Proteins binding NKG2d, CD16 and tumor-associated antigen

Abstract

To provide: multi-specific binding proteins that bind the Natural killer group 2 member D (NKG2D), cluster of differentiation 16 (CD16), and a tumor-associated antigen; and pharmaceutical compositions and therapeutic methods useful for the treatment of cancer.SOLUTION: A protein comprises: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds an antigen selected from the group consisting of EpCAM, Cancer Antigen 125, sodium / phosphate cotransporter 2B, Nectin cell adhesion molecule 4, Fucosyl-GM1, disintegrin and metalloproteinase domain-containing protein 8, disintegrin and metalloproteinase domain-containing protein 9, solute carrier family 44 member 4, and sialylated Lewis a antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.SELECTED DRAWING: 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 / 555,110, filed on September 7, 2017 , and U.S. Provisional Patent Application No. 62 / 566,824, filed on October 2, 2017 , and the entire disclosure of each is hereby incorporated by reference herein for all purposes . Sequence Listing

[0002] This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference herein in its entirety. The ASCII copy was created on September 6, 2018 , and has the name DFY - 038WO_SL.txt and a size of 321,395 bytes .

[0003] The present invention relates to a multispecific binding protein that binds to NKG2D, CD16, and tumor - associated antigens .

Background Art

[0004] Cancer remains a major health problem despite considerable research efforts and scientific progress reported in the literature for treating this disease. Among the most frequently diagnosed cancers are prostate cancer, breast cancer, lung cancer, and colorectal cancer. Prostate cancer is the most common form of cancer in men . Breast cancer remains a leading cause of death in women . Blood and bone marrow cancers, including multiple myeloma, leukemia, and lymphoma, are also frequently diagnosed cancer types. Current treatment options for these cancers are not satisfactory for all patients . ​not effective, and / or may have substantial adverse side effects Other types of cancer still remain difficult to treat using existing treatment options .

[0005] Cancer immunotherapies are desirable because they are highly specific and can use the patient's own immune system to promote the destruction of cancer cells Fusion proteins, such as bispecific T cell 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 certain tumor-associated antigens and certain immune cells are described in the literature. See, for example, WO2016 / 134371 and WO2015 / 095412 WO2015 / 095412 .

[0006] Natural killer (NK) cells are components of the innate immune system and make up approximately 15% of circulating lymphocytes NK cells infiltrate substantially all tissues and are initially characterized by their ability to effectively kill tumor cells without the need for prior sensitization Activated NK cells kill target cells by means similar to those of cytotoxic T cells, i.e., via cytotoxic granules containing perforin and granzyme, 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

[0007] NK cells respond to signals via various activating and inhibitory receptors on their surface For example, when NK cells encounter healthy self-cells, their activity is inhibited by ​​It is inhibited by the activation of the lymphocyte immunoglobulin-like receptor (KIR). Alternatively, when NK cells encounter foreign or cancer cells, they are activated via their activating receptors (e.g., , natural killer group 2 member D (NKG2D), NCR, DNAM1). NK cells are also activated by the constant region of several immunoglobulins via their CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of the stimulatory and inhibitory signals.

[0008] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates Ca 2+ -independent homotypic cell-cell adhesion. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Furthermore, EpCAM has the potential to form tumors due to its ability to upregulate c-myc, e-fabp, as well as cyclin A and E. Since EpCAM is expressed exclusively in epithelia and epithelial-derived neoplasms, EpCAM can be used as a diagnostic marker for various cancers such as head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer. EpCAM appears to play a role in tumor formation and cancer metastasis, and thus EpCAM can also function as a potential prognostic marker and as a potential target for immunotherapy strategies.

[0009] CA125, also known as mucin 16, is a member of the mucin family of glycoproteins. CA-125 is a specific type of marker for ovarian cancer, endometrial cancer, and pancreatic cancer, among others. Tumor markers or biomarkers that may be elevated in the blood of some patients with cancer and are applied as . CA-125 suppresses the response of natural killer cells, thereby protecting cancer cells from the immune response through several different mechanisms, including and enabling cell growth and promoting cell motility, and has been shown to play a role in tumor formation and tumor progression.

[0010] Sodium-dependent phosphate transport protein 2b (NaPi2b) is involved in the active transport of phosphate into cells by Na+ cotransport. For example, NaPi2b is a major phosphate transport protein in the brush border membrane of the intestine and has a role in the synthesis of surfactant in the alveoli. NaPi2b is also an antigen expressed in various cancers such as lung cancer, ovarian cancer, and thyroid cancer.

[0011] Nectin4 is a member of the nectin family of cell adhesion molecules involved in Ca 2+ -independent cell adhesion. Nectins are ubiquitously expressed and have a role in adhesion in a wide range of tissues, such as epithelial adhesion junctions or chemical synapses in neuronal tissue. Nectins are also tumor-associated antigens and are expressed in cancers such as bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and lung cancer.

[0012] Gangliosides are associated with many physiological processes, including growth, differentiation, migration, and apoptosis, by modulating both cell signaling processes and interactions between cells and between cells and the extracellular matrix. GM1 is a gangliosid is a fucosyl-GM1, and fucosyl-GM1 is a ganglioside having a unique structure in which the terminal galactose is α-1,2-fucosylated at the non-reducing end. Fucosyl-GM1 is expressed in a very small number of normal tissues, but is present in various cancers such as small cell lung cancer, neuroblastoma, and liver cancer. Therefore, fucosyl-GM1 is considered a candidate as a target antigen in antibody immunotherapy for tumor markers and small cell lung cancer, neuroblastoma, and liver cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer.

[0013] ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer. ADAM (a disintegrin and metalloprotease) proteins play a dominant role in the shedding of the protein ectodomain of membrane-bound molecules. ADAM proteins are considered important regulators of intercellular signaling and homeostasis during development, and their dysregulation contributes to pathologies such as cancers. ADAM8 is a member of the ADAM family and is overexpressed in pancreatic cancer, breast cancer, lung cancer, and kidney cancer. ADAM9 has been shown to cleave and release several molecules important in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1, and VE-cadherin. ADAM9 is overexpressed in kidney cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, and brain cancer.

