Antibody variable domain targeting the NKG2D receptor

Abstract

Antibody heavy chain variable domains that can pair with antibody light chain variable domains to form antigen-binding sites that target the NKG2D receptor on natural killer cells have been described. Proteins containing NKG2D antigen-binding sites, pharmaceutical compositions thereof, and therapeutic methods thereof, including cancer treatment, have also been described. Antibodies against NKG2D have been identified to offer important advantages in the design of therapeutic agents. For example, some of these antibodies not only bind to human NKG2D receptors, but also have one or more additional advantages, such as the ability to stimulate the receptor, the ability to compete with natural ligands for receptor binding, and / or the ability to cross-react with NKG2D from other species, such as cynomolgus monkeys. These advantages can be achieved across the entire range of NKG2D affinities. [Selection diagram] None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 628,161, filed on Feb. 8, 2018 (the disclosure of which is hereby incorporated by reference in its entirety for all purposes) and U.S. Provisional Patent Application No. 62 / 716,259, filed on Aug. 8, 2018.

[0002] Field of the Invention The present invention provides a protein having an antibody heavy - chain variable domain and an antibody light - chain variable domain that can pair to form an antigen - binding site targeting the natural killer group 2D (NKG2D) receptor on natural killer cells, a pharmaceutical composition comprising such a protein, and a method of treatment using such a protein and pharmaceutical composition for the treatment of cancer.

Background Art

[0003] Cancer remains a serious health problem despite substantial research efforts and scientific progress 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 a leading cause of death in women. Current treatment options for these cancers are not effective for all patients and / or have substantial side effects. Treating other types of cancer using existing treatment options remains difficult.

[0004] Cancer immunotherapy is desirable because it is highly specific and can use a patient's own immune system to promote the destruction of cancer cells. Fusion proteins, such as bispecific T - cell engagers, are cancer immunotherapies 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 infiltrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for priming to distinguish tumor cells from T cells. Activated NK cells kill target cells in a similar manner to cytotoxic T cells, i.e., via cytotoxic granules containing perforin and granzymes, and via the death receptor pathway. Activated NK cells also secrete inflammatory cytokines such as IFN-gamma, and chemokines that promote the recruitment of other white blood cells to the target tissue. NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by the activation of killer cell immunoglobulin-like receptors (KIR). Alternatively, when NK cells encounter cancer cells, they are activated via 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 the stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein expressed by essentially all natural killer cells in which NKG2D functions as an activating receptor. The function of regulating NK cell function via NKG2D is useful in various therapeutic situations including malignant tumors.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Means for Solving the Problems

[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 also have one or more additional advantages such as the ability to stimulate the receptor, the ability to compete with natural ligands for binding to the receptor, and / or the ability to cross-react with NKG2D from other species such as cynomolgus monkeys. These advantages can be achieved across the entire range of NKG2D affinities.

[0008] Accordingly, one aspect of the present 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, as the first complementarity determining region 1 (“CDR1”), the amino acid sequence YTFTSYYMH (SEQ ID NO: 11), as the second CDR (“CDR2”), IINPSGGSTSYAQKFQG (SEQ ID NO: 12), and as the third CDR (“CDR3”), ARGAPNYGDTTHDYYYMDV (SEQ ID NO: 13), of SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence SYYMH (SEQ ID NO: 45), as CDR2, IINPSGGSTSYAQKFQG (SEQ ID NO: 12), and as CDR3, GAPNYGDTTHDYYYMDV (SEQ ID NO: 68), 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, as the first complementarity-determining region ("CDR1"), the amino acid sequence YTFTGYYMH (SEQ ID NO: 17), as the second CDR ("CDR2"), the amino acid sequence WINPNSGGTNYAQKFQG (SEQ ID NO: 18), and as the third CDR ("CDR3"), the amino acid sequence ARDTGEYYDTDDHGMDV (SEQ ID NO: 19), of SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence GYYMH (SEQ ID NO: 92), as CDR2, the amino acid sequence WINPNSGGTNYAQKFQG (SEQ ID NO: 18), and as CDR3, the amino acid sequence DTGEYYDTDDHGMDV (SEQ ID NO: 69), of SEQ ID NO: 3.

[0010] Another aspect of the present 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, as the first complementarity determining region (“CDR1”), the amino acid sequence FTFSSYAMS (SEQ ID NO: 23), as the second CDR (“CDR2”), AISGSGGSTYYADSVKG (SEQ ID NO: 24), and as the third CDR (“CDR3”), AKDGGYYDSGAGDY (SEQ ID NO: 25), of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence SYAMS (SEQ ID NO: 47), as CDR2, AISGSGGSTYYADSVKG (SEQ ID NO: 24), and as CDR3, DGGYYDSGAGDY (SEQ ID NO: 70), 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, as the first complementarity determining region ("CDR1"), the amino acid sequence FTFSSYSMN (SEQ ID NO: 29), as the second CDR ("CDR2"), the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 30), and as the third CDR ("CDR3"), the amino acid sequence ARGAPMGAAAGWFDP (SEQ ID NO: 31), of SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence SYSMN (SEQ ID NO: 48), as CDR2, the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 30), and as CDR3, the amino acid sequence GAPMGAAAGWFDP (SEQ ID NO: 71), of SEQ ID NO: 7.

[0012] Another aspect of the present 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, as CDR1, the amino acid sequence FTFSSYSMN (SEQ ID NO: 29), as CDR2, the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 30), and as CDR3, the amino acid sequence ARGAPIGAAAGWFDP (SEQ ID NO: 77) of SEQ ID NO: 85. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence SYSMN (SEQ ID NO: 48), as CDR2, the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 30), and as CDR3, the amino acid sequence GAPIGAAAGWFDP (SEQ ID NO: 78) 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, as the first complementarity determining region (“CDR1”), the amino acid sequence YTFTSYYMH (SEQ ID NO: 35), as the second CDR (“CDR2”), the amino acid sequence IINPSGGSTSYAQKFQG (SEQ ID NO: 36), and as the third CDR (“CDR3”), the amino acid sequence AREGAGFAYGMDYYYMDV (SEQ ID NO: 37) of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises, as CDR1, the amino acid sequence SYYMH (SEQ ID NO: 45), as CDR2, the amino acid sequence IINPSGGSTSYAQKFQG (SEQ ID NO: 36), and as CDR3, the amino acid sequence EGAGFAYGMDYYYMDV (SEQ ID NO: 72) of SEQ ID NO: 9.

[0014] The variable domain of the heavy chain of the present invention can optionally be coupled to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region containing a hinge, CH2 domain, and CH3 domain, with or without an antibody constant region such as 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 rabbit, dog, cat, mouse, or horse. One or more variants can be included in the constant region at, for example, 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 compared 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 can 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 can be amino acids E123, F116, S176, V163, S174, and / or T164.

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

[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 pair 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 the first complementarity determining region (“CDR”), GASTRAT (SEQ ID NO: 21) as the second CDR, and QQDDYWPPT (SEQ ID NO: 22) as the third CDR.

