1 constant region, respectively. (Item 48) The amino acid sequence of the first antibody constant region is Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409 , T411, or K439, or any combination thereof, 48. The protein according to item 47, wherein the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, or K439, or any combination thereof. (Item 49) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, L368, or Y407, or any combination thereof. (Item 50) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, L368, or Y407, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366. (Item 51) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at E357, K360, Q362, S364, L368, K370, T394, D401, F405, or T411, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at Y349, E357, S364, L368, K370, T394, D401, F405, or T411, or any combination thereof. (Item 52) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at Y349, E357, S364, L368, K370, T394, D401, F405, or T411, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at E357, K360, Q362, S364, L368, K370, T394, D401, F405, or T411, or any combination thereof. (Item 53) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at L351, D399, S400, or Y407, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at T366, N390, K392, K409, or T411, or any combination thereof. (Item 54) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at T366, N390, K392, K409, or T411, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at L351, D399, S400, or Y407, or any combination thereof. (Item 55) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at Q347, Y349, K360, or K409, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at Q347, E357, D399, or F405, or any combination thereof. (Item 56) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at Q347, E357, D399, or F405, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at Y349, K360, Q347, or K409, or any combination thereof. (Item 57) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at K370, K392, K409, or K439, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at D356, E357, or D399, or any combination thereof. (Item 58) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at D356, E357, or D399, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at K370, K392, K409, or K439, or any combination thereof. (Item 59) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at L351, E356, T366, or D399, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at Y349, L351, L368, K392, or K409, or any combination thereof. (Item 60) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at Y349, L351, L368, K392, or K409, or any combination thereof, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at L351, E356, T366, or D399, or any combination thereof. (Item 61) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by a S354C substitution and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution. (Item 62) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by a S354C substitution. (Item 63) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V, and F405T substitutions. (Item 64) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V, and F405T substitutions, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions. (Item 65) The amino acid sequence of the first antibody constant region differs from that of the IgG1 constant region by a T366W substitution, and the amino acid sequence of the second antibody constant region differs from that of the IgG1 constant region by a T366S, T 48. The protein according to item 47, which differs from the amino acid sequence of the IgG1 constant region by substitutions Y407V, Y368A, and Y407V. (Item 66) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366S, T368A, and Y407V substitution and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution. (Item 67) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L, and T394W substitutions. (Item 68) 48. The protein according to item 47, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L, and T394W substitutions, and the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions. (Item 69) 43. The protein according to any one of items 40 to 42, wherein the protein further comprises an antigen-binding site capable of binding to CD16. (Item 70) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:7 and a light chain variable region having the amino acid sequence of SEQ ID NO:8. (Item 71) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. (Item 72) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2. (Item 73) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 4. (Item 74) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:5 and a light chain variable region having the amino acid sequence of SEQ ID NO:6. (Item 75) A protein comprising an antigen-binding site that competes for binding to human and / or cynomolgus monkey NKG2D with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20. (Item 76) A formulation comprising the protein according to any one of items 40 to 75 and a pharma- ceutically acceptable carrier. (Item 77) 76. A cell comprising one or more nucleic acids encoding the protein according to any one of items 40 to 75. (Item 78) 76. A method for enhancing tumor cell death, comprising exposing tumor and natural killer cells to a protein according to any one of items 40 to 75. (Item 79) A method for treating cancer, comprising administering to a patient the protein according to any one of items 40 to 75 or the formulation according to item 76. (Item 80) 80. The method of claim 79, 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 tumors, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated with myeloid derived suppressor cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with angiogenesis. [Brief description of the drawings]