[0014] SLC44A4, also known as CTL4, is a member of the family of solute carrier proteins known as choline transporter-like proteins (CTL1-5). Although SLC44A4 has not been shown to be involved in choline transport, it is involved in acetylcholine synthesis and transport. SLC44A4, also known as CTL4, is a member of the family of solute carrier proteins known as choline transporter-like proteins (CTL1-5). Although SLC44A4 has not been shown to be involved in choline transport, it is involved in acetylcholine synthesis and transport. SLC44A4, also known as CTL4, is a member of the family of solute carrier proteins known as choline transporter-like proteins (CTL1-5). Although SLC44A4 has not been shown to be involved in choline transport, it is involved in acetylcholine synthesis and transport.​ Regarding the transport and uptake of thiamine pyrophosphate, the phosphorylated form of vitamin B1, SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but , significantly upregulated in various epithelial tumors, among them pancreatic, prostate, and gastric cancers will be done. CA19-9 is the general term for the carbohydrate antigen sialyl Lewis a. It is overexpressed in gastrointestinal cancers such as pancreatic cancer, colorectal cancer, cholangiocarcinoma, and hepatic cancer. Therefore, CA19-9 is the most frequently applied for the diagnosis of these above cancers. It is a serum tumor marker. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention [Means for solving the problem]

[0016] The present invention relates to a tumor-associated antigen (selected from any one of the antigens presented in Table 11). ), and binds to the NKG2D and CD16 receptors on natural killer cells. The present invention provides a multispecific binding protein that binds two or more NK activating proteins. It can associate with the receptor and block the binding of the natural ligand to NKG2D. In certain embodiments, the protein is expressed in humans and other animals, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are further described below. Please describe in detail.

[0017] Thus, one aspect of the invention is a first antigen-binding site that binds to NKG2D and EpC AM, cancer antigen 125 (CA125), sodium / phosphate cotransporter 2B (NaPi2b ), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (monosialotetra hexosylganglioside), disintegrin and metalloproteinase domain containing protein 8 (ADAM8), disintegrin and metalloproteinase domain containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC 44A4), and a second antigen-binding site that binds to an antigen selected from sialyl Lewis a antigen (CA19-9), as well as an antibody Fc domain sufficient to bind to CD16, a portion thereof, or a third antigen-binding site that binds to CD16, provided as a protein incorporating the same. Each antigen-binding site may incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged like an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may be a single domain antibody such as a VH antibody like those of camels or a V antibody such as those found in cartilaginous fish. The invention relates to the NKG2D receptor, CD16, and EpCAM, cancer antigen 125 (C A125), sodium / phosphate cotransporter 2B (NaPi2b), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (monosialotetrahexosylganglioside ), disintegrin and metalloproteinase domain-containing protein 8 (ADA H H antibody or a single domain NAR antibody such as a V antibody.

[0018] A125), sodium / phosphate cotransporter 2B (NaPi2b), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (monosialotetrahexosylganglioside ), disintegrin and metalloproteinase domain-containing protein 8 (ADA M8). M8), disintegrin and metalloproteinase domain-containing protein 9 (A DAM9), solute carrier family 44 member 4 (SLC44A4), and sialyl Lewis a antigen (CA19-9). A multispecific binding protein that binds to an antigen selected from the group consisting of

[0019] Some proteins of the present disclosure also have an antibody Fc domain sufficient to bind to CD16 or a portion thereof or a Fab fragment linked to a third antigen-binding site that binds to CD16 is included.

[0020] Some proteins of the present disclosure include a Fab fragment, wherein the heavy chain portion of the Fab fragment includes a heavy chain variable domain and a CH1 domain, and the heavy chain variable domain is linked to the CH1 domain.

[0021] Some proteins of the present disclosure include a Fab fragment linked to an antibody Fc domain.

[0022] In one aspect, the present invention provides a protein comprising: (a) a first antigen-binding site comprising a Fab fragment that binds to NKG2D; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds to EpCAM; and (c) an antibody Fc domain sufficient to bind to CD16 or a portion thereof or a third antigen-binding site that binds to CD16. The present invention provides a protein wherein the first antigen-binding site that binds to NKG2D is a Fab fragment and the second antigen-binding site that binds to the tumor-associated antigen EpCAM is an scFv.

[0023] Certain proteins described in the present disclosure have a heavy chain variable domain and a light chain variable domain ​comprises an scFv targeting EpCAM linked via a hinge containing Ala-Ser or Gly-Ala-Ser to an antibody Fc domain or a portion thereof that is sufficient to bind CD16 or a third antigen-binding site that binds CD16. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM linked via a hinge containing Ala-Ser or Gly-Ala-Ser to an antibody Fc domain or a portion thereof that is sufficient to bind CD16 or a third antigen-binding site that binds CD16. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM linked via a hinge containing Ala-Ser or Gly-Ala-Ser to an antibody Fc domain or a portion thereof that is sufficient to bind CD16 or a third antigen-binding site that binds CD16. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM linked to an antibody Fc domain. Some of the proteins of the present disclosure comprise the heavy chain variable domain of an scFv targeting EpCAM, where the heavy chain variable domain forms a disulfide bridge with the light chain variable domain of the scFv. comprise the heavy chain variable domain of an scFv targeting EpCAM, where the heavy chain variable domain forms a disulfide bridge with the light chain variable domain of the scFv. formed.

[0024] Some of the proteins of the present disclosure comprise an scFv targeting EpCAM in which a disulfide bridge is formed between C44 of the heavy chain variable domain and C 100 of the light chain variable domain. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM in which a disulfide bridge is formed between C44 of the heavy chain variable domain and C

[0025] Some of the proteins of the present disclosure comprise an scFv targeting EpCAM linked to an antibody Fc domain, where the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv and is linked to the heavy chain variable domain of the scFv via a flexible linker (Gly Some of the proteins of the present disclosure comprise an scFv targeting EpCAM linked to an antibody Fc domain, where the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv and is linked to the heavy chain variable domain of the scFv via a flexible linker (Gly GlyGlyGlySer)4 (G4S)4) (SEQ ID NO: 206), and a Fab fragment that binds NKG2D is linked to the antibody Fc domain. GlyGlyGlySer)4 (G4S)4) (SEQ ID NO: 206), and a Fab fragment that binds NKG2D is linked to the antibody Fc domain. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM, where the heavy chain variable domain is located at the N-terminus or C-terminus of the light chain variable domain of the scFv.

[0026] Some of the proteins of the present disclosure comprise an scFv targeting EpCAM, where the heavy chain variable domain is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. Some of the proteins of the present disclosure comprise an scFv targeting EpCAM, where

[0027] the heavy chain variable domain is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. The light chain variable domain is located at the N-terminus of the heavy chain variable domain of the scFv.

[0028] In one aspect of the invention, provided is a protein comprising: (a) a single-chain variable fragment (scFv) that binds to NKG2D, a first antigen-binding site; (b) a second antigen-binding site that binds to EpCAM; and (c) an antibody Fc domain or a portion thereof that is sufficient to bind to CD 16 or a third antigen-binding site that binds to CD16. In certain embodiments, the protein of the present disclosure further comprises an additional antigen-binding site that binds to EpCAM. In certain embodiments, the second antigen-binding site of the protein described in the present disclosure binds to EpCAM and is a Fab fragment. In certain embodiments, the second and additional antigen-binding sites of the protein described in the present disclosure are Fab fragments that bind to EpCAM.