[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 pair 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 pair 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 the first complementarity determining region ("CDR"), DASNRAT (SEQ ID NO: 39) as the second CDR, and QQSDNWPFT (SEQ ID NO: 40) as the third CDR.

[0021] When the heavy chain variable domain is combined with the light chain variable domain to form an antigen-binding site capable of binding to NKG2D, the antigen-binding site can be incorporated into various structures, such as a typical antibody structure having two identical heavy chains and two identical light chains, to form a pair of antigen-binding sites capable of binding to NKG2D, bispecific, trispecific, tetra-specific, or other multi-specific antibodies, or smaller structures such as scFv (where the heavy chain variable domain is linked to the light chain variable domain).

[0022] In some embodiments, any NKG2D antigen-binding site disclosed in the present invention is included in a protein that also includes a separate antigen-binding site that binds to a tumor-associated antigen, enabling the protein to interact with NK cells and tumor cells simultaneously. The tumor-associated antigen can be, for example, CD33, HER2, EpCAM, CD2, CD3, CD8, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD37, CD38, CD40, CD45RO, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CSAp, CA-125, TAG-72, EFGR / ERBB1, IGF1R, HER2, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA, and IL-6, MAGE-A3, B7.1, B7.2, CTLA4, or PD1.

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

[0024] Another aspect of the present invention provides a method of enhancing tumor cell death in a patient and treating cancer. The method includes administering to a patient in need of treatment a therapeutically effective amount of the protein described herein to treat 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 is 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, and the antibody heavy chain variable domain according to Item 1. (Item 3) The amino acid sequence is 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, and the antibody heavy chain variable domain according to Item 1. (Item 4) The amino acid sequence is 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, and the antibody heavy chain variable domain according to Item 1. (Item 5) The amino acid sequence is 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, and the antibody heavy chain variable domain according to Item 1. (Item 6) The amino acid sequence is 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, and the antibody heavy chain variable domain according to Item 1. (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 is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 13, and the antibody heavy chain variable domain according to Item 7. (Item 9) The amino acid sequence is 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, The antibody heavy chain variable domain according to item 7, comprising the 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 is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 19, and is the antibody heavy chain variable domain according to item 10. (Item 12) The amino acid sequence is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 69, and is the antibody heavy chain variable domain according to item 10. (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 is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 25, and is the antibody heavy chain variable domain according to item 13. (Item 15) The amino acid sequence is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 70, and is the antibody heavy chain variable domain according to item 13. (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 is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 37, and is the antibody heavy chain variable domain according to item 16. (Item 18) The amino acid sequence is 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, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 72, and is the antibody heavy chain variable domain according to item 16. (Item 19) An antibody heavy chain comprising an 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) The antibody heavy chain according to item 19, wherein the antibody constant region is a human IgG constant region comprising a hinge, CH2, and CH3 domains. (Item 21) The antibody heavy chain according to item 20, wherein the antibody constant region further comprises a CH1 domain and is a human IgG constant region. (Item 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) The antibody heavy chain according to item 22, wherein the amino acid sequence that is at least 90% identical to the antibody constant region is different from the amino acid sequence of the IgG1 constant region in 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 antibody heavy chain according to item 23, wherein the amino acid sequence that is at least 90% identical to the antibody constant region is different from the amino acid sequence of the IgG1 constant region by a substituent selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E, or any combination thereof. (Item 25) An antigen-binding site comprising an 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) The antigen-binding site according to item 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) As measured by surface plasmon resonance, having a K of 2 to 120 nM D The antigen-binding site according to item 25 or 26. (Item 28) An antigen-binding site comprising an 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) The antigen-binding site according to 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) As measured by surface plasmon resonance, having a K of 5 to 500 nM D The antigen-binding site according to item 28 or 29. (Item 31) An antigen-binding site comprising an 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) The antigen-binding site according to item 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) As measured by surface plasmon resonance, having a K of 6 to 600 nM D The antigen-binding site according to item 31 or 32. (Item 34) An antigen-binding site comprising an 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) The antigen-binding site according to item 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) As measured by surface plasmon resonance, K of 1 to 100 nM D The antigen-binding site according to item 34 or 35, having (Item 37) An antigen-binding site comprising an 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) The antigen-binding site according to item 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) As measured by surface plasmon resonance, K of 6 to 600 nM D The antigen-binding site according to item 37 or 38, having (Item 40) A protein comprising an antigen-binding site according to any one of items 25 to 39 that binds to NKG2D and a further antigen-binding site. (Item 41) The protein according to item 40, wherein the further antigen-binding site binds to a tumor-associated antigen. (Item 42) 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. The protein according to item 41. (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. 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. The protein according to any one of items 40 to 42. (Item 44) The protein according to item 43, wherein the first antibody constant region and the second antibody constant region form a complex capable of binding to CD16. (Item 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, CH2, and CH3 domain. (Item 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) 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 each at least 90% identical to the human IgG1 constant region. (Item 48) The amino acid sequence of the first 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, K392, T394, D399, S400, D401, F405, Y407, K409, T411, or K439, or any combination thereof, The protein according to item 47, wherein the amino acid sequence of the second antibody constant region is different 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) The protein according to item 47, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region at T366, L368, or Y407, or any combination thereof. (Item 50) The protein according to item 47, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at T366, L368, or Y407, or any combination thereof, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366. (Item 51) The amino acid sequence of the first antibody constant region is different 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 is different 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. The protein according to item 47. (Item 52) The amino acid sequence of the first antibody constant region is different 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 is different 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. The protein according to item 47. (Item 53) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at T366, N390, K392, K409, or T411, or any combination thereof. The protein according to item 47. (Item 54) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at L351, D399, S400, or Y407, or any combination thereof. The protein according to item 47. (Item 55) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at Q347, E357, D399, or F405, or any combination thereof, the protein according to item 47. (Item 56) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at Y349, K360, Q347, or K409, or any combination thereof, the protein according to item 47. (Item 57) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at D356, E357, or D399, or any combination thereof, the protein according to item 47. (Item 58) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at K370, K392, K409, or K439, or any combination thereof, the protein according to item 47. (Item 59) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at Y349, L351, L368, K392, or K409, or any combination thereof, the protein according to item 47. (Item 60) The amino acid sequence of the first antibody constant region is different 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 is different from the amino acid sequence of the IgG1 constant region at L351, E356, T366, or D399, or any combination thereof. The protein according to item 47. (Item 61) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region by the S354C substituent, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region by the Y349C substituent. The protein according to item 47. (Item 62) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region by the Y349C substituent, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region by the S354C substituent. The protein according to item 47. (Item 63) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region by the K360E and K409W substituents, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region by the O347R, D399V, and F405T substituents. The protein according to item 47. (Item 64) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region by the O347R, D399V, and F405T substituents, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region by the K360E and K409W substituents. The protein according to item 47. (Item 65) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region by the T366W substituent, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region by the T366S, T368A, and Y407V substituents. The protein according to item 47. (Item 66) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region with T366S, T368A, and Y407V substituents, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region with T366W substituent. The protein according to item 47. (Item 67) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region with T350V, L351Y, F405A, and Y407V substituents, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region with T350V, T366L, K392L, and T394W substituents. The protein according to item 47. (Item 68) The amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region with T350V, T366L, K392L, and T394W substituents, and the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region with T350V, L351Y, F405A, and Y407V substituents. The protein according to item 47. (Item 69) The protein according to any one of items 40 to 42, further comprising an antigen-binding site capable of binding to CD16. (Item 70) A protein comprising an antigen-binding site that competes with the binding of 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 to human and / or cynomolgus macaque NKG2D. (Item 71) A protein comprising an antigen-binding site that competes with the binding of 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 to human and / or cynomolgus macaque NKG2D. (Item 72) A protein comprising an antigen-binding site that competes with the binding of 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 to human and / or cynomolgus macaque NKG2D. (Item 73) A protein comprising an antigen-binding site that competes with the binding of 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 to human and / or cynomolgus macaque NKG2D. (Item 74) A protein comprising an antigen-binding site that competes 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 for binding to human and / or cynomolgus macaque NKG2D. (Item 75) A protein comprising an antigen-binding site that competes 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 for binding to human and / or cynomolgus macaque NKG2D. (Item 76) A pharmaceutical formulation comprising the protein according to any one of Items 40 to 75 and a pharmaceutically acceptable carrier. (Item 77) A cell comprising one or more nucleic acids encoding the protein according to any one of Items 40 to 75. (Item 78) A method for enhancing tumor cell death, comprising exposing a tumor and natural killer cells to the 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 pharmaceutical formulation according to Item 76. (Item 80) The method according to Item 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 sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated by bone marrow-derived suppressor cells, cancer associated with extracellular matrix deposition, cancer associated with a high level of reactive stroma, and cancer associated with angiogenesis.