[0025] [Figure 1] Schematic diagram of a multispecific binding protein comprising an NKG2D-binding domain (right arm), a tumor-associated antigen-binding domain (left arm), and an Fc domain or portion thereof that binds to CD16. [Diagram 2]A representation of a multispecific binding protein comprising an NKG2D-binding domain or a tumor-associated antigen-binding domain, either one of which may be in scFv format, and an Fc domain or portion thereof that binds to CD16. [Diagram 3] Figures 3A-3F are profiles of NKG2D binding affinity of NKG2D binding domains measured by surface plasmon resonance. Figure 3A is the NKG2D binding affinity of NKG2D binding domain ADI-27744 measured by surface plasmon resonance, Figure 3B is the NKG2D binding affinity of NKG2D binding domain ADI-29379 measured by surface plasmon resonance, Figure 3C is the NKG2D binding affinity of NKG2D binding domain ADI-27749 measured by surface plasmon resonance, Figure 3D is the NKG2D binding affinity of NKG2D binding domain ADI-29463 measured by surface plasmon resonance, and Figure 3E is the NKG2D binding affinity of NKG2D binding domain ADI-29378 measured by surface plasmon resonance. [Figure 4]4A-4H are profiles of competitive NKG2D binding by NKG2D binding domain ADI-27744 (A44) and ULBP6 or other NKG2D antibodies measured by surface plasmon resonance. FIG. 4A shows a profile of NKG2D monoclonal antibodies including ADI-27744 injected onto immobilized NKG2D followed by ULBP6 injection. FIG. 4B shows a profile of ULBP6 injected onto immobilized NKG2D followed by NKG2D monoclonal antibodies including ADI-27744 injection. FIG. 4C shows a profile of MS monoclonal antibodies injected onto immobilized NKG2D followed by ULBP6 injection. FIG. 4D shows a profile of MS injected onto immobilized NKG2D followed by NKG2D monoclonal antibodies including ADI-27744 injection. Figure 4E shows the profile of 1D11 injected onto immobilized NKG2D followed by injection of NKG2D monoclonal antibodies including ADI-27744. Figure 4F shows the profile of MAB139 injected onto immobilized NKG2D followed by injection of NKG2D monoclonal antibodies including ADI-27744. Figure 4G shows the profile of NKG2D monoclonal antibodies including ADI-27744 injected onto immobilized NKG2D followed by injection of NKG2D monoclonal antibodies including ADI-27749 (A49). Figure 4H shows the profile of NKG2D monoclonal antibodies including ADI-27744 injected onto immobilized NKG2D followed by injection of NKG2D monoclonal antibodies including F47. [Diagram 5] 1 is a line graph showing the binding profile of CD33-targeted TriNKET to NKG2D expressed on EL4 cells. [Figure 6] 1 is a line graph showing the binding profile of HER2-targeted TriNKET to NKG2D expressed in EL4 cells. [Figure 7] 1 is a bar graph showing the binding profile of CD33-targeted TriNKET to CD33 expressed on Mv4-11 cells. [Figure 8]1 is a bar graph showing the binding profile of HER2-targeted TriNKET to HER2 expressed in 786-O cells. [Figure 9] 1 is a bar graph showing the binding profile of HER2-targeted TriNKET to HER2 expressed in NCI-H661 cells. [Figure 10] 1 is a bar graph showing that TriNKET targeting HER2 mediates activation of human NK cells co-cultured with HER2-expressing NCI-H661 cells. [Figure 11] 1 is a bar graph showing that TriNKET, which targets HER2, mediates activation of human NK cells co-cultured with HER2-expressing SkBr-3 cells. [Figure 12] 1 is a bar graph showing that TriNKET targeting CD33 mediates activation of human NK cells co-cultured with CD33-expressing human AML Mv4-11 cells. [Figure 13] 1 is a line graph showing that TriNKET targeting CD33 potentiates the cytotoxicity of resting NK cells against CD33-expressing Molm-13 cancer cells. [Figure 14] 1 is a line graph showing that TriNKET targeting CD33 enables activated NK cell cytotoxicity against CD33-expressing Molm-13 cancer cells. [Figure 15] 1 is a bar graph showing that TriNKET targeting HER2 potentiates resting NK cell cytotoxicity against HER2-expressing 786-O cancer cells. [Figure 16] 1 is a bar graph showing that TriNKET targeting HER2 enables activated NK cell cytotoxicity against HER2-expressing 786-O cancer cells. [Figure 17]Schematic diagram of the triomab format of TriNKET, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light and one heavy chain, derived from two parent antibodies. The triomab format is a heterodimeric construct containing 1 / 2 rat antibody and 1 / 2 mouse antibody. [Figure 18] Schematic diagram of KiH common light chain (LC) form of TriNKET with knob-into-hole (KIH) technology. KiH is a heterodimer containing two Fabs that bind target 1 and target 2, and an Fc stabilized by a heterodimerization mutation. KiH form of TriNKET is a heterodimeric construct with two fabs that bind target 1 and target 2, containing two different heavy chains and a common light chain that pairs with both HCs. [Figure 19] Schematic representation of TriNKET in the form of a Dual Variable Domain Immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies via a flexible, naturally occurring linker to generate a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the antigen 2 targeting variable domain is fused to the N-terminus of the antigen 1 targeting Fab variable domain, and the construct contains a normal Fc. [Figure 20] Schematic diagram of TriNKET in orthogonal Fab interface (Ortho-Fab) form, a heterodimeric construct containing two Fabs that bind target 1 and target 2 fused to Fc. LC-HC pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc. [Figure 21] Schematic representation of TrinKET in its two-in-one Ig format. [Figure 22] Schematic representation of TriNKET in ES form, a heterodimeric construct containing two different Fabs binding target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc. [Diagram 23]Fab arm swapped form of TriNKET: Schematic diagram of an antibody that has had its Fab arms swapped by swapping the heavy and attached light chains (half molecules) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm swapped form (cFae) is a heterodimer containing two Fabs that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation. [Figure 24] Schematic diagram of the SEED Body form of TriNKET, a heterodimer containing two Fabs that bind to target 1 and target 2, and an Fc stabilized by a heterodimerization mutation. [Diagram 25] Schematic diagram of the LuZ-Y form of TriNKET, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs that bind targets 1 and 2 fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc. [Figure 26] Schematic diagram of TriNKET in Cov-X-Body form. [Figure 27] 27A-B are schematic diagrams of TriNKET in the form of a Kλ-Body, a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations, where antigen 1 targeting Fab1 contains a kappa LC, while antigen 2 targeting the second Fab contains a lambda LC. Figure 27A is an exemplary diagram of one form of Kλ-Body, and Figure 27B is an exemplary diagram of the other Kλ-Body. [Figure 28] This is an Oasc-Fab heterodimer construct that contains a Fab that binds target 1 and a scFab that binds target 2 fused to an Fc. Heterodimerization is ensured by mutations in the Fc. [Figure 29] DuetMab is a heterodimeric construct that contains two different Fabs that bind antigen 1 and antigen 2, and an Fc stabilized by a heterodimerization mutation. Fab1 and Fab2 contain specific S-S bridges that ensure correct LC and HC pairing. [Diagram 30] CrossmAb is a heterodimeric construct with two different Fabs that bind target 1 and target 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains, as well as the VH and VL domains, are swapped, e.g., CH1 is fused in-line with the VL, while CL is fused in-line with the VH. [Diagram 31] Fit-Ig is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of the HC of a Fab that binds antigen 1. This construct contains a wild-type Fc. [Diagram 32] 1 is a series of line graphs showing the binding of TriNKET A* and TriNKET A to human NKG2D as tested by SPR. The top panel represents the dynamic fit and the bottom panel represents the steady-state affinity fit. [Diagram 33] 1 is a line graph showing the efficacy of TriNKET A and TriNKET A* in mediating NK cell cytotoxicity against target cells. [Diagram 34] 1 is a line graph showing the efficacy of TriNKET A and TriNKET A* in mediating NK cell cytotoxicity against target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides proteins having antibody heavy chain domains and light chain variable domains that can pair to form an antigen-binding site that targets the natural killer group 2D (NKG2D) receptor on natural killer cells, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer. Various aspects of the invention are described in the following sections; however, aspects of the invention described in a particular section are not limited to any particular section.

[0027] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0028] As used herein, the words "a" and "an" mean "one or more" and include the plural forms unless the context is inappropriate.

[0029] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0030] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches in the V regions of the heavy and light chains, called "hypervariable regions", are inserted between more conserved adjacent stretches known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to this three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single domain antibody." The antigen-binding site may be present in an intact antibody, an antigen-binding fragment of an antibody that retains the antigen-binding surface, or a recombinant polypeptide such as an scFv, using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0031] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a specific formulation or route of administration. As used herein, the term "treat" includes any effect that results in the improvement of a condition, disease, disorder, etc., such as, for example, lowering, reducing, regulating, improving, or eliminating, or ameliorating the symptoms thereof.

[0032] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that inherently renders the composition suitable for in vivo or ex vivo therapeutic use.

[0033] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions, etc.), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and preservatives, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0034] Throughout this description, where compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is additionally contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods of the invention that consist essentially of or consist of the recited process steps.

[0035] Generally, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of the variable is controlling. NKG2D antigen binding site

[0036] The present invention provides antigen-binding sites that bind to NKG2D, and antigen heavy chain variable domains that can be used to generate such antigen-binding sites.