[0029] In certain embodiments, the second and additional antigen-binding sites of the protein described in the present disclosure are scFvs that bind to EpCAM. In certain embodiments, the heavy chain variable domain of the scFv that binds to NKG2D is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. In certain embodiments, the light chain variable domain is located at the N-terminus of the heavy chain variable domain of the scFv that binds to NKG2D.

[0030] In certain embodiments, the scFv that binds to NKG2D is linked to an antibody Fc domain or a portion thereof that is sufficient to bind to CD16 or a third antigen-binding site that binds to CD16. In certain embodiments, the scFv that binds to NKG2D is linked to an antibody Fc domain or a portion thereof that is sufficient to bind to CD16 or a third antigen-binding site that binds to CD16. ​ In the third antigen-binding site, Ala-Ser (e.g., EpCAM, CA125, NaPi2 b, Nectin4, fucosyl-GM1, ADAM8, ADAM9, SLC44A4, or also in TriNKET containing an additional antigen-binding site that binds to CA19-9) or Gly-Ala-Ser (e.g., EpCAM, CA125, NaPi2b, Nec tin4, fucosyl-GM1, ADAM8, ADAM9, SLC44A4, or in TriNKET that does not contain an additional antigen-binding site that binds to CA 19-9) is linked via a hinge. In certain embodiments, the scFv that binds to NKG2D is linked via a flexible linker containing G4S to the C-terminus of an antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen-binding site that binds to CD16 . In certain embodiments, the C-terminus of the antibody Fc domain is linked to the N-terminus of the light chain variable domain of the scFv that binds to NKG2D. In certain embodiments, within the scFv that binds to NKG2D, a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv. In a certain embodiment, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain.

[0031]

[0032] Some proteins of the present disclosure include a sequence selected from SEQ ID NO: 210 and SEQ ID NO: 211.

[0033] Some proteins of the present disclosure include an scFv linked to an antibody Fc domain, where The scFv linked to the antibody Fc domain is represented by the sequences selected from SEQ ID NO: 208 and SEQ ID NO: 209. It is represented by the sequences selected.

[0034] Some proteins of the present disclosure include the sequences of SEQ ID NO: 205 and SEQ ID NO: 213. .

[0035] Some proteins of the present disclosure include a sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO: 210 and SEQ ID NO: 211.

[0036] Some proteins of the present disclosure include a sequence that is at least 95% identical to the amino acid sequence selected from SEQ ID NO: 210 and SEQ ID NO: 211.

[0037] Some proteins of the present disclosure include a sequence that is at least 99% identical to the amino acid sequence selected from SEQ ID NO: 210 and SEQ ID NO: 211.

[0038] Some proteins of the present disclosure include the amino acid sequence of SEQ ID NO: 203.

[0039] Some proteins of the present disclosure include the amino acid sequences of SEQ ID NO: 203 and SEQ ID NO: 204.

[0040] Some proteins of the present disclosure include an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 203. Some proteins of the present disclosure include an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 203. Some proteins of the present disclosure include an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 203.

[0041] In some embodiments, the first antigen-binding site that binds to NKG2D is, for example, SEQ ID NO: Having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100%) identical amino acid sequence, and / or incorporating an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 105), CDR2 sequence (SEQ ID NO: 106), and CDR3 sequence (SEQ ID NO: 107) of SEQ ID NO: 1, it may incorporate a heavy chain variable domain related to SEQ ID NO: 1. The heavy chain variable domain related to SEQ ID NO: 1 can be linked to various light chain variable domains to form an NKG2D binding site. For example, the first antigen-binding site incorporating the heavy chain variable domain related to SEQ ID NO: 1 may further incorporate a light chain variable domain selected from any one of the sequences related to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. For example, the first antigen-binding site has a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1 and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of the sequences selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. Alternatively, the first antigen-binding site has a heavy chain variable domain related to SEQ ID NO: 41 and a light chain selected from any one of the sequences related to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, and having an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 %) identical to the selected sequence.

[0042] Alternatively, the first antigen-binding site has a heavy chain variable domain related to SEQ ID NO: 41 and a light chain It may incorporate a light chain variable domain related to No. 42. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% identical to SEQ ID NO: 41 (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 00%), and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 43), CDR2 sequence (SEQ ID NO: 44), and CDR3 sequence (SEQ ID NO: 45) of SEQ ID NO: 41. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% identical to SEQ ID NO: 42 (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%), and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 46), CDR2 sequence (SEQ ID NO: 47), and CDR3 sequence (SEQ ID NO: 48) of SEQ ID NO: 42.

[0043] In other embodiments, the first antigen binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 49 and a light chain variable domain related to SEQ ID NO: 50. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% identical to SEQ ID NO: 49 (e.g., 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 00%), and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 51), CDR2 sequence (SEQ ID NO: 52), and CDR3 sequence (SEQ ID NO: 53) of SEQ ID NO: 49. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% identical to SEQ ID NO: 50 (e.g., 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 100%), and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 54), CDR2 sequence (SEQ ID NO: 55), and CDR3 sequence (SEQ ID NO: 56) of SEQ ID NO: 50. identical, e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 54), the CDR2 sequence (SEQ ID NO: 55), and the CDR3 sequence (SEQ ID NO: 56) of SEQ ID NO: 50.

[0044] Alternatively, the first antigen-binding site may incorporate, for example, an amino acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to SEQ ID NO: 57 and an amino acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 58, thereby incorporating a heavy chain variable domain associated with SEQ ID NO: 57 and a light chain variable domain associated with SEQ ID NO: 58, respectively.

[0045] In another embodiment, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 59 and a light chain variable domain associated with SEQ ID NO: 60. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% identical (e.g., 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to SEQ ID NO: 59 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 109), the CDR2 sequence (SEQ ID NO: 110), and the CDR3 sequence (SEQ ID NO: 111) of SEQ ID NO: 59. Similarly, the light chain variable do main of the second antigen-binding site may be at least 90% identical (e.g., 90%, 91%, 92%, 93% to SEQ ID NO: 60. ​​, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical and well, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 112), CDR2 sequence (SEQ ID NO: 113), and CDR3 sequence (SEQ ID NO: 114) of SEQ ID NO: 60. It may be.

[0046] In some embodiments, the first antigen-binding site that binds to NKG2D may incorporate a heavy chain variable domain associated with SEQ ID NO: 61 and a light chain variable domain associated with SEQ ID NO: 62. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100%) identical to SEQ ID NO: 61, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 63), CDR2 sequence (SEQ ID NO: 64), and CDR3 sequence ( SEQ ID NO: 65) of SEQ ID NO: 61. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 %) identical to SEQ ID NO: 62, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 66), C DR2 sequence (SEQ ID NO: 67), and CDR3 sequence (SEQ ID NO: 68) of SEQ ID NO: 62. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 %) identical to SEQ ID NO: 69, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 66), C DR2 sequence (SEQ ID NO: 67), and CDR3 sequence (SEQ ID NO: 68) of SEQ ID NO: 62.