Brief Description of the Drawings

[0025]

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[0026] The present invention provides a protein having an antibody heavy chain domain and a light chain variable domain that can pair to form an antigen-binding site targeting the natural killer group 2D (NKG2D) receptor on natural killer cells, a pharmaceutical composition containing such a protein, and a method of treatment using such a protein and pharmaceutical composition for the treatment of cancer, including. Various aspects of the present invention are described in the following chapters. However, the aspects of the present invention described in a particular chapter are not limited to any particular chapter.

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

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

[0029] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), more preferably humans.

[0030] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, 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 found naturally between and adjacent to hypervariable regions within an immunoglobulin. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and each of the three hypervariable regions of the heavy and light chains is called a "complementary determining region" or "CDR." In certain animals such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain that provides a "single-domain antibody." The antigen-binding site can 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 light chain variable domain to the heavy 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 particular formulation or route of administration. As used herein, the term "treating" includes any effect that results in the improvement of a condition, disorder, disease, etc., e.g., any effect such as reducing, lessening, modulating, ameliorating, or eliminating, or improving the symptoms thereof.

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

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

[1975] .

[0034] Throughout this description, when a composition is described as having, including, or comprising a particular component, or a process and method are described as having, including, or comprising particular steps, it is also contemplated that there exist compositions of the invention that consist essentially of, or consist of, the recited components, as well as processes and methods according to the invention that consist essentially of, or consist of, the recited process steps.

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

[0036] The present invention provides an antigen-binding site that binds to NKG2D and an antigen heavy chain variable domain that can be used to create such an antigen-binding site.

[0037] The antibody heavy chain variable domain and light chain variable domain that pair to form an antigen-binding site capable of binding and activating the NKG2D receptor are identified and provided in Table 1 below. Unless otherwise specified, 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 or more advantages of the above antibody heavy chain variable domain amino acid sequences are that they can bind to human and cynomolgus NKG2D, stimulate the receptor, and compete with the natural ligand for binding to the receptor. Additional antigen-binding sites that bind to NKG2D and share one or more of these properties are also particularly useful and can be identified by binding competition assays known in the art. For example, additional antigen-binding sites can be identified by competition with ADI-29379, ADI-29463, ADI-27744, ADI-27749, or ADI-29378 for binding to both human and optionally cynomolgus NKG2D.

[0039] Another advantage of the NKG2D binding site comprising the above-mentioned 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 binds to NKG2D having a K D of 0.1 to 1000 nM. In some embodiments, the NKG2D binding site binds to NKG2D having a K D of 1 to 500 nM. In some embodiments, the NKG2D binding site binds to NKG2D having a K D of 5 to 100 nM. In some embodiments, the NKG2D binding site binds to NKG2D having a K D of 10 to 62 nM.

[0040] In certain embodiments, the present 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, the 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: 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, the antigen-binding site comprising an antibody 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 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 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 antibody 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: 4. In certain embodiments, the 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: 3 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 92, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 18, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 69. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 present invention provides 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: 5, and an antibody 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: 6. In certain embodiments, the 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: 5 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 93, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 24, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 70. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 present invention provides 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: 7, and an antibody 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: 8. In certain embodiments, the 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: 7 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 71. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 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 present invention provides 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: 83, and an antibody 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: 8. In certain embodiments, the 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: 83 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence 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, the antigen-binding site comprising an antibody 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: 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 the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence 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 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 present 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 the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence 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 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, the antigen-binding site comprising an antibody 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: 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 present 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 the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antigen-binding site comprising an antibody heavy chain variable domain having an amino acid sequence 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 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 94, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In certain embodiments, the antigen-binding site comprising an antibody light chain variable domain having an amino acid sequence 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 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 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 an antibody 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: 10. In certain embodiments, the 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: 9 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 91, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 36, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 72. In certain embodiments, the antigen-binding site comprising an antibody light-chain variable domain having an amino acid sequence 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 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 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 antibody 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: 6, which does not block the binding of anti-NKG2D antibodies MS, 1D11, and MAB139 to NKG2D.

[0052] In an embodiment, the present invention provides 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: 7, 83, 84, 85, 86, or 87, and an antibody 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: 8, which does not block the binding of 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: 5 to NKG2D, and an antibody 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: 6.

[0053] 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: 5, and an antibody 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: 6, binds to a specific epitope on NKG2D that is different from the MS, 1D11, MAB139, ADI-27749, and F47 binding epitope(s).

[0054] Antibodies and Multispecific Binding Proteins In some embodiments of the invention, the NKG2D antigen-binding site formed by pairing an antibody heavy chain variable domain with the light chain variable domains described herein can be included in larger proteins such as intact antibodies, multispecific binding proteins, or multispecific binding antibodies. 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-125, TAG-72, EFGR / ERBB1, IGF1R, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA, and IL-6, MAGE-A3, B7.1, B7.2, CTLA4, or PD1. The binding of NKG2D and the multispecific protein to tumor-associated antigens on cancer cells brings the cancer cells closer to natural killer cells and promotes the direct or indirect destruction of cancer cells by 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 the antibody Fc region, with or without the CH1 domain. In some embodiments, the antibody Fc region is derived from the Fc region of human and / or other mammalian immunoglobulins. Within the Fc region, binding of CD16 is known to be mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mediated by amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al, Nature, 406(6793):267-273). Based on known domains and amino acid residues, in some embodiments, mutations can be selected within the CD16 binding domain to enhance or decrease the binding affinity for CD16. The selection method is a method well known in the art, such as a phage display library or a yeast surface display cDNA library. Based on the known three-dimensional structure of the interaction by those skilled in the art, an appropriate selection method can also be designed.