[0037] Antibody heavy and light chain variable domains that pair to form an antigen binding site capable of activating binding to the NKG2D receptor have been identified and are provided below in Table 1. Unless otherwise stated, the CDR sequences provided in Table 1 are determined under Kabat. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0038] One of the advantages of one or more of the above antibody heavy chain variable domain amino acid sequences is that they can bind to human and cynomolgus NKG2D, stimulate the receptor, and compete with natural ligands for binding to the receptor.Additional antigen-binding sites that bind to NKG2D and share one or more of these characteristics 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 to bind to both human and optionally cynomolgus NKG2D.

[0039] 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 to NKG2D with high affinity. In some embodiments, the NKG2D binding site has a K D In some embodiments, the NKG2D binding site binds to NKG2D having a K D In some embodiments, the NKG2D binding site binds to NKG2D having a K D In some embodiments, the NKG2D binding site binds to NKG2D having a K D It binds to NKG2D having the

[0040] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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 SEQ ID NO:68. In certain embodiments, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0041] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:3, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:4. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0042] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:6. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0043] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0044] The amino acid residue M at position 102 of SEQ ID NO:7, located in the CDR3 of the heavy chain variable domain, can be mutated. In certain embodiments, M102 is substituted with an uncharged residue. In certain embodiments, M102 is substituted with a hydrophobic residue (Gly, Ala, Val, Leu, Ile, Pro, Phe, or Trp). In certain embodiments, M102 is substituted with a polar residue (Ser, Thr, Cys, Asn, Gln, or Tyr). In certain embodiments, M102 is substituted with Leu, Ile, Val, Gln, or Phe.

[0045] Thus, in certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:83, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0046] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:84, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0047] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:85, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0048] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:86, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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 SEQ ID NO:80. In certain embodiments, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0049] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:41, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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.

[0050] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:9, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10. In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: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, an antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 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.

[0051] In certain embodiments, the invention provides an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5, and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6, which does not block binding of the anti-NKG2D antibodies MS, 1D11, and MAB139 to NKG2D.

[0052] In embodiments, the invention provides an antibody comprising an antibody heavy chain variable domain comprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86, or 87, and an antibody light chain variable domain comprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. The antigen-binding site does not block binding to NKG2D of an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5 and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6.

[0053] In certain embodiments, an antigen-binding site comprising an antibody heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5 and an antibody light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6 binds to a specific epitope on NKG2D distinct from the MS-, 1D11-, MAB139-, ADI-27749-, and F47-binding epitope(s).

[0054] Antibodies and multispecific binding proteins In some embodiments of the present invention, the NKG2D antigen-binding site formed by pairing an antibody heavy chain variable domain with a light chain variable domain described herein can be included in a larger protein, such as an intact 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 to 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-12 5, 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. Binding of the multispecific protein to NKG2D and tumor-associated antigens on cancer cells brings the cancer cells into proximity with natural killer cells, facilitating direct or indirect destruction of the cancer cells by the natural killer cells.

[0055] In some embodiments, in addition to the NKG2D-binding site and the tumor-associated antigen-binding site, the multispecific binding protein can further comprise 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 can comprise 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 an antibody Fc region with or without a CH1 domain. In some embodiments, the antibody Fc region is derived from the Fc region of a human and / or other mammalian immunoglobulin. Within the Fc region, binding of CD16 is known to be mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mediated by amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 in the CH2 domain, and carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al, Nature, 406(6793):267-273). Based on the known domains and amino acid residues, in some embodiments, mutations can be selected in the CD16 binding domain to enhance or decrease the binding affinity to CD16. Selection methods are well known in the art, such as phage display library or yeast surface display cDNA library. Appropriate selection methods can also be designed based on the known three-dimensional structure of the interaction by those skilled in the art.

[0056] The multispecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain comprises a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen binding site that binds to a tumor antigen. Both the first Fc domain and the second Fc domain can bind to CD16 (Figure 1).

[0057] Another exemplary format includes a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused via a linker or antibody hinge to a single chain variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain that pairs with and 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 CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to a tumor-associated antigen. Both the first Fc domain and the second Fc domain can bind to CD16 (Figure 2). Further formats of polyspecific binding proteins can be devised by combining various formats of the NKG2D-binding fragments described herein.

[0058] One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the antigen-binding site may be a single chain or a disulfide-stabilized variable region (scFv), or may form a tetravalent or trivalent molecule.

[0059] In some embodiments, the multispecific binding protein is a triomab format, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain, derived from two parent antibodies. The triomab format is a heterodimeric construct containing 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0060] In some embodiments, the polyspecific binding protein is a KiH common light chain (LC) format that includes knob-into-hole (KIH) technology. KIH is a method for creating "knobs" or "holes" in each heavy chain to promote heterodimerization. H The concept behind the “knobs-into-holes (KiH)” Fc technology involves engineering three domains to replace small residues with bulky residues (i.e., T366W in EU numbering). CH3A The goal of this study was to introduce a "knob" into one CH3 domain (CH3A) by replacing the adjacent residues closest to the knob with smaller ones (i.e., T366S / L368A / Y407V) in the other CH3 domain (CH3B) to accommodate the "knob." CH3B) The "hole" mutation was optimized by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol (1997) 270 (1): 26-35). X-ray crystal structure of KiH Fc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) 426 (9): 1947-57, Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol Immunol (2014) 58(1):132-8) showed that heterodimerization is thermodynamically supported by hydrophobic interactions driven by steric complementarity at the core interface between the CH3 domains, whereas knob-knob and hole-hole interfaces do not support homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0061] In some embodiments, the polyspecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies via a flexible naturally occurring linker to generate a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the antigen 2 targeting variable domain is fused to the N-terminus of the antigen 1 targeting Fab variable domain, and the construct contains a normal Fc.

[0062] In some embodiments, the polyspecific binding protein is in an orthogonal Fab interface (Ortho-Fab) form, which is a heterodimeric construct containing two Fabs that bind target 1 and target 2 fused to an Fc. The LC-HC pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc. In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191-8), structure-based regional design ensures that the LC and HC VH-CH1 Complementary mutations are introduced into the interface of only one Fab without making any changes to the other Fab.

[0063] In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in an ES format, which is a heterodimeric construct containing two different Fabs that bind target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0064] In some embodiments, the polyspecific binding protein is a heterodimeric construct with two different Fabs fused to an Fc stabilized by a heterodimerization mutation. K Antigen 1, which is in the λ-Body format and targets Fab1, contains a kappa LC, whereas antigen 2, which targets the second Fab, contains a lambda LC.

[0065] In some embodiments, the polyspecific binding protein is in a Fab arm exchanged form (an antibody that exchanges Fab arms by swapping the heavy chain and attached light chain (half molecule) with a heavy light chain pair from another molecule, resulting in a bispecific antibody). The Fab arm exchanged form (cFae) is a heterodimer that contains two Fabs that bind target 1 and target 2, and an Fc stabilized by a heterodimerization mutation.