[0047] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the first 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or may be identical to 100%), and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 71), CDR2 sequence (SEQ ID NO: 72), and CDR3 sequence (SEQ ID NO: 73) of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 70 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 74), CDR2 sequence (SEQ ID NO: 75), and CDR3 sequence (SEQ ID NO: 76) of SEQ ID NO: 70. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 77 and a light chain variable domain associated with SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 79), CDR2 sequence (SEQ ID NO: 80), and CDR3 sequence (SEQ ID NO: 81) of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 78 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 82), CDR2 sequence (SEQ ID NO: 83), and CDR3 sequence (SEQ ID NO: 84) of SEQ ID NO: 78.

[0048] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 77 and a light chain variable domain associated with SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 79), CDR2 sequence (SEQ ID NO: 80), and CDR3 sequence (SEQ ID NO: 81) of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 78 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 82), CDR2 sequence (SEQ ID NO: 83), and CDR3 sequence (SEQ ID NO: 84) of SEQ ID NO: 78. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 77 and a light chain variable domain associated with SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 79), CDR2 sequence (SEQ ID NO: 80), and CDR3 sequence (SEQ ID NO: 81) of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 78 and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 82), CDR2 sequence (SEQ ID NO: 83), and even if it incorporates an amino acid sequence identical to the CDR3 sequence (SEQ ID NO: 84) is acceptable.

[0049] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 85 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% identical to SEQ ID NO: 85 (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 87), CDR2 sequence (SEQ ID NO: 88), and CDR3 sequence (SEQ ID NO: 89) of SEQ ID NO: 85. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 86 (e.g., 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 90), CDR2 sequence (SEQ ID NO: 91), and CDR3 sequence (SEQ ID NO: 92) of SEQ ID NO: 86. is acceptable. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 93 and a light chain variable domain related to SEQ ID NO: 94. For example, the

[0050] heavy chain variable domain of the first antigen-binding site may be at least 90% identical to SEQ ID NO: 93 (e.g., 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 93) of SEQ ID NO: 93, CDR2 sequence (SEQ ID NO: 94), and CDR3 sequence (SEQ ID NO: identical to the VH CDR1 sequence (SEQ ID NO: 95), CDR2 sequence (SEQ ID NO: 96), and CDR3 sequence (SEQ ID NO: 97). It may incorporate an amino acid sequence that is the same. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 94 (e.g., 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100%) and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 98), CDR2 sequence (SEQ ID NO: 99), and CDR3 sequence (SEQ ID NO: 100) of SEQ ID NO: 94.

[0051] In some embodiments, the first antigen-binding site has, for example, an amino acid sequence that is at least 90 % identical to SEQ ID NO: 101 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100%) and an amino acid sequence that is at least 90 % identical to SEQ ID NO: 102 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100%), thereby incorporating a heavy chain variable domain related to SEQ ID NO: 101 and a light chain variable domain related to SEQ ID NO: 102, respectively.

[0052] In some embodiments, the first antigen-binding site has, for example, an amino acid sequence that is at least 90 % identical to SEQ ID NO: 103 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100%) and an amino acid sequence that is at least 90 % identical to SEQ ID NO: 104 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100%), thereby incorporating a heavy chain variable domain related to SEQ ID NO: 103 and a light chain variable domain related to SEQ ID NO: 104, respectively. ​​​​The heavy chain variable domain related to column number 103 and the light chain variable domain related to SEQ ID NO: 104 may be incorporated. It may incorporate.

[0053] In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain related to SEQ ID NO: 115 and the light chain variable domain related to SEQ ID NO: 119. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 115, and / or may incorporate the amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 116), CDR2 sequence (SEQ ID NO: 117 ), and CDR3 sequence (SEQ ID NO: 118) of SEQ ID NO: 115. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100%) identical to SEQ ID NO: 119, and / or may incorporate the amino acid sequence identical to the C DR1 sequence (SEQ ID NO: 120), CDR2 sequence (SEQ ID NO: 121), and CDR3 sequence (SEQ ID NO: 122) of SEQ ID NO: 119. In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain related to SEQ ID NO: 123 and the light chain variable domain related to SEQ ID NO: 127. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 123, and / or may incorporate the amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 124), CDR2 sequence (SEQ ID NO: 125 ), and CDR3 sequence (SEQ ID NO: 126) of SEQ ID NO: 123. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9

[0054] In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain related to SEQ ID NO: 123 and the light chain variable domain related to SEQ ID NO: 127. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 123, and / or may incorporate the amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 124), CDR2 sequence (SEQ ID NO: 125 ), and CDR3 sequence (SEQ ID NO: 126) of SEQ ID NO: 123. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 identical, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 124), CDR2 sequence (SEQ ID NO: 125), and CDR3 sequence (SEQ ID NO: 126) of SEQ ID NO: 123, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 127, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 128), CDR2 sequence (SEQ ID NO: 129), and CDR3 sequence (SEQ ID NO: 130) of SEQ ID NO: 127. The CDR1 sequence (SEQ ID NO: 124), CDR2 sequence (SEQ ID NO: 125), and CDR3 sequence (SEQ ID NO: 126) of SEQ ID NO: 123 may be incorporated. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 127, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 128), CDR2 sequence (SEQ ID NO: 129), and CDR3 sequence (SEQ ID NO: 130) of SEQ ID NO: 127. In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain associated with SEQ ID NO: 131 and the light chain variable domain associated with SEQ ID NO: 135. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 131, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 132), CDR2 sequence (SEQ ID NO: 133), and CDR3 sequence (SEQ ID NO: 134) of SEQ ID NO: 131. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 135, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 136), CDR2 sequence (SEQ ID NO: 137), and CDR3 sequence (SEQ ID NO: 138) of SEQ ID NO: 135.

[0055] In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain associated with SEQ ID NO: 131 and the light chain variable domain associated with SEQ ID NO: 135. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 131, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 132), CDR2 sequence (SEQ ID NO: 133), and CDR3 sequence (SEQ ID NO: 134) of SEQ ID NO: 131. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 135, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 136), CDR2 sequence (SEQ ID NO: 137), and CDR3 sequence (SEQ ID NO: 138) of SEQ ID NO: 135. In some embodiments, the second antigen-binding site can bind to EpCAM and may incorporate the heavy chain variable domain associated with SEQ ID NO: 131 and the light chain variable domain associated with SEQ ID NO: 135. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 131, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 132), CDR2 sequence (SEQ ID NO: 133), and CDR3 sequence (SEQ ID NO: 134) of SEQ ID NO: 131. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 135, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 136), CDR2 sequence (SEQ ID NO: 137), and CDR3 sequence (SEQ ID NO: 138) of SEQ ID NO: 135. The DR1 sequence (SEQ ID NO: 136), the CDR2 sequence (SEQ ID NO: 137), and the CDR3 sequence may incorporate the same amino acid sequence as (SEQ ID NO: 138).