[0056] The multispecific binding proteins described herein can take various forms. For example, one form 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 includes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain includes a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, 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 form 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 includes a first Fc (hinge-CH2-CH3) domain fused to a single-chain variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain that pair and bind to NKG2D via a linker or an antibody hinge. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The immunoglobulin light chain includes 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 forms of the multispecific binding protein can be devised by combining various forms of the NKG2D-binding fragments described herein.

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

[0059] In some embodiments, the multispecific binding protein is in the form of a trimab, 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 parental antibodies. The trimab form is a heterodimeric construct that includes 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0060] In some embodiments, the multispecific binding protein is in the form of a Knob-into-Hole (KIH) common light chain (LC) that includes KIH technology. KIH involves engineering the C H 3 domain to create a "knob" or "hole" in each heavy chain to facilitate heterodimerization. The concept behind the "Knob-into-Hole (KiH)" Fc technology was to introduce a "knob" into one CH3 domain (CH3A) by replacing a small residue with a bulky residue (i.e., T366W in EU numbering CH3A ). To create a complementary "hole" surface, the other CH3 domain (CH3B) was engineered to have small adjacent residues closest to the knob (i.e., T366S / L368A / Y407V CH3B) by replacement. The "hole" mutation was optimized by structured guide 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). The X-ray crystal structures of the KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) 426(9):1947 - 57, Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol Immunol (2014) 58(1):132 - 8) showed that heterodimerization is thermodynamically supported by hydrophobic interactions driven by steric complementarity at the core interface between the CH3 domains, whereas the 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 multispecific binding protein is in the form of a Dual Variable Domain Immunoglobulin (DVD-Ig™), which combines the target binding domains of two monoclonal antibodies via a flexible, naturally occurring linker to generate a tetravalent IgG-like molecule. The DVD-Ig™ is a homodimeric construct in which the antigen 2 targeting the variable domain is fused to the N-terminus of the variable domain of the antigen 1 targeting the Fab, and the construct contains a normal Fc.

[0062] In some embodiments, the multispecific binding protein is in the form of an Orthogonal Fab (Ortho-Fab), a heterodimeric construct comprising two Fabs that bind to target 1 and target 2 fused to the Fc. The LC-HC pairing is ensured by the orthogonal interface. The heterodimerization is ensured by a mutation of 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), in the structure-based regional design, a mutation complementary only to one Fab is introduced into the interface of the LC and HC VH-CH1 without making any changes to the other Fab.

[0063] In some embodiments, the multispecific binding protein is in the 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in the ES form, a heterodimeric construct comprising two different Fabs that bind to target 1 and target 2 fused to the Fc. The heterodimerization is ensured by electrostatic steering of a mutation of the Fc.

[0064] In some embodiments, the multispecific binding protein is a heterodimeric construct having two different Fabs fused to an Fc stabilized by a heterodimerization mutation. K In the λ-Body format, antigen 1 that targets Fab1 contains kappa LC, while antigen 2 that targets the second Fab contains lambda LC.

[0065] In some embodiments, the multispecific binding protein is in the Fab-arm exchange format (an antibody that results in a bispecific antibody by exchanging Fab arms by exchanging the heavy chain and the attached light chain (half molecule) with a heavy-light chain pair from another molecule). The Fab-arm exchange format (cFae) is a heterodimer containing two Fabs that bind to target 1 and target 2, and an Fc stabilized by a heterodimerization mutation.

[0066] In some embodiments, the multispecific binding protein is in the SEED Body format, which is a heterodimer containing two Fabs that bind to target 1 and target 2, and an Fc stabilized by a heterodimerization mutation. The strand exchange engineered domain (SEED) platform was designed to generate asymmetric bispecific antibody-like molecules with functions that expand the therapeutic use 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 enables the efficient generation of AG / GA heterodimers while avoiding the 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, in which the 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 containing two different scFabs that bind to 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 multispecific binding protein is in the Cov-X-Body form (in bispecific CovX-Bodies, two different peptides are linked using a branched azetidinone linker and site-specifically fused to a scaffold antibody under mild conditions. The pharmacophore is involved in functional activity, while the antibody scaffold provides a long half-life and an Ig-like distribution. By chemically optimizing the pharmacophore or replacing it with another pharmacophore, an optimized or unique bispecific antibody can be generated. (Doppalapudi VR et al., PNAS (2010), 107(52);22611-22616).

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

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

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

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

[0073] Heterodimeric antibody heavy chain Assembly of the heterodimeric antibody heavy chain 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. Preferential assembly of the heterodimeric heavy chains within the multispecific binding proteins described herein can be facilitated by incorporating different pairs of amino acid substitutions into the first CH3 domain within the first heavy chain polypeptide and the second CH3 domain within the second heavy chain polypeptide, and as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, US14 / 830336, these two chains can selectively heterodimerize with each other. In some embodiments, the multispecific binding protein includes the Fc domain of human IgG1. Various examples of amino acid substitutions within pairs of human IgG1 Fc domains are shown below. Each position of the amino acid substitution is numbered according to the EU index, similar to Kabat.

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

[0075] Alternatively, the amino acid substitution can be selected from the following series of substitutions shown in Table 2.

Table 2

[0076] Alternatively, the amino acid substitution can be selected from the following series of substitutions shown in Table 3.

Table 3

[0077] Alternatively, the amino acid substitution can be selected from the following series of substitutions shown in Table 4.

Table 4

[0078] Alternatively, at least one amino acid substitution in each polypeptide chain can 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) shown in the first polypeptide sequence is / are replaced with a known negatively charged amino acid, and the position(s) shown in the second polypeptide sequence is / 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) shown in the first polypeptide sequence is / are replaced with a known positively charged amino acid, and the position(s) shown in the second polypeptide sequence is / are replaced with a known negatively charged amino acid. [Table 7]

[0081] Alternatively, or in addition, the structural stability of the heterodimeric heavy chain within the multispecific binding protein can be increased by introducing S354C into either the first or second polypeptide chain and introducing Y349C into the opposite polypeptide chain to form an artificial disulfide bridge within the boundary of the two polypeptides. [Table 8]

[0082] Alternatively, or in addition, the structural stability of the heterodimeric heavy chain within the multispecific binding protein can be increased by introducing S354C into either the first or second polypeptide chain and introducing Y349C into the opposite polypeptide chain to form an artificial disulfide bridge within the boundary of the two polypeptides.