[0066] In some embodiments, the polyspecific binding protein is in the form of a SEED Body, a heterodimer containing two Fabs that bind target 1 and target 2, and an Fc stabilized by a heterodimerization mutation. The Strand Exchange Recombinant Domain (SEED) platform was designed to generate asymmetric bispecific antibody-like molecules, a feature that expands the therapeutic applications of naturally occurring antibodies. This protein recombination platform is based on the exchange of structurally related sequences of immunoglobulins within the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while avoiding homodimerization of the SEED CH3 domains of AG and GA. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

[0067] In some embodiments, the multispecific binding protein is in the LuZ-Y form, where a leucine zipper is used to induce heterodimerization of two different HCs. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9). The LuZ-Y form is a heterodimer that contains two different scFabs that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of Fc.

[0068] In some embodiments, the polyspecific binding proteins are in the form of Cov-X-Body (In bispecific CovX-Bodies, two different peptides are linked using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. The antibody scaffold confers a long half-life and Ig-like distribution, while the pharmacophore is responsible for the functional activity. Optimized or unique bispecific antibodies can be generated by chemically optimizing or replacing the pharmacophore with another pharmacophore. (Doppalapudi VR et al., PNAS (2010), 107(52); 22611-22616).

[0069] In some embodiments, the polyspecific binding protein is in an Oasc-Fab heterodimer construct comprising a Fab that binds target 1 and a scFab that binds target 2 fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0070] In some embodiments, the polyspecific binding protein is in a DuetMab format that contains two different Fabs that bind antigen 1 and antigen 2, and an Fc stabilized by a heterodimerization mutation. Fab1 and Fab2 contain specific S-S bridges that ensure correct LC and HC pairing.

[0071] In some embodiments, the polyspecific binding protein is in a CrossmAb format, which is a heterodimeric construct with two different Fabs that bind target 1 and target 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains, as well as the VH and VL domains, are swapped, e.g., CH1 is fused in-line with the VL, while CL is fused in-line with the VH.

[0072] In some embodiments, the polyspecific binding protein is in a CrossmAb format, which is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of the HC of a Fab that binds antigen 1. This construct includes the wild type.

[0073] Heterodimeric antibody heavy chain The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. The preferential assembly of heterodimeric heavy chains in the multispecific binding proteins described herein can be promoted by incorporating different pairs of amino acid substitutions into the first CH3 domain in the first heavy chain polypeptide and the second CH3 domain in the second heavy chain polypeptide, such that these two chains can 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. Various examples of intrapair amino acid substitutions of human IgG1 Fc domains to promote heterodimerization of the two heavy chains are shown below. Each position of the amino acid substitution is numbered according to the EU index as in Kabat.

[0074] In one scenario, the amino acid substitutions in the first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid, and the amino acid substitutions in the second polypeptide replace the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V), such that the amino acid substitution (the protuberance) fits into the surface of the smaller amino acid substitution (the cavity). For example, one polypeptide can include a T366W substitution and the other can include three substitutions including T366S, L368A, and Y407V.

[0075] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 2. [Table 2]

[0076] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 3. [Table 3]

[0077] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 4]

[0078] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 5. [Table 5]

[0079] Alternatively, at least one amino acid substitution may be selected from the following series of substitutions in Table 6, where the position(s) set forth in the first polypeptide row are replaced with a known negatively charged amino acid and the position(s) set forth in the second polypeptide row are replaced with a known positively charged amino acid. [Table 6]

[0080] Alternatively, at least one amino acid substitution may be selected from the following series of substitutions in Table 7, where the position(s) set forth in the first polypeptide row are replaced with a known positively charged amino acid and the position(s) set forth in the second polypeptide row are replaced with a known negatively charged amino acid. [Table 7]

[0081] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 8. [Table 8]

[0082] Alternatively, or in addition, the structural stability of the heterodimer heavy chain in a multispecific binding protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposing polypeptide chain forming an artificial disulfide bridge within the interface of the two polypeptides.

[0083] The multispecific binding protein can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, a third nucleic acid sequence encoding a first immunoglobulin light chain can be cloned into a third expression vector, and 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 transfected together into a host cell to produce a multimeric protein.

[0084] To achieve the highest yield of 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 host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

[0085] The clones can be cultured under conditions suitable for scale-up in a bioreactor to maintain expression of the multispecific proteins. The multispecific binding proteins can be isolated or 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.

[0086] Proteins that contain an antigen-binding site that competes with the NKG2D-binding site described herein In certain embodiments, the present invention provides proteins that comprise an antigen-binding site that binds to NKG2D in competition with the NKG2D-binding site described herein. The NKG2D-binding sites described herein include the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6, the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8, the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10, the amino acid sequences of SEQ ID NO:83 and SEQ ID NO:8, the amino acid sequences of SEQ ID NO:84 and SEQ ID NO:8, the amino acid sequences of SEQ ID NO:85 and SEQ ID NO:8, the amino acid sequences of SEQ ID NO:86 and SEQ ID NO:8, or the amino acid sequences of SEQ ID NO:87 and SEQ ID NO:8. These NKG2D-binding sites can bind to different epitopes on NKG2D that have been mapped by surface plasmon resonance. For example, as shown in Example 2, ADI-27744 binds to a different epitope on NKG2D than ADI-27749 and other existing NKG2D antibodies.

[0087] 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 that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and a light chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical 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 that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:3, and a light chain variable domain having an amino acid sequence that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical 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 that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5, and a light chain variable domain having an amino acid sequence that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical 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 that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable domain having an amino acid sequence that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical 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 that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:9, and a light chain variable domain having an amino acid sequence that is at least at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.

[0088] In some embodiments, the protein comprising an antigen-binding site that competes with the NKG2D-binding site described herein further comprises a second antigen-binding site that binds to 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 can bind to CD16. In some embodiments, the CD16-binding site comprises a human IgG1 Fc domain.

[0089] Cells for expressing proteins In one embodiment, the disclosure provides an NKG2D binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:2, a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5. a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:6; a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86, or 87; or a NKG2D-binding site having a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO:9; and a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO:10;or 100%) identical to the nucleic acid sequence of ...

[0090] Therapeutic Applications The present invention provides methods for enhancing tumor cell death and / or treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat a variety of cancers. The type of cancer being treated desirably matches the type of cancer cells to which the protein binds. Further aspects and embodiments of the treatment methods are described below.