[0056] In some embodiments, the second antigen-binding site can bind to EpCAM and has a sequence that may incorporate the heavy chain variable domain associated with SEQ ID NO: 139 and the light chain variable domain associated with SEQ ID NO: 143. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 139 and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 140), the CDR2 sequence (SEQ ID NO: 141 ), and the CDR3 sequence (SEQ ID NO: 142) of SEQ ID NO: 139. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100%) identical to SEQ ID NO: 143 and / or may incorporate the same amino acid sequence as the C DR1 sequence (SEQ ID NO: 144), the CDR2 sequence (SEQ ID NO: 145), and the CDR3 sequence of SEQ ID NO: 143 (SEQ ID NO: 146). In some embodiments, the second antigen-binding site can bind to CA125 and has a sequence that may incorporate the heavy chain variable domain associated with SEQ ID NO: 155 and the light chain variable domain associated with SEQ ID NO: 159.

[0057] For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 155 and may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 160), the CDR2 sequence (SEQ ID NO: 161 ), and the CDR3 sequence (SEQ ID NO: 162) of SEQ ID NO: 155. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 identical, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 156), CDR2 sequence (SEQ ID NO: 157), and CDR3 sequence (SEQ ID NO: 158) of SEQ ID NO: 155, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 159, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 160), CDR2 sequence (SEQ ID NO: 161), and CDR3 sequence (SEQ ID NO: 162) of SEQ ID NO: 159. In some embodiments, the second antigen-binding site can bind to CA125 and may incorporate the heavy chain variable domain associated with SEQ ID NO: 163 and the light chain variable domain associated with SEQ ID NO: 167. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 163, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 164), CDR2 sequence (SEQ ID NO: 165), and CDR3 sequence (SEQ ID NO: 166) of SEQ ID NO: 163, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 167, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 168), CDR2 sequence (SEQ ID NO: 169), and CDR3 sequence (SEQ ID NO: 170) of SEQ ID NO: 167. and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 156), CDR2 sequence (SEQ ID NO: 157 ), and CDR3 sequence (SEQ ID NO: 158) of SEQ ID NO: 155. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100%) identical to SEQ ID NO: 159, and / or may incorporate an amino acid sequence identical to the C DR1 sequence (SEQ ID NO: 160), CDR2 sequence (SEQ ID NO: 161), and CDR3 sequence (SEQ ID NO: 162) of SEQ ID NO: 159.

[0058] In some embodiments, the second antigen-binding site can bind to CA125 and may incorporate the heavy chain variable domain associated with SEQ ID NO: 163 and the light chain variable domain associated with SEQ ID NO: 167. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 163, and / or may incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 164), CDR2 sequence (SEQ ID NO: 165 ), and CDR3 sequence (SEQ ID NO: 166) of SEQ ID NO: 163. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 167, and / or may incorporate an amino acid sequence identical to the C DR1 sequence (SEQ ID NO: 168), CDR2 sequence (SEQ ID NO: 169), and CDR3 sequence (SEQ ID NO: 170) of SEQ ID NO: 167. Similarly, the light chain variable domain of the second antigen-binding site may be at least 9 0% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100%) identical to SEQ ID NO: 167, and / or may incorporate an amino acid sequence identical to the C The DR1 sequence (SEQ ID NO: 168), the CDR2 sequence (SEQ ID NO: 169), and the CDR3 sequence may incorporate the same amino acid sequence as (SEQ ID NO: 170).

[0059] In some embodiments, the second antigen-binding site can bind to NaPi2b and may incorporate the heavy chain variable domain related to SEQ ID NO: 171 and the light chain variable domain related to SEQ ID NO: 175. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 171 and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 172), the CDR2 sequence (SEQ ID NO: 17 3), and the CDR3 sequence (SEQ ID NO: 174) of SEQ ID NO: 171. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 175 and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 176), the CDR2 sequence (SEQ ID NO: 177), and the CDR3 sequence (SEQ ID NO: 178) of SEQ ID NO: 175. 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 176), the CDR2 sequence (SEQ ID NO: 177), and the CDR3 sequence (SEQ ID NO: 178) of SEQ ID NO: 175.

[0060] In some embodiments, the second antigen-binding site can bind to Nectin4 and may incorporate the heavy chain variable domain related to SEQ ID NO: 179 and the light chain variable domain related to SEQ ID NO: 183. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 179 and may incorporate the amino acid sequence of SEQ ID NO: 179 and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 172), the CDR2 sequence (SEQ ID NO: 17 be the same as 95%, 96%, 97%, 98%, 99%, or 100%, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 180), CDR2 sequence (SEQ ID NO: 1 81), and CDR3 sequence (SEQ ID NO: 182) of SEQ ID NO: 179. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 183 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100%) and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 184), CDR2 sequence (SEQ ID NO: 185), and CDR3 sequence (SEQ ID NO: 186) of SEQ ID NO: 183.

[0061] In some embodiments, the second antigen-binding site can bind to fucosyl-GM1 and may incorporate the heavy chain variable domain related to SEQ ID NO: 187 and the light chain variable domain related to SEQ ID NO: 191. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 187 (e.g., 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100%) and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 188), CDR2 sequence (SEQ ID NO: 189), and CDR3 sequence (SEQ ID NO: 190) of SEQ ID NO: 187. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% identical to SEQ ID NO: 191 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100%) and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 192), CDR2 sequence (SEQ ID NO: 193), and CDR3 sequence (SEQ ID NO: 194) of SEQ ID NO: 191, and / or incorporate an amino acid sequence identical to the CDR1 sequence (SEQ ID NO: 195), CDR2 sequence (SEQ ID NO: 196), and CDR3 sequence (SEQ ID NO: 197) of SEQ ID NO: 191. The CDR1 sequence of 1 (SEQ ID NO: 192), the CDR2 sequence (SEQ ID NO: 193), and the CDR 3 sequence (SEQ ID NO: 194) may incorporate the same amino acid sequence.

[0062] In some embodiments, the second antigen-binding site can bind to SLC44A4, The heavy chain variable domain related to SEQ ID NO: 195 and the light chain variable do main related to SEQ ID NO: 199 may be incorporated. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 195, and / or may incorporate the same amino acid sequence as the CDR1 sequence (SEQ ID NO: 196), CDR2 sequence (SEQ ID NO: 1 97), and CDR3 sequence (SEQ ID NO: 198) of SEQ ID NO: 195. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%

[0063] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having the same amino acid sequence as the amino acid sequence of the light chain variable domain present in the first antigen-binding site.