[0083] The above-mentioned 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, 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 the multispecific binding protein, different ratios of the first, second, third, and fourth expression vectors can be explored to determine the optimal ratio for transfection into the host cell. After transfection, 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 the expression of the multispecific protein. The multispecific binding protein 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] A protein comprising an antigen-binding site that competes with the NKG2D binding site described herein In certain embodiments, the invention provides a protein comprising an antigen-binding site that binds to NKG2D, competing with the NKG2D-binding sites 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 mapped by surface plasmon resonance. For example, as shown in Example 2, ADI-27744 binds to an epitope on NKG2D that is different from that of 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 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 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 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 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 the protein In one aspect, the present disclosure provides an NKG2D binding site having a heavy chain variable domain with 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 with 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 NKG2D binding site having a heavy chain variable domain with 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 a light chain variable domain with 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 NKG2D binding site having a heavy chain variable domain with 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 a light chain variable domain with 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; an NKG2D binding site having a heavy chain variable domain with 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 and a light chain variable domain with 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; or an NKG2D binding site having a heavy chain variable domain with an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orA cell comprising one or more nucleic acids encoding a protein comprising an NKG2D binding site having a light chain variable domain having an amino acid sequence that is identical (or 100%) thereto.

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

[0091] Pharmaceutical compositions In one aspect, the present 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 the NKG2D binding site described herein, and a pharmaceutically 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 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. In certain embodiments, the formulation comprises a protein comprising an antigen-binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, 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: 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 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 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: 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 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, 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: 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 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 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: 10.

[0093] The compositions can be formulated for use in a variety of drug delivery systems. The one or more physiologically acceptable excipients or carriers can include compositions for proper 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 a protein in a buffer solution forming the formulation. Aqueous carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (such as phosphate buffered saline), sterile aqueous saline solutions, Ringer's solutions, or dextrose solutions. In certain embodiments, the aqueous formulation comprising the protein disclosed herein is prepared in a pH buffer solution. The pH of the preparation will typically be from 3 to 11, more preferably from 5 to 9 or 6 to 8, most preferably from 7 to 8, such as from 7 to 7.5. Intermediate ranges of the above pH are also intended to be part of the present disclosure. For example, as upper and / or lower limits, ranges of values using any combination of the above values are intended to be included. Examples of buffers that control pH within this range include acetates (such as sodium acetate), succinates (such as sodium succinate), gluconic acid, histidine, citrates, and other organic acid buffers. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / ml (such as 1.305 mg / ml) of citric acid, about 0.3 mg / ml (such as 0.305 mg / ml) of sodium citrate, about 1.5 mg / ml (such as 1.53 mg / ml) of disodium hydrogen phosphate dihydrate, about 0.9 mg / ml (such as 0.86) of sodium dihydrogen phosphate dihydrate, and about 6.2 mg / ml (such as 6.165 mg / ml) of sodium chloride. In certain embodiments, the buffer system comprises 1 to 1.5 mg / ml of citric acid, 0.25 to 0.5 mg / ml of sodium citrate, 1.25 to 1.75 mg / ml of disodium hydrogen phosphate dihydrate, 0.7 to 1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / ml of sodium chloride. The pH of the liquid formulation can be set by the addition of pharmaceutically acceptable acids and / or bases. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0095] In some embodiments, the formulation comprises an aqueous carrier that is pharmaceutically 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), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solutions.

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

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

[0098] In certain embodiments, the liquid formulations of the present disclosure can be prepared as a 10 mg / mL concentrated solution in combination with a stabilizing level of sugar. In certain embodiments, the liquid formulation can be prepared in an aqueous carrier. In certain embodiments, the stabilizer can be added in an amount that does not result in a viscosity that is undesirable or inappropriate for intravenous administration. In certain embodiments, the sugar can be a disaccharide such as sucrose. In certain embodiments, the liquid formulation 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 multi-use (multiple dose) formulations.

[0099] In some embodiments, the present disclosure provides a formulation with an extended shelf life comprising the protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen 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, harvest / cell purification, purification, storage of drug substances / drug products, and sample analysis. Deamidation is the loss of NH3 from a protein that forms a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17 Da mass loss from the parent peptide. Subsequent hydrolysis results in an 18 u mass increase. Isolation of the succinimide intermediate is difficult because it is unstable under aqueous conditions. Therefore, deamidation is usually detectable when there is a 1 u mass increase. Deamidation of asparagine results in either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, dielectric constant of the solvent, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser following Asn in the protein sequence are more sensitive to deamidation. In certain embodiments, the liquid formulations of the present disclosure can 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 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 an 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 the protein 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 such that the weight ratio of protein to sucrose or maltose is at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

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

[0104] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates 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 formulations of the present invention may include such bulking agents.

[0105] In certain embodiments, the lyophilized protein product is composed of an aqueous carrier. The intended aqueous carrier is pharmaceutically acceptable herein (e.g., safe and non-toxic for administration to humans) and useful for the preparation of a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile aqueous saline solutions, Ringer's solution, or dextrose solutions. In certain embodiments, the lyophilized formulations of the present disclosure are reconstituted with either sterile water for injection, USP (SWFI), or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder dissolves in the 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] The protein composition can be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for use as is or may be lyophilized, and the lyophilized preparation is mixed with a sterile aqueous carrier prior to administration. The resulting composition in solid form may be packaged in multiple single-dose units, each containing a fixed amount of the above-described agent(s). The composition in solid form can also be packaged in a container for variable amounts.

[0107] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can 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 specific dosage can be a uniform dosage for each patient, for example, 50 to 5000 mg of protein. Alternatively, the dosage for a patient can be adjusted according to the approximate body weight or surface area of the patient. Other factors in determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely done by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining the dosage to be used in conjunction with appropriate dose-response data. The dosage for an individual patient can be adjusted while observing the progression of the disease. It is possible to measure the blood concentration of the targetable construct or complex in the patient to confirm whether it is necessary to adjust the dosage to reach or maintain the 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] Generally, the dosage based on body weight is from about 0.01 μg to about 100 mg / kg body weight, for example, 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 / kg body weight, from about 0.1 μg to about 100 mg / kg body weight, from about 0.1 μg to about 50 mg / kg body weight, from about 0.1 μg to about 10 mg / kg body weight, from about 0.1 μg to about 1 mg / kg body weight, from about 0.1 μg to about 100 μg / kg body weight, from about 0.1 μg to about 10 μg / kg body weight, from about 0.1 μg to about 1 μg / kg body weight, from about 1 μg to about 100 mg / kg body weight, from about 1 μg to about 50 mg / kg body weight, from about 1 μg to about 10 mg / kg body weight, from about 1 μg to about 1 mg / kg body weight, from about 1 μg to about 100 μg / kg body weight, from about 1 μg to about 50 μg / kg body weight, from about 1 μg to about 10 μg / kg body weight, from about 10 μg to about 100 mg / kg body weight, from about 10 μg to about 50 mg / kg body weight, from about 10 μg to about 10 mg / kg body weight, from about 10 μg to about 1 mg / kg body weight, from about 10 μg to about 100 μg / kg body weight, from about 10 μg to about 50 μg / kg body weight, from about 50 μg to about 100 mg / kg body weight, from about 50 μg to about 50 mg / kg body weight, from about 50 μg to about 10 mg / kg body weight, from about 50 μg to about 1 mg / kg body weight, from about 50 μg to about 100 μg / kg body weight, from about 100 μg to about 100 mg / kg body weight, from about 100 μg to about 50 mg / kg body weight, from about 100 μg to about 10 mg / kg body weight, from about 100 μg to about 1 mg / kg body weight, from about 1 mg to about 100 mg / kg body weight, from about 1 mg to about 50 mg / kg body weight, from about 1 mg to about 10 mg / kg body weight, from about 10 mg to about 100 mg / kg body weight, from about 10 mg to about 50 mg / kg body weight, from about 50 mg to about 100 mg / kg body weight. The dosing can be carried out once or multiple times daily, weekly, monthly, or annually, or once every 2 to 20 years. One of ordinary skill in the art can easily predict the dosing repetition rate 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 can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, or intracavitary by perfusion through a catheter or direct intralesional injection. This may be administered more than once a day, more than once a week, more than once a month, and more than once a year.