[0091] Pharmaceutical Compositions In one aspect, the disclosure also features a pharmaceutical composition comprising an effective amount of a protein comprising an NKG2D binding site described herein or an NKG2D binding site that competes with an NKG2D binding site described herein, and a pharma- ceutically acceptable carrier.

[0092] In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:3, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:6. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86, or 87, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:8.In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:9, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.

[0093] The composition can be formulated for use in various drug delivery systems.One or more physiologically acceptable excipients or carriers can be included in the composition for suitable formulation.Formulations suitable for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985.For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533) 1990.

[0094] For example, the present disclosure may be present in an aqueous pharmaceutical formulation comprising a therapeutically effective amount of the protein in a buffer solution forming the formulation. Aqueous carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. In certain embodiments, an aqueous formulation comprising a protein disclosed herein is prepared in a pH buffered solution. The pH of the preparation will typically be 3-11, more preferably 5-9 or 6-8, most preferably 7-8, e.g., 7-7.5. Intermediate pH ranges above are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination 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 acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / ml (e.g., 1.305 mg / ml) citric acid, about 0.3 mg / ml (e.g., 0.305 mg / ml) sodium citrate, about 1.5 mg / ml (e.g., 1.53 mg / ml) disodium phosphate dihydrate, about 0.9 mg / ml (e.g., 0.86) sodium dihydrogen phosphate dihydrate, and about 6.2 mg / ml (e.g., 6.165 mg / ml) sodium chloride. In certain embodiments, the buffer system comprises 1-1.5 mg / ml citric acid, 0.25-0.5 mg / ml sodium citrate, 1.25-1.75 mg / ml disodium phosphate dihydrate, 0.7-1.1 mg / ml sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / ml sodium chloride. The pH of the liquid formulation may be set by the addition of a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0095] In some embodiments, the formulation comprises an aqueous carrier that is pharma- ceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0096] A polyol may also be included in the formulation, which may function as an isotonicity agent and stabilize the antibody. The polyol is added to the formulation in an amount that may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary with respect to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that may be used in the formulation as an isotonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / ml. In certain embodiments, the mannitol concentration may be about 7.5 to 15 mg / ml. In certain embodiments, the mannitol concentration may be about 10 to 14 mg / ml. In certain embodiments, the mannitol concentration may be about 12 mg / ml. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0097] Detergents or surfactants may also be added to the formulation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may include the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (Fiedler, Lexikon der Hifsstoffe, Editio Cantor (See Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain about 0.1 to about 10 mg / mL of polysorbate 80, or about 0.5 mg / mL to about 5 mg / mL of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0098] In certain embodiments, the liquid formulations of the present disclosure can be prepared as a concentrated solution of 10 mg / mL in combination with a stabilizing level of sugar. In certain embodiments, the liquid formulations can be prepared in an aqueous carrier. In certain embodiments, the stabilizer can be added in an amount that would result in an undesirable or unsuitable viscosity for intravenous administration. In certain embodiments, the sugar can be a disaccharide, such as sucrose. In certain embodiments, the liquid formulations can also include one or more of a buffer, a surfactant, and a preservative, which are added to the formulations herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multi-dose) formulation.

[0099] In some embodiments, the disclosure provides an extended shelf life formulation comprising a protein of the disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0100] Deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, storage of drug substance / drug product, and during 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 mass loss of 17 Da from the parent peptide. Subsequent hydrolysis results in a mass gain of 18 u. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is usually detectable when there is an increase in mass of 1 u. Deamidation of asparagine results in either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain affects the rate of deamidation. Gly and Ser following Asn in the protein sequence are more susceptible to deamidation. In certain embodiments, the liquid formulations of the present disclosure may be stored under conditions of pH and humidity to prevent deamination of the protein product.

[0101] In some embodiments, the formulation is a lyophilized formulation. In certain embodiments, the formulation is freeze-dried (lyophilized) and is contained in about 12 to 60 vials. In certain embodiments, the formulation is lyophilized and 45 mg of the lyophilized formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the lyophilized formulation is contained in one vial. In certain embodiments, lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. The formulation may 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.

[0102] In some embodiments, the lyophilized formulation comprises a protein as described herein and a cryoprotectant. The cryoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the cryoprotectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative. The amount of sucrose or maltose useful for stabilizing the lyophilized formulation can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be 1:2 to 1:5.

[0103] In certain embodiments, the pH of the formulation prior to lyophilization may be set by the addition of a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma- ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharma- ceutically acceptable base may be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range of the lyophilized drug product may be between 7 and 8.

[0104] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially homogeneous lyophilized cake that maintains an open-pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulation of the present invention may include such a bulking agent.

[0105] In certain embodiments, the lyophilized protein product is composed of an aqueous carrier. The intended aqueous carrier herein is pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. In certain embodiments, the lyophilized formulation of the present disclosure is reconstituted with either sterile water for injection, USP (SWFI), or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder dissolves in solution. In certain embodiments, the lyophilized protein product of the present disclosure is composed of about 4.5 mL of water for injection and diluted with 0.9% saline (sodium chloride solution).

[0106] 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 preparation is mixed with a sterile aqueous carrier before administration. The resulting composition in solid form can be packaged in a number of single dose units, each containing a fixed amount of the above-mentioned drug(s). The solid form composition can also be packaged in a container for variable amounts.

[0107] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0108] A particular dosage can be a uniform dosage per patient, for example 50-5000 mg of protein. Alternatively, a patient's dosage can be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage can include the disease or condition being treated or prevented, the severity of the disease, the route of administration, as well as the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those of skill in the art, especially in light of the dosage information and assays disclosed herein. Dosages can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients can be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex in the patient can be measured to ascertain whether dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are likely to be most 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).

[0109] In general, dosages based on body weight are from about 0.01 μg to about 100 mg / kg body weight, e.g., from about 0.01 μg to about 100 mg / kg body weight, from about 0.01 μg to about 50 mg / kg body weight, from about 0.01 μg to about 10 mg / kg body weight, from about 0.01 μg to about 1 mg / kg body weight, from about 0.01 μg to about 100 μg / kg body weight, from about 0.01 μg to about 50 μg / kg body weight, from about 0.01 μg to about 10 μg / kg body weight, from about 0.01 μg to about 1 μg / kg body weight, from about 0.01 μg to about 0.1 μg g / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg~about 10μg / kg body weight, about 0.1μg~about 1μg / kg body weight, about 1μg~about 100mg / kg body weight, about 1μg~about 50mg / kg body weight, about 1μg~about 10mg / kg body weight, about 1μg~about 1mg / kg body weight, about 1μg~about 1 00 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight. Dosing can be done once or multiple times daily, weekly, monthly, or yearly, or every 2 to 20 years. Those skilled in the art can easily predict the repetition rate of dosing based on the measured residence time and concentration of the targetable construct or complex in the body fluid or tissue.Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion or direct intralesional injection, and may be administered once or more per day, once or more per week, once or more per month, and once or more per year.