[0064] In some embodiments, the protein has an antibody Fc domain sufficient to bind to CD16 incorporates a portion thereof, wherein the antibody Fc domain is the hinge and CH2 domains, and / or at least 90% identical to the amino acid sequence 234-332 of a human IgG antibody and comprises an amino acid sequence.

[0065] Some of the proteins of the present disclosure bind to NKG2D with a K of 10 nM D or weaker affinity.

[0066] A formulation containing one of these proteins; one or a plurality of cells containing nucleic acids expressing these proteins, and methods of enhancing tumor cell death using these proteins are also provided.

[0067] Another aspect of the invention provides a method of treating cancer in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of the multispecific binding protein described herein. Exemplary cancers treated using the multispecific binding protein include, for example, head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0111] Detailed Description The present invention relates to EPCAM in cancer cells and NKG in natural killer cells Multiple specificities that bind to 2D and CD16 receptors and activate natural killer cells The present invention relates to a method for the preparation of a multispecific binding protein comprising administering to a subject a pharmaceutical composition comprising the multispecific binding protein, Therapeutic methods using such multispecific proteins and pharmaceutical compositions, such as for the treatment of cancer. Various aspects of the invention are described below in sections. However, embodiments of the invention described in one particular section may be combined with any of the embodiments described in any particular section. The present invention is not limited to the above.

[0112] To facilitate the understanding of this invention, several terms and phrases are defined below.

[0113] As used herein, the terms "a" and "an" refer to "one or more" and includes plural unless the context is inappropriate.

[0114] As used herein, the term "antigen-binding site" refers to the immunological site involved in antigen binding. In human antibodies, the antigen-binding site is composed of heavy ("H") chains and The heavy chain is formed by amino acid residues from the N-terminal variable ("V") region of the heavy ("L") chain. Three highly branched stretches within the V regions of the hepatic and light chains are called "framework regions" or "FR" are intervening stretches of more conserved flanking sequences. The term "FR" is therefore used to refer to the "hypervariable region" of an immunoglobulin. It refers to the amino acid sequence found naturally between and adjacent to the hypervariable regions in human In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are responsible for antigen binding. The antigen-binding surface is a surface that is formed by binding is complementary to the three-dimensional surface of the combined antigen, and each of the three hypervariable regions of the heavy and light chains is referred to as a "complementary determining region" or "CDR". In certain animals such as camels and cartilaginous fish, the antigen-binding site is provided by a single antibody chain that provides a "single-domain antibody". The antigen-binding site is present in the intact antibody, in the antigen-binding fragment of the antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as scFv, and the heavy-chain variable domain can be linked to the light-chain variable domain in a single polypeptide using a peptide linker.

[0115] As used herein, the term "tumor-associated antigen" means any antigen, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, and lipids, associated with cancer. Such antigens can be expressed in malignant cells or in the tumor microenvironment, such as in tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates.

[0116] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0117] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to achieve a beneficial or desired result. The effective amount may be administered in one or more doses, applications, or dosages and is not limited to a particular formulation or route of administration. is not intended to. As used herein, the term "treating" means any effect, such as reduction, diminution, modulation, improvement or elimination, or amelioration of those symptoms, which results in the amelioration of a

[0118] condition, disease, disorder, etc. As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that renders the composition particularly suitable for

[0119] diagnostic or therapeutic use in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsion (such as oil / water or water / oil emulsion, etc.), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and

[0120] adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] . As used herein, the term "pharmaceutically acceptable salt" of a compound of the present invention refers to any pharmaceutically acceptable salt (e.g., acid or base) of the compound of the present invention that can be administered to a subject and provides the compound of the present n-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. are included Other acids such as oxalic acid are not pharmaceutically acceptable by themselves, but are useful salts for the preparation of the compounds of the present invention and their pharmaceutically acceptable acid addition salts. They can be used.

[0121] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of the formula NW4 + (in the formula wherein W is C 1~4 alkyl).

[0122] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmitate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, etc. Other examples of salts include Na + , NH4 + and NW4 + (in the formula, W is C + (wherein W is C1~4 such as an alkyl group) The anion of the compound of the present invention formulated with a suitable cation is included.

[0123] For therapeutic use, the salts of the compounds of the present invention are intended to be pharmaceutically acceptable. However salts of acids and bases that are not pharmaceutically acceptable can also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0124] Throughout the description where a composition is described as having, including, or comprising a particular component, or a process and method are described as having, including, or comprising a particular step, it is also intended that there are compositions of the present invention consisting essentially of or consisting of the recited components, and processes and methods according to the present invention consisting essentially of or consisting of the recited process steps.

[0125] In general, compositions specifying percentages are by weight unless otherwise indicated. Further, when a variable is not accompanied by a definition, the previous definition of the variable prevails. I. Protein

[0126] The present invention provides a multispecific binding protein that binds to the NKG2D receptor and the CD16 receptor in natural killer cells, as well as to a tumor-associated antigen selected from any one of the antigens presented in Table 11. The multispecific binding protein is useful in the pharmaceutical compositions and treatment methods described herein. NKG in natural killer cells ​​​​​​​​​When a multispecific binding protein binds to the 2D receptor and the CD16 receptor, the destruction of tumor cells expressing a tumor-associated antigen selected from any one of the antigens shown in Table 11 is enhanced by the activation of natural killer cells. When a multispecific binding protein binds to cells expressing a tumor-associated antigen, the cancer cells are brought close to natural killer cells, facilitating the direct and indirect destruction of cancer cells by natural killer cells. Further description of several exemplary multispecific binding proteins is provided below.

[0127] The first component of the multispecific binding protein binds to cells expressing the NKG2D receptor, which may include but is not limited to NK cells, γδ T cells, and CD8 + αβ T cells. When the multispecific binding protein binds to NKG2D, it can block the binding of natural ligands such as ULBP6 (UL16-binding protein 6) and MICA (major histocompatibility complex class I chain-related A) to NKG2D and the activation of the NKG2D receptor.

[0128] The second component of the multispecific binding protein binds to a tumor-associated antigen selected from any one of the antigens presented in Table 11. For example, cells expressing a tumor-associated antigen found in leukemias such as acute myeloid leukemia and T-cell leukemia.