[0110] Enhancement of tumor cell death and cancer treatment The present invention provides a method for enhancing tumor cell death and / or treating cancer in a patient. In some embodiments, the method comprises exposing a tumor and natural killer cells to a multispecific binding protein disclosed herein. In some embodiments, the method comprises 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 has 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, an antigen binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, 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: 4, an antigen binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, 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: 6, an antigen binding site having a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, 83, 84, 85, 86, or 87, 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%,An antigen-binding site having a light chain variable domain with an amino acid sequence that is identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 9, or 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, 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: 10 may be included.

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

[0112] In some embodiments, the patient to be 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 to be treated has a solid cancer, such as a 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, stomach cancer, testicular cancer, or uterine cancer.In still other embodiments, the cancer is an angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenocarcinoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectum cancer, astrocytic tumor, Bartholin gland adenocarcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, bile duct cancer, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometroid adenocarcinoma, endothelial cell carcinoma, epithelioma, epithelial cell carcinoma, Ewing sarcoma, eye and orbit cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatocellular adenoma, hepatocellular adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, malignant lentigo melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningiocarcinoma, mesothelioma, metastatic cancer, intraoral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin lymphoma, oat cell carcinoma, anaplastic glioma, 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, leiomyosarcoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, mesothelioma, 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 being treated has a non-Hodgkin lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin lymphoma is 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, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thiamiprine, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, besnilone, aminoglutethimide, amsacrine, proglymidine, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, buthionine, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, iproplatin, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone releasing factor, and variations of the foregoing agents that can exhibit different binding to its cognate receptor and increase or decrease the serum half-life are included.

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

[0116] Still other agents that can 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] Still other categories of anti-cancer agents include, for example, (i) inhibitors selected from ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton 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, 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) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS, and (iii) IL-12, IL-15, GM-CSF, and G-CSF.

[0118] The protein of the present invention can also be used to assist in the surgical removal of primary lesions.

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

[0120] The foregoing description illustrates various aspects and embodiments of the invention. This patent application expressly contemplates all combinations and permutations of aspects and embodiments.

Examples

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

[0122] Example 1 - Binding Affinities of Various NKG2D Binding Domains The reaction rates and affinities of various NKG2D-binding domains were evaluated by surface plasmon resonance using a Biacore 8K instrument (GE Healthcare). Anti-human Fc antibody was immobilized on a CM5 chip using standard amine coupling chemistry. Human monoclonal antibodies containing various NKG2D-binding domains were captured on the anti-human Fc chip at a density of approximately 100 RU. Solutions containing soluble mouse Fc-human NKG2D dimers at 0.411 - 100 nM were injected at 37 °C at 30 μl / min over the captured NKG2D antibodies and control surfaces. The surfaces were regenerated between cycles by rapid injection of 10 mM glycine, pH 1.8. To obtain the kinetic rate constants, double-reference data were fitted to a 1:1 interaction model using Biacore 8K Evaluation software (GE Healthcare). The equilibrium binding constant K D is the ratio of the dissociation constant k d and the association constant k a (k d / k a ). As shown in Table 9 and Figure 3, the binding affinities of the NKG2D-binding domains for NKG2D are in the range of 10 - 62 nM.

Table 9

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

Table 10

[0124] ULBP amino acid sequence number 67 RRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSG

[0125] Figure 4A shows the profile in which an NKG2D monoclonal antibody containing ADI-27744 was injected onto immobilized NKG2D, followed by injection of ULBP6. Figure 4B shows the profile of ULBP6 injected onto immobilized NKG2D, followed by injection of an NKG2D monoclonal antibody containing ADI-27744. These results indicate that the NKG2D monoclonal antibody containing the ADI-27744 antigen-binding site does not block the binding of ULBP6 to NKG2D, that is, ADI-27744 binds to a different epitope of NKG2D from ULBP6.

[0126] Figure 4C shows the profile in which an MS monoclonal antibody was injected onto NKG2D, followed by injection of ULBP6. The MS monoclonal antibody blocks ULBP6 from binding to NKG2D. Figures 4D - 4F show the profiles in which MS, 1D11, or MAB139 was injected onto immobilized NKG2D, followed by injection of an NKG2D monoclonal antibody containing ADI-27744. Figures 4G - 4H show the profiles in which an NKG2D monoclonal antibody containing ADI-27744 was injected onto immobilized NKG2D, followed by injection of an NKG2D monoclonal antibody containing ADI-27749 or F47. ADI-27744 does not block the binding of MS, 1D11, and MAB139 to NKG2D. ADI-27749 and F47 do not block the binding of ADI-27744 to NKG2D. These results indicate that ADI-27744 binds to a unique epitope on NKG2D that is different from the MS, 1D11, MAB139, ADI-27749, and F47 binding epitope(s).

[0127] Example 3 - The trispecific binding protein binds to NKG2D The EL4 mouse lymphoma cell line was genetically engineered to express human NKG2D. As shown in Figure 1, a trispecific binding protein (TriNKET) containing an NKG2D binding domain, a tumor-associated antigen binding domain (such as a CD33 or HER2 binding domain), and an Fc domain that binds to CD16, respectively, was tested for its affinity for extracellular NKG2D expressed on EL4 cells. Binding of the multispecific binding protein to NKG2D was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and a fold-over-background (FOB) relative to background was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[0128] The TriNKETs tested included CD33-TriNKET-A44 (ADI-27744 and CD33 binding domain), CD33-TriNKET-A49 (ADI-27749 and CD33 binding domain), CD33-TriNKET-F63 (ADI-29463 and CD33 binding domain), HER2-TriNKET-A44 (ADI-27744 and CD33 binding domain), HER2-TriNKET-A49 (ADI-27749 and HER2 binding domain), HER2-TriNKET-F63 (ADI-29463 and HER binding domain), and HER2-TriNKET-E79 (ADI-29379 and HER2 binding domain). The HER2 binding domain consists of the heavy chain variable domain and the light chain variable domain of trastuzumab. The CD33 binding domain consists of the heavy chain variable domain and the light chain variable domain listed below. SEQ ID NO: 49

Chemical formula

Chemical formula

[0129] All TriNKETs bind to NKG2D of 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 affinities for each clone were similar between cells expressing human and mouse NKG2D (Figs. 5-6).