[0110] Enhancement of tumor cell death and cancer therapy The present invention provides methods of enhancing tumor cell death and / or treating cancer in a patient. In some embodiments, the methods include exposing tumor and natural killer cells to a multispecific binding protein disclosed herein. In some embodiments, the methods include administering to a patient in need of treatment a therapeutically effective amount of a protein described herein and / or a desired formulation thereof. In these embodiments, the multispecific binding protein comprises a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1, and a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:2. an antigen binding site having a light chain variable domain having the sequence: a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:3; and an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4. an antigen binding site having a light chain variable domain having the sequence: a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5; and an antigen binding site having a light chain variable domain having the sequence: an antigen binding site having a light chain variable domain having the sequence: a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7, 83, 84, 85, 86, or 87;or an antigen-binding site having a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:9, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.

[0111] The type of cancer being treated desirably matches the type of cancer cells to which the multispecific binding proteins disclosed herein bind. Treatment of cancers that express epithelial cell adhesion molecule (EpCAM), e.g., colon cancer, which expresses EpCAM, is desirably treated using the multispecific binding proteins described herein that bind to EpCAM and NKG2D.

[0112] In some embodiments, the patient being treated comprises cancer cells that express one or more of 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 being treated has a solid cancer, such as brain cancer, bladder cancer, breast cancer, lung cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, or uterine cancer.In yet other embodiments, the cancer is angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid carcinoma, biliary tract carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, anorectal carcinoma, anorectal cancer ... cancer), astrocytic tumor, Bartholin adenocarcinoma, basal cell carcinoma, cholangiocarcinoma, bone cancer, bone marrow cancer, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, chorionic plexus papilloma / carcinoma, chronic cancer. lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, Skin cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antral carcinoma, gastric fundus carcinoma, gastrinoma, glioblastoma, glucagonoma , cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatocellular adenoma, hepatocellular adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasm neoplasia), interepithelial squamous cell neoplasm neoplasia), intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, malignant lentigo melanoma, lymphoma, male genital cancer, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, 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, paranasal sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyocarcinoma, mesothelial carcinoma, 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.

[0113] In some embodiments, the patient to be treated has a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0114] In some embodiments, the proteins described herein are used in combination with additional therapeutic agents to treat patients suffering from cancer.Exemplary therapeutic agents that can be used as part of a combination therapy in treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, Included are streptozocin, nimustine, vindesine, flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone-releasing factor, as well as variations of the foregoing agents that may exhibit differential binding to their cognate receptors and have increased or decreased serum half-lives.

[0115] An additional class of drugs that may be used as part of a combination therapy in treating cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. Food and Drug Administration for the treatment of melanoma.

[0116] Still other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0117] Further categories of anti-cancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors and 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors agents, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) inhibitors selected from OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS agonists; and (iii) IL-12, IL-15, GM-CSF, and G-CSF.

[0118] The proteins of the invention may also be used as an adjunct to surgical removal of the primary lesion.

[0119] The amounts of the protein and additional therapeutic agent and the relative timing of administration can be selected to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or pharmaceutical composition(s) containing the therapeutic agents, can be administered in any order, e.g., sequentially, concurrently, together, simultaneously. Furthermore, for example, the protein described herein can be administered during the time that the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa.

[0120] The above description describes multiple aspects and embodiments of the present invention. This patent application expressly contemplates all combinations and permutations of the aspects and embodiments. EXAMPLES

[0121] The invention generally described herein will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0122] Example 1 - Binding affinity of various NKG2D binding domains The kinetics and affinity of the various NKG2D binding domains were evaluated by surface plasmon resonance using a Biacore 8K instrument (GE Healthcare). Anti-human Fc antibodies were 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 dimers were injected over the captured NKG2D antibody and control surfaces at 30 μl / min at 37 °C. The surfaces were regenerated between cycles by rapid injection of 10 mM glycine, pH 1.8. To obtain kinetic rate constants, the double-referenced data were fitted to a 1:1 interaction model using Biacore 8K Evaluation software (GE Healthcare). The equilibrium binding constant K D is the dissociation constant k d and the coupling constant k a The ratio (k d / k a ) As shown in Table 9 and FIG. 3, the binding affinity of the NKG2D-binding domain to NKG2D is in the range of 10 to 62 nM. [Table 9]

[0123] Example 2 - Binding epitope binning of ADI-27744 clones Binning of the ADI-27744 (A44) NKG2D binding domain was performed using Biacore Surface plasmon resonance was performed on a series of antibodies and ULBP6 (NKG2D natural ligand) using an 8K instrument. Briefly, mouse Fc-human NKG2D was captured using anti-mouse Fc antibody immobilized on a CM5 chip at a density of approximately 100 RU. This was followed by continuous injection of antibodies, including NKG2D monoclonal antibodies, including ADI-27744, ADI-27749, F47 (sequences listed below), or 1D11 (commercially available monoclonal NKG2D antibody), ULBP6 (sequences listed below), MS (NKG2D antibody from Novo Nordisk, sequences listed below), and MAB139 (NKG2D antibody from R&D system, clone 149810), at 25° C., 30 μl / min. Biacore 8K evaluation software was used for all data analysis. [Table 10]

[0124] ULBP amino acid sequence ID No. 67 RRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSG

[0125] Figure 4A shows the profile of NKG2D monoclonal antibodies, including ADI-27744, injected over immobilized NKG2D, followed by injection of ULBP6. Figure 4B shows the profile of ULBP6 injected over immobilized NKG2D, followed by injection of NKG2D monoclonal antibodies, including ADI-27744. These results indicate that NKG2D monoclonal antibodies, including the ADI-27744 antigen-binding site, do not block the binding of ULBP6 to NKG2D, i.e., ADI-27744 binds to a different epitope on NKG2D from ULBP6.

[0126] FIG. 4C shows a profile in which MS monoclonal antibody was injected onto NKG2D, followed by ULBP6. MS monoclonal antibody blocks ULBP6 from binding to NKG2D. FIG. 4D-FIG. 4F shows a profile in which MS, 1D11, or MAB139 was injected onto immobilized NKG2D, followed by NKG2D monoclonal antibody, including ADI-27744. FIG. 4G-FIG. 4H shows a profile in which NKG2D monoclonal antibody, including ADI-27744, was injected onto immobilized NKG2D, followed by NKG2D monoclonal antibody, including 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 that is distinct from the MS, 1D11, MAB139, ADI-27749, and F47 binding epitope(s).