[0129] The third component of the multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0130] ​​​​​​​​​​​ The multispecific binding proteins described herein can take various formats For example, one format is a heterodimeric multispecific antibody (Figure 1) that includes a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc (hinge -CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, 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-associated antigen selected from any one of the antigens presented in Table 11 The first Fc domain and the second Fc domain can together bind to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain Another exemplary format relates to a heterodimeric multispecific antibody (Figure 2) that includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain. The first immunoglobulin heavy chain pairs and binds to NKG2D or is presented in Table 11 The first Fc domain and the second Fc domain can together bind to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain Another exemplary format relates to a heterodimeric multispecific antibody (Figure 2) that includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain

[0131] Another exemplary format relates to a heterodimeric multispecific antibody (Figure 2) that includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain. The first immunoglobulin heavy chain pairs and binds to NKG2D or is presented in Table 11 The first immunoglobulin heavy chain pairs and binds to NKG2D or is presented in Table 11 A heavy chain variable domain that binds to a tumor-associated antigen selected from any one of the antigens present, and a single-chain variable fragment (scFv) composed of a light chain variable domain are fused via either a linker or an antibody hinge to include a first Fc (hinge-CH2-CH3) domain. 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 NKG2D or binds to a tumor-associated antigen selected from any one of the antigens presented in Table 11. The first Fc domain and the second Fc domain can bind to CD16 together (Figure 2). One or more additional binding motifs can be fused to the C-terminus of the constant region CH3 domain via a linker sequence, if desired. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) that forms a tetravalent or trivalent molecule. In some embodiments, the multispecific binding protein is in the Triomab format, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. In some embodiments, the multispecific binding protein is in the KiH common light chain (LC) format using the knob-into-hole (KIH) technology. KIH promotes heterodimerization by In some embodiments, the multispecific binding protein is in the Triomab format, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. The 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 NKG2D or binds to a tumor-associated antigen selected from any one of the antigens presented in Table 11. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to NKG2D or binds to a tumor-associated antigen selected from any one of the antigens presented in Table 11. The first Fc domain and the second Fc domain can bind to CD16 together (Figure 2). The first Fc domain and the second Fc domain can bind to CD16 together (Figure 2).

[0132] One or more additional binding motifs can be fused to the C-terminus of the constant region CH3 domain via a linker sequence, if desired. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) that forms a tetravalent or trivalent molecule. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) that forms a tetravalent or trivalent molecule. One or more additional binding motifs can be fused to the C-terminus of the constant region CH3 domain via a linker sequence, if desired. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) that forms a tetravalent or trivalent molecule. One or more additional binding motifs can be fused to the C-terminus of the constant region CH3 domain via a linker sequence, if desired. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) that forms a tetravalent or trivalent molecule.

[0133] In some embodiments, the multispecific binding protein is in the Triomab format, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain. This chimera consists of two half-antibodies derived from two parental antibodies, each half-antibody having one light chain and one heavy chain.

[0134] In some embodiments, the multispecific binding protein is in the KiH common light chain (LC) format using the knob-into-hole (KIH) technology. KIH promotes heterodimerization by In some embodiments, the multispecific binding protein is in the KiH common light chain (LC) format using the knob-into-hole (KIH) technology. KIH promotes heterodimerization by In some embodiments, the multispecific binding protein is in the KiH common light chain (LC) format using the knob-into-hole (KIH) technology. KIH promotes heterodimerization byH Engineering the 3 domains to create either a "knob" or a "hole" in each heavy chain is involved. The concept behind the "knob-into-hole (KiH)" Fc technology was to introduce a "knob" (e.g., T366W in EU numbering ) into one CH3 domain (CH3A CH3A ) by replacing a small residue with a bulky residue. To accommodate this "knob", in the other CH3 domain (CH3B), the residue closest to the knob was replaced with a smaller residue (i.e., T366S / L368A / Y 407V CH3B ) to create a complementary "hole" surface. The "hole" mutations were optimized by structure-based phage display library screening (Atwell S, Ridgway JB, We lls JA, Carter P., Stable heterodimers from remodeling the domain interf ace of a homodimer using a phage display library, J Mol Biol (1997 year) 270(1):26 - 35). The X-ray crystal structure of the KiH Fc mutant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C et al., Ant iparallel conformation of knob and hole aglycosylated half-antibody homo dimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) 426(9):1947 - 57, Mimoto F, Kadono S, Katada (2014) 426(9):1947 - 57, Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmet rically engineered Fc variant with improved affinity for FcγRs. Mol Immunol (2014) 58(1):132 - 138) showed that at the core interface between CH3 domains, hydrophobic interactions driven by steric complementarity contribute thermodynamically favorably to heterodimerization, while the knob - knob and hole - hole interfaces do not contribute favorably to homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0135] In some embodiments, the multispecific binding protein combines the target binding domains of two monoclonal antibodies via a naturally - occurring flexible linker to form a tetravalent IgG - like molecule in the form of a dual - variable domain immunoglobulin (DVD - Ig™).

[0136] In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (ortho - Fab). 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 - 198) showed that through structure - based domain design, only at the interface between the LC and HC VH-CH1 in one Fab fragment ​Complementary mutations are introduced and the other Fab does not change.

[0137] In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format In some embodiments, the multispecific binding protein is a heterodimeric construct that contains two different Fab fragments that bind to target 1 and target 2 and are fused to the Fc and is in the ES form. Heterodimerization is ensured by electrostatic steering mutations in the Fc

[0138] In some embodiments, the multispecific binding protein is a heterodimeric construct having two different Fab fragments fused to an Fc stabilized by heterodimerization mutations and is in the κ λ-Body form. Fab fragment 1 targeting antigen 1 contains a kappa LC and the second Fab fragment targeting antigen 2 contains a lambda LC. Figure 30A is an exemplary diagram of one form of the κ λ-Body and Figure 30B is an exemplary diagram of another κλ-Body

[0139] In some embodiments, the multispecific binding protein is in the Fab arm exchange form (an antibody that becomes a bispecific antibody by swapping the Fab arms by swapping the heavy chain and associated light chain (half molecule) with the heavy chain-light chain pair of another molecule)

[0140] In some embodiments, the multispecific binding protein is in the SEED Body form The SEED (strand-exchange engineered do main) platform is an asymmetric and bispecific ​​​​​Designed to generate bispecific antibody-like molecules. This protein engineering operation plan The toform is based on the exchange of immunoglobulin structurally related sequences in the conserved CH3 domain. The SEED design enables the effective generation of AG / GA heterodimers while avoiding the homodimerization of AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54). ) )

[0141] In some embodiments, the multispecific binding protein is in the LuZ-Y form that uses a leucine zipper to induce the heterodimerization of two different HCs ( Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9). )

[0142] In some embodiments, the multispecific binding protein is in the Cov-X-Body form. In the bispecific CovX-Body, a branched azetidinone linker is used to link two different peptides together and fused to the scaffold antibody in a site-specific manner under mild conditions. It is the pharmacophore that is involved in the functional activity, but the antibody scaffold provides a long half-life and an Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with other pharmacophores (Doppalapudi VR et al., PNAS (20 10), 107(52);22611-22616). Use to bind two different peptides together and fuse them to the scaffold antibody in a site-specific manner under mild conditions. It is the pharmacophore that is involved in the functional activity, but the antibody scaffold provides a long half-life and an Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with other pharmacophores (Doppalapudi VR et al., PNAS (20 10), 107(52);22611-22616). The antibody scaffold provides a long half-life and an Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with other pharmacophores (Doppalapudi VR et al., PNAS (20 10), 107(52);22611-22616). 10), 107(52);22611-22616). )

[0143] In some embodiments, the multispecific binding protein is fused to Fc and binds to target 1 An Oasc-Fab heterodimer comprising a Fab fragment that binds to antigen 1 and an scFab that binds to antigen 2 is in the form. Heterodimerization is ensured by mutations in the Fc .