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

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

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

[0133] HER2 - TriNKET - A44, HER2 - TriNKET - A49, and HER2 - TriNKET - F63 show binding levels comparable to those of 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 protein activates 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 the isolated NK cells was usually over 90%. The isolated NK cells were cultured in medium containing 100 ng / mL of IL - 2 for activation or rested overnight without cytokines. NK cells activated with IL - 2 were used within 24 - 48 hours after activation.

[0135] Human cancer cells expressing tumor antigens were harvested and resuspended in culture medium at 2×10 6 cells / mL. Monoclonal antibodies or TriNKET targeting tumor antigens were diluted in the culture medium. Activated NK cells were harvested, washed, and resuspended at 2×10 6They were resuspended in the culture medium at cells / mL. Then, the cancer cells were 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 culture to block protein transport to the extracellular space for intracellular cytokine staining. Fluorophore-conjugated anti-CD107a was added to the mixed culture, and after incubating this culture for 4 hours, samples were prepared for FACS analysis using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD107a and IFN-gamma staining was analyzed in CD3 - CD56 + cells. An increase in CD107a / IFN-gamma double-positive cells indicates better NK cell activation due to the involvement of two activating receptors rather than one receptor.

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

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

[0138] Example 6 - The trispecific binding protein enables cytotoxicity against target 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 PBMC, and the purity of the isolated NK cells was usually over 90%. The isolated NK cells were cultured in medium containing 100 ng / mL of IL-2 for activation or rested overnight without cytokines. NK cells activated or rested with IL-2 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 resuspended in culture medium at 1 - 2×10 5 cells / mL. Rested and / or activated NK cells were harvested, washed, and resuspended in the same culture medium as the cancer cell culture medium at 10 5 -2.0×10 6 cells / mL. In each well of a 96-well plate, 50 μl of the cancer cell suspension was mixed with 50 μl of the NK cell suspension with or without TriNKET targeting the tumor antigen expressed by the cancer cells. At 37°C, 5% CO 2 , after incubation for 3 hours and 15 minutes, 10-fold lysis buffer was added to the wells containing cancer cells only, wells containing medium only for maximum lysis, and negative reagent controls, respectively. The plate was then returned to the incubator for an additional 45 minutes to reach a total incubation of 4 hours. The cells were then pelleted, and the culture supernatant was transferred to a new 96-well plate and mixed with the substrate for development. The new plate was incubated at room temperature for 30 minutes, and the absorbance 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) / (Maximum lysis - Negative reagent control)) * 100%

[0140] TriNKET mediates the cytotoxicity of human NK cells against CD33-positive Molm-13 human AML cell line. As shown in Figure 13, resting human NK cells were mixed with Molm-13 cancer cells, and TriNKET was able to enhance the cytotoxic activity of resting human NK cells in a dose-responsive manner against cancer cells. The dotted line indicates the cytotoxic activity of resting NK cells without using TriNKET. As shown in Figure 14, resting human NK cells were mixed with Molm-13 cancer cells, and TriNKET further enhanced 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 HER2-positive 786-O human renal cell carcinoma cell line. As shown in Figure 15, resting human NK cells were mixed with 786-O cancer cells, and TriNKET was able to enhance the cytotoxic activity of resting human NK cells in a dose-responsive manner against cancer cells (each TriNKET was added at 5, 1, 0.2 μg / ml in the assay, and these results are represented in the three columns from left to right for each TriNKET in Figures 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 Figure 16, activated human NK cells were mixed with 786-O cells, and TriNKET further enhanced 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 TriNKET containing the variants As described above, ADI-27749 (A49) includes, inter alia, a heavy chain CDR3 having the amino acid sequence of GAPMGAAAGWFDP (SEQ ID NO: 71). Met at position 102 of SEQ ID NO: 7 (i.e., position 4 of this CDR3 sequence) is replaced by Gln, Leu, Ile, Phe, or Val, whereby the NKG2D antibodies A49MQ, A49ML, A49MI, A49MF, and A49MV having the corresponding heavy chain variable region, light chain variable region, and CDR sequences provided in Table 1 can be generated, respectively.

[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. Using the Protein Builder module, the residues were mutated and after connecting all the residues, the whole Fab was minimized. Dynamic specific sampling was performed using the lowMD protocol of BIOMOE. As shown in Table 11, these mutations did not substantially adversely affect the predicted hydrophobicity of A49 Fab.

Table 11

[0144] The hydrophobicity of TriNKET containing A49 (「TriNKET A」) and mutant forms of TriNKET A having 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 at pH 6.0 by differential scanning calorimetry (DSC) with 20 mM histidine, 260 mM sucrose, and 0.005% PS-80. T m values are shown in Table 13, and T m is the mid-transition temperature of the individual domains. The M102 mutation had a small effect by shifting the T m3 and T m4 values of the two most stable transitions (T m ) 0.6 and 0.7 °C lower compared to TriNKET A. The previous transitions (T m1 and T m2 ) were not affected. Thus, the M102 mutation had only a minor effect on the overall thermal stability of TriNKET A. [Table 13]

[0146] The binding of TriNKET A and TriNKET A* to the fusion protein of human NKG2D and mouse Fc (「mFc-hNKG2D」) was characterized by surface plasmon resonance (SPR) at 37 °C. To obtain equilibrium affinity data, two different fittings, steady-state affinity fitting and kinetics fitting, were used (Figure 32). Rate constants and equilibrium affinity constants were calculated, and data from two independent experiments for TriNKET A* and data from three independent experiments for TriNKET A were averaged. [Table 14]

[0147] As shown in Table 14, the equilibrium affinity constants (K D ) obtained from both affinity fitting and kinetics fitting are very similar between replicates, suggesting high reliability in the measured parameters. The M102 variant has less than half the affinity for human NKG2D compared to TriNKET A. The K Dwas (6.87 ± 0.16) × 10 -7 M, but the K of TriNKET A D was (4.87 ± 0.83) × 10 -7 M (calculated from affinity fitting). When K D was calculated from kinetic fitting, similar differences in affinity were observed. 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 to one molecule of TriNKET A*. This suggests that the M102 mutation had little effect on the binding of TriNKET containing A49 to human NKG2D.

[0148] Finally, the effect of the M102 mutation on the potency of TriNKET was evaluated in a cytotoxicity assay. Briefly, KHYG-1 cells expressing the high-affinity variant 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 of human IL-2. To prepare KHYG-1-CD16V cells for use as effectors in the cytotoxicity assay, cells were harvested from the culture, pelleted, washed three times in culture medium without IL-2, resuspended in culture without IL-2, and rested for 24 hours.