[0127] Example 3 - Trispecific binding proteins bind to NKG2D The EL4 mouse lymphoma cell line was genetically engineered to express human NKG2D. As shown in Figure 1, trispecific binding proteins (TriNKETs) each 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 to CD16 were tested for their affinity to extracellular NKG2D expressed on EL4 cells. Binding of the multispecific binding proteins to NKG2D was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells was used to calculate the fold-over-background (FOB).

[0128] The TriNKETs tested include 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 and light chain variable domains of trastuzumab. The CD33 binding domain consists of a heavy chain variable domain and a light chain variable domain as listed below. SEQ ID NO:49 [ka] SEQ ID NO:50 [ka]

[0129] All TriNKETs bind to NKG2D on EL4 cells, but with different affinities. CD33-TriNKET-A44 shows the same binding profile as HER2-TriNKET-A44, CD33-TriNKET-A49 shows the same binding profile as HER2-TriNKET-A49, and CD33-TriNKET-F63 shows the same binding profile as HER2-TriNKET-F63. The NKG2D binding affinity for each clone was similar between cells expressing human and mouse NKG2D (Figures 5-6).

[0130] Example 4 - Trispecific binding proteins bind to human tumor antigens Trispecific binding proteins bind to CD33 The CD33-expressing human AML cell line MV4-11 was used to assay binding of TriNKET to tumor-associated antigens. TriNKET and parental CD33 monoclonal antibodies were incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and fold over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and parental CD33 antibody normalized to secondary antibody control.

[0131] CD33-TriNKET-A44, CD33-TriNKET-A49, and CD33-TriNKET-F63 show comparable levels of binding to CD33 compared to the parental CD33 antibody (Figure 7).

[0132] Trispecific binding proteins bind to HER2 Binding of TriNKET to tumor-associated antigens was assayed using human cancer cell lines expressing HER2. 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 optionally a parental HER2 monoclonal antibody (trastuzumab) were incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and fold over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and trastuzumab normalized to secondary antibody control.

[0133] HER2-TriNKET-A44, HER2-TriNKET-A49, and HER2-TriNKET-F63 show comparable levels of binding to HER2 expressed in 786-O cells compared to trastuzumab (Figure 8). Binding to HER2 expressed in NCI-H661 cells by HER2-TriNKET-E79 is shown (Figure 9).

[0134] Example 5 - Trispecific binding proteins activate NK cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated using negative selection with magnetic beads from PBMCs, and the purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. IL-2-activated NK cells were used within 24–48 h after activation.

[0135] Human cancer cells expressing tumor antigens were harvested and cultured at 2 × 10 6 Activated NK cells were harvested, washed, and resuspended in culture medium at 2 × 10 cells / mL. Monoclonal antibodies targeting tumor antigens or TriNKET were diluted in culture medium. 6 The tumor cells were resuspended in culture medium at 1000 cells / mL. The cancer cells were then mixed with monoclonal antibody / TriNKET and activated NK cells in the presence of IL-2. Brefeldin A and monensin were also added to the mixed cultures to block protein transport out of the cells for intracellular cytokine staining. Fluorophore-conjugated anti-CD107a was added to the mixed cultures, and the cultures were incubated for 4 hours before samples were prepared for FACS analysis using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD107a and IFN-gamma staining were used to assess NK cell activation by CD3. - CD56 + The increase in CD107a / IFN-gamma double positive cells indicates better NK cell activation by engaging two activating receptors instead of one.

[0136] TriNKET mediates activation of human NK cells co-cultured with HER2-expressing NCI-H661 cells (Figure 10) and SkBr-3 cells (Figure 11), respectively, as indicated by increased CD107a degranulation and IFN-gamma production. Compared to the monoclonal antibody trastuzumab, TriNKET shows superior activation of human NK cells in the presence of human cancer cells.

[0137] TriNKET mediates activation of human NK cells co-cultured with human AML Mv4-11 cells expressing CD33, as indicated by increased CD107a degranulation and IFN-gamma production, respectively (Figure 12). Compared to monoclonal anti-CD33 antibodies, TriNKET shows superior activation of human NK cells in the presence of human cancer cells.

[0138] Example 6 - Trispecific binding proteins enable cytotoxicity of targeted cancer cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated using negative selection with magnetic beads from PBMCs, and the purity of isolated NK cells was routinely >90%. Isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

[0139] To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, the cytoTox 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 and lysed at 1-2 × 10 5 Resting and / or activated NK cells were harvested, washed, and resuspended in culture medium at 10 cells / mL. 5 -2.0×10 6The NK cells were resuspended in the same culture medium as the cancer cells at 1000 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 with or without TriNKET, which targets tumor antigens expressed on the cancer cells. After 3 hours and 15 minutes of incubation at 37°C and 5% CO2, 10x lysis buffer was added to wells containing only cancer cells, wells containing only medium for maximum lysis, and negative reagent controls, respectively. The plates were then returned to the incubator for an additional 45 minutes, reaching 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 substrate for development. The new plate was incubated at room temperature for 30 minutes and the absorbance at 492 nm was read on a SpectraMax i3x. The percentage of specific lysis of cancer cells was calculated as follows: specific lysis (%) = ((experimental lysis - spontaneous lysis from NK cells only - spontaneous lysis from cancer cells only) / (maximal lysis - negative reagent control)) * 100%.

[0140] TriNKET mediates the cytotoxicity of human NK cells against the CD33 positive Molm-13 human AML cell line. As shown in FIG. 13, resting human NK cells are mixed with Molm-13 cancer cells, and TriNKET can enhance the cytotoxic activity of resting human NK cells in a dose-responsive manner against cancer cells. The dotted line shows the cytotoxic activity of resting NK cells without TriNKET. As shown in FIG. 14, resting human NK cells are mixed with Molm-13 cancer cells, and TriNKET further enhances the cytotoxic activity of activated human NK cells in a dose-responsive manner against cancer cells.

[0141] TriNKET mediates the cytotoxicity of human NK cells against the HER2-positive 786-O human renal cell carcinoma cell line. As shown in FIG. 15, resting human NK cells are mixed with 786-O cancer cells, and TriNKET can enhance the cytotoxic activity of resting human NK cells in a dose-responsive manner against cancer cells (each TriNKET is added at 5, 1, 0.2 μg / ml in the assay, and these results are represented in three columns from left to right for each TriNKET in FIG. 15-16). The dotted line indicates the cytotoxic activity of resting NK cells against 786-O cells in the absence of TriNKET. As shown in FIG. 16, activated human NK cells are mixed with 786-O cells, and TriNKET further enhances the cytotoxic activity of activated human NK cells in a dose-responsive manner against cancer cells. The dotted line indicates the cytotoxic activity of activated NK cells against 786-O cells in the absence of TriNKET.