[0149] Human cancer cell lines expressing the target of interest were harvested from the culture. Cells were washed with HBS and resuspended in growth medium at 10 6 cells / mL for labeling with the BATDA reagent (Perkin Elmer C136-100). Labeling of the target cells was performed according to the manufacturer's instructions. After labeling, cells were washed three times with HBS and resuspended in culture medium at 0.5 × 10 5 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 the diluted TriNKET was added to each well. Resting NK cells were harvested from the culture, washed, and resuspended at 1.0×10 6 cells / mL in culture medium. 50 μl of NK cells was added to each well of the plate to obtain a desired E:T ratio of 10:1, and a total of 200 μl of culture was prepared in each well. The plate was incubated at 37 °C in 5% CO 2 for 2 - 3 hours.

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

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

[0153] To measure the activities of TriNKET A and TriNKET A*, a cell line expressing the first tumor antigen was selected as the target cell. Two different lots of TriNKET A were used for comparison. The values of specific lysis (%) were plotted in Figure 33, and the EC50 and the maximum percentage of specific lysis (%) were summarized in Table 15. The EC50 and the 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 the effect of the M102 mutation on TriNKET activity was not tumor antigen-specific, TriNKET A and TriNKET A* that bind to a second different tumor antigen were constructed. The activities of the two TriNKETs were compared in a cytotoxicity assay using a cell line expressing the second tumor antigen as the target cell and KHYG-1-CD16V cells as the effector cells. As shown in Figure 34, TriNKET A* showed activity equivalent to that of TriNKET A.

[0155] Incorporation by reference All disclosures of each of the patent documents and scientific papers referred to herein are hereby incorporated by reference for all purposes.

[0156] Equivalents The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are not intended to limit the invention described herein, but should be regarded as illustrative in all respects. Accordingly, the scope of the present invention is indicated by the appended claims rather than the foregoing description, and all modifications within the meaning and scope of equivalents of the claims are intended to be included therein.

Claims

**Claim 1** A multispecific binding protein, comprising: (a) a Fab that binds to NKG2D, wherein the Fab comprises: (i) a heavy chain portion comprising an antibody heavy chain variable domain and a CH1 domain; and (ii) a light chain portion comprising an antibody light chain variable domain and a constant light chain domain wherein: (A) the antibody heavy chain variable domain comprises the complementarity determining region 1 (CDR1) amino acid sequence of SEQ ID NO: 48, the complementarity determining region 2 (CDR2) amino acid sequence of SEQ ID NO: 30, and the complementarity determining region 3 (CDR3) amino acid sequence of SEQ ID NO: 44; (B) the antibody light chain variable domain comprises the CDR1 amino acid sequence of SEQ ID NO: 32, the CDR2 amino acid sequence of SEQ ID NO: 33, and the CDR3 amino acid sequence of SEQ ID NO: 34; the Fab; (b) a single-chain variable fragment (scFv) or an additional Fab that binds to a tumor-associated antigen; and (c) a first Fc antibody constant domain and a second Fc antibody constant domain that both bind to CD16, wherein the N-terminus of the first Fc antibody constant domain of (c) is fused to the C-terminus of the scFv or the additional Fab of (b), and the N-terminus of the second Fc antibody constant domain of (c) is fused to the C-terminus of the CH1 domain of (a). **Claim 2** The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of (a) comprises the CDR1 amino acid sequence of SEQ ID NO: 29, the CDR2 amino acid sequence of SEQ ID NO: 30, and the CDR3 amino acid sequence of SEQ ID NO:

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

71. **Claim 4** The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of (a) comprises the CDR1 amino acid sequence of SEQ ID NO: 29, the CDR2 amino acid sequence of SEQ ID NO: 30, and the CDR3 amino acid sequence of SEQ ID NO:

77. **Claim 5** The multispecific binding protein according to claim 1, wherein the antibody heavy chain variable domain of (a) comprises the CDR1 amino acid sequence of SEQ ID NO: 48, the CDR2 amino acid sequence of SEQ ID NO: 30, and the CDR3 amino acid sequence of SEQ ID NO:

78. **Claim 6** ​ The anti - body heavy - chain variable domain of (a) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and the antibody light - chain variable domain of (a) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

8. The multispecific binding protein according to any one of claims 1 to 5.

7. When the Fab of (a) is measured by surface plasmon resonance, it binds to NKG2D at a K of 2 to 120 nM Ｄ The multispecific binding protein according to any one of claims 1 to 6, which binds to NKG2D at K

8. The first Fc antibody constant domain and the second Fc antibody constant domain each comprise a hinge, CH2, and CH3 domains. The multispecific binding protein according to any one of claims 1 to 7.

9. The amino acid sequences of the first Fc antibody constant domain and the second Fc antibody constant domain are each at least 90% identical to the human IgG1 constant region. The multispecific binding protein according to claim 8.

10. The first Fc antibody constant domain and the second Fc antibody constant domain contain mutations with respect to the IgG1 constant region to promote heterodimerization. The multispecific binding protein according to claim 8 or 9.

11. (a) The amino acid sequence of the first Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by K360E and K409W substituents, and the amino acid sequence of the second Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by Q347R, D399V, and F405T substituents; or, (b) The amino acid sequence of the first Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by Q347R, D399V, and F405T substituents, and the amino acid sequence of the second Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by K360E and K409W substituents; or, (c) The amino acid sequence of the first Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by K360E and K409W substituents, and the amino acid sequence of the second Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region by Q347R, D399V, and F405T substituents, and, (i) The amino acid sequence of the first Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region by an S354C substituent, and the amino acid sequence of the second Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region by a Y349C substituent; or, (ii) The amino acid sequence of the first Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the Y349C substitution group, and the amino acid sequence of the second Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the S354C substitution group; or, (d) The amino acid sequence of the first Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region due to the Q347R, D399V, and F405T substitution groups, and the amino acid sequence of the second Fc antibody constant domain is different from the amino acid sequence of the IgG1 constant region due to the K360E and K409W substitution groups, and, (i) The amino acid sequence of the first Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the S354C substitution group, and the amino acid sequence of the second Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the Y349C substitution group; or, (ii) The amino acid sequence of the first Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the Y349C substitution group, and the amino acid sequence of the second Fc antibody constant domain is further different from the amino acid sequence of the IgG1 constant region due to the S354C substitution group, The multispecific binding protein according to claim 10.

12. The multispecific binding protein according to any one of claims 1 to 7, wherein (b) is a single-chain variable fragment (scFv).

13. A formulation comprising the multispecific binding protein according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

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

15. A composition comprising the multispecific binding protein according to any one of claims 1 to 12 for use in therapy or the formulation according to claim 13.

16. A composition comprising the multispecific binding protein according to any one of claims 1 to 12 for enhancing the cell death of tumor cells expressing the tumor-associated antigen.

17. The composition according to claim 16, wherein the tumor cells and natural killer cells are exposed to the multispecific binding protein.

18. A composition comprising the multispecific binding protein according to any one of claims 1 to 12, or a formulation according to claim 13, for treating cancer expressing the tumor-associated antigen.

19. The composition for use according to claim 18, 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 sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T cell lymphoma, testicular cancer, thymic cancer, thyroid cancer, urothelial cancer, cancer infiltrated by bone marrow-derived suppressor cells, cancer with extracellular matrix deposition, cancer with a high level of reactive stroma, and cancer with angiogenesis.

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