[0142] Example 7 - Variants of ADI-27749 and TriNKETs containing variants As noted above, ADI-27749 (A49) comprises, inter alia, a heavy chain CDR3 having the amino acid sequence of 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 to generate the NKG2D antibodies A49MQ, A49ML, A49MI, A49MF, and A49MV, respectively, having the corresponding heavy chain variable region, light chain variable region, and CDR sequences provided in Table 1.

[0143] The effect of these mutations on hydrophobicity was analyzed using the MOE2018.01 program with the parameter settings of avg_pro_patch_cdr_hyd. The protein builder module was used to mutate residues and minimize the entire Fab after stitching together all residues. Dynamic specific sampling was performed using the lowMD protocol in BIOMOE. As shown in Table 11, these mutations did not have a substantial negative effect on the predicted hydrophobicity of the A49 Fab. [Table 11]

[0144] The hydrophobicity of TriNKET containing A49 ("TriNKET A") and a mutant form of TriNKET A with substitutions of Ile, Leu, Val, Gln, or Phe for Met ("TriNKET A*") was tested by analytical hydrophobic interaction chromatography (HIC). Each of the TriNKETs also bound to the first tumor antigen. As shown in Table 12, the retention time of TriNKET A* was similar to that of TriNKET A. [Table 12]

[0145] The thermal stability of TriNKET A and TriNKET A* was examined by differential scanning calorimetry (DSC) in 20 mM histidine, 260 mM sucrose, and 0.005% PS-80 at pH 6.0. m The values ​​of are shown in Table 13. m are the midpoint transition temperatures of the individual domains. The M102 mutation shifts the two most stable transitions (T m3 and T m4 )T m The previous transition (T m1 and T m2) was not affected. Thus, the M102 mutation had only a minor effect on the overall thermostability of TriNKET A. [Table 13]

[0146] Binding of TriNKET A and TriNKET A* to a fusion protein of human NKG2D and mouse Fc ("mFc-hNKG2D") was characterized by surface plasmon resonance (SPR) at 37°C. Two different fits were used to obtain equilibrium affinity data: a steady-state affinity fit and a kinetic fit (Figure 32). Rate and equilibrium affinity constants were calculated and averaged from data from two independent experiments for TriNKET A* and from three independent experiments for TriNKET A. [Table 14]

[0147] As shown in Table 14, the equilibrium affinity constants (K D ) are highly similar between replicates, suggesting high confidence in the measured parameters. The M102 variant has less than 2-fold lower affinity for human NKG2D compared to TriNKET A. The K D is (6.87±0.16)×10 -7 M, but TriNKET A's K D is (4.87±0.83)×10 -7 M (calculated from affinity fit). DSimilar differences in affinity were observed when A49-containing TriNKET A* was calculated from the kinetic fits. The stoichiometry of NKG2D binding to TriNKET A* was 0.85 ± 0.12, similar to 1.01 ± 0.11 for TriNKET A, confirming that each NKG2D dimer binds one molecule of TriNKET A*. This suggests that the M102 mutation had only a minor effect on the binding of A49-containing TriNKET to human NKG2D.

[0148] Finally, the effect of the M102 mutation on TriNKET potency was evaluated in a cytotoxicity assay. Briefly, KHYG-1 cells expressing a high-affinity variant of CD16a(158V) were generated by retroviral transduction. After transduction, 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, cells were harvested from culture, pelleted, washed three times in culture medium without IL-2, resuspended in culture medium without IL-2, and rested for 24 h.

[0149] Human cancer cell lines expressing the target of interest were harvested from culture. Cells were washed with HBS and incubated for 10 min for labeling with BATDA reagent (Perkin Elmer C136-100). 6 The cells were resuspended in growth medium at 0.5 × 10 cells / mL. The manufacturer's instructions were followed for labeling of target cells. After labeling, cells were washed three times with HBS and resuspended in culture medium at 0.5 × 10 5 Resuspended at 100 cells / mL. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate.

[0150] TriNKET was serially diluted in culture medium and 50 μl of diluted TriNKET was added to each well. Resting NK cells were harvested from the culture, washed, and diluted to 1.0 × 10 6The NK cells were resuspended at 100 cells / mL. 50 μl of NK cells were added to each well of the plate to obtain a desired E:T ratio of 10:1, making a total of 200 μl of culture in each well. Plates were incubated at 37°C, 5% CO2 for 2-3 hours.

[0151] After incubation, plates were removed from the incubator and cells were pelleted by centrifugation at 200 × g for 5 min. 20 μl of culture supernatant was transferred to a sterile microplate provided by the manufacturer. Supernatants from labeled cells incubated alone without NK cells were used to measure spontaneous release of TDA. Supernatants from labeled cells incubated with 1% Triton-X were used to measure maximum lysis of target cells. Supernatants from labeled cells prior to 2-3 h of incubation were used to measure background and for quality control purposes.

[0152] 200 μl of room temperature europium solution (Perkin Elmer C135-100) was added to each well containing culture supernatant. Plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. Fluorescence was measured using a SpectraMax i3X instrument. Fluorescence levels represented the lysis of target cells. Values ​​for specific lysis (%) were calculated as follows: specific lysis (%) = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) x 100%.

[0153] To measure the activity of TriNKET A and TriNKET A*, cell lines expressing the first tumor antigen were selected as target cells. Two different lots of TriNKET A were used for comparison. The values ​​of specific lysis (%) were plotted in FIG. 33, and the EC50 and maximum percentage of specific lysis (%) were summarized in Table 15. The EC50 and maximum percentage of specific lysis (%) of TriNKET A* were similar to those of TriNKET A, suggesting that the M102 mutation did not affect the biological activity of TriNKET A. [Table 15]

[0154] To confirm that the lack of effect of the M102 mutation on TriNKET activity was not tumor antigen specific, TriNKET A and TriNKET A* were constructed, which bind to a second, different tumor antigen. The activity of the two TriNKETs was compared in a cytotoxicity assay using a cell line expressing a second tumor antigen as the target cell and KHYG-1-CD16V cells as the effector cell. As shown in Figure 34, TriNKET A* showed activity comparable to that of TriNKET A.

[0155] Incorporation by Reference The entire disclosures of each of the patent documents and scientific articles referenced herein are incorporated by reference for all purposes.

[0156] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

[Claim 1] The invention described in the specification.

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