[0144] In some embodiments, the multispecific binding protein is a DuetMab form, which is a heterodimeric construct containing two different Fab fragments that bind to antigens 1 and 2, as well as an Fc stabilized by a heterodimerization mutation. Fab fragments 1 and 2 contain specific S-S bridges that ensure the correct pairing of the LC and HC . is in the form. In some embodiments, the multispecific binding protein is a CrossmAb form, which is a heterodimeric construct having two different Fab fragments that bind to antigens 1 and 2, fused to an Fc stabilized by heterodimerization. The CL domain and the CH1 domain are switched with the VH domain and the VL domain. For example, CH1 is fused in-line with VL, and CL is fused in-line with VH

[0145] In some embodiments, the multispecific binding protein is a Fit-Ig form, 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 fragment that binds to antigen 1. This construct contains a wild-type Fc . is in the form. The CL domain and the CH1 domain are switched with the VH domain and the VL domain. For example, CH1 is fused in-line with VL, and CL is fused in-line with VH .

[0146] In some embodiments, the multispecific binding protein is a Fit-Ig form, 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 fragment that binds to antigen 1. This construct contains a wild-type Fc . is in the form.

[0147] Table 1 lists the peptide sequences of the heavy chain variable domains and the light chain variable domains that can combine to bind to NKG2D. The NKG2D binding domain is specific for NKG2D . Although the binding affinities thereof can vary, all activate human NKG2D and NK cells.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

[0148] Alternatively, as described in US9,273,136, the heavy chain variable domain represented by SEQ ID NO: 101 may be paired with the light chain variable domain represented by SEQ ID NO: 102 to form an antigen-binding site capable of binding to NKG2D.

Chemical Structure

[0149] Alternatively, as described in US7,879,985, the heavy chain variable domain represented by SEQ ID NO: 103 may be paired with the light chain variable domain represented by SEQ ID NO: 104 to form an antigen-binding site capable of binding to NKG2D.

Chemical Structure

[0150] Table 2 lists the peptide sequences of the heavy-chain variable domain and the light-chain variable domain that can combine to bind to EpCAM. [Table 2-1] [Table 2-2] [Table 2-3]

[0151] Alternatively, a novel antigen-binding site capable of binding to EpCAM can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 147. [Chemical formula]

[0152] An antigen-binding site capable of binding to the tumor-associated antigen CA125 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 148. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula]

[0153] An antigen-binding site capable of binding to the tumor-associated antigen NaPi2b can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 149. [Chemical formula]

[0154] An antigen-binding site capable of binding to the tumor-associated antigen Nectin4 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 150. [Chemical formula]

[0155] An antigen-binding site capable of binding to the tumor-associated antigen fucosyl-GM1 can be identified by screening for binding to monosialotetrahexosylganglioside.

[0156] An antigen-binding site capable of binding to the tumor-associated antigen ADAM8 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 151. [Chemical formula]

[0157] An antigen-binding site capable of binding to the tumor-associated antigen ADAM9 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 152. ​​​​​​​​​ It is possible.

Chem.

Chem.

[0158] An antigen-binding site capable of binding to the tumor-associated antigen SLC44A4 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 153 It can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 153 It is possible.

Chem.

[0159] An antigen-binding site capable of binding to the tumor-associated antigen CA19-9 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 154 It can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 154 It is possible.

Chem.

[0160] Alternatively, Table 3 lists the peptide sequences of the heavy-chain variable domain and the light-chain variable domain that can bind, when combined, to CA125 (Abagovomab, Sofituzumab), NaPi2b (Refastuzumab), Nectin4 (Enfortumab), fucosyl-G M1 (described in US Patent Application Publication No. 20130142789, the specific sequence of which is incorporated herein by reference), or SLC44A4 (described in International Application Publication No. WO2010111 018, the specific sequence of which is incorporated herein by reference). It can bind to the peptide sequences of the heavy-chain variable domain and the light-chain variable domain that can bind, when combined, to CA125 (Abagovomab, Sofituzumab),

Table 3-1

Table 3-2

Table 3-3

[0161] linked to an antibody constant region that also contains mutations that allow heterodimerization of two polypeptide chains Examples of scFvs are listed below. The heavy chain variable domain (V H ) and light chain variable domain (V L ) containing scFvs are used in the preparation of the multispecific proteins of the present disclosure. Each sequence represents V -(G4S)4-V L -hinge (AS or GAS)-Fc with heterodimerization mutations (underlined). V H -V -hinge (AS or GAS)-Fc with heterodimerization mutations (underlined). V L and V H are 100V L -44 V H containing an S-S bridge (underlined) and may be derived from any antibody that targets a tumor or binds to NKG2D. Ala-Ser (AS, bold and underlined) is included in the elbow hinge region sequence to balance flexibility and optimal geometry. In certain embodiments, an additional Gly is added to the N-terminus of the AS sequence, thereby generating a hinge having the sequence Gly-Ala-Ser (GAS, bold and underlined). In certain embodiments, an additional sequence Thr-Lys-Gly can be added to the AS sequence of the hinge. The (G4S)4 linker is underlined in the sequences listed in the following paragraphs. For balance, Ala-Ser (AS, bold and underlined) is included in the elbow hinge region sequence. In certain embodiments, an additional Gly is added to the N-terminus of the AS sequence, thereby generating a hinge having the sequence Gly-Ala-Ser (GAS, bold and underlined). In certain embodiments, an additional sequence Thr-Lys-Gly can be added to the AS sequence of the hinge. The (G4S)4 linker is underlined in the sequences listed in the following paragraphs. certain embodiments, an additional Gly is added to the N-terminus of the AS sequence, thereby generating a hinge having the sequence Gly-Ala-Ser (GAS, bold and underlined). In certain embodiments, an additional sequence Thr-Lys-Gly can be added to the AS sequence of the hinge. The (G4S)4 linker is underlined in the sequences listed in the following paragraphs. In certain embodiments, an additional sequence Thr-Lys-Gly can be added to the AS sequence of the hinge. The (G4S)4 linker is underlined in the sequences listed in the following paragraphs. linker is underlined in the sequences listed in the following paragraphs.

[0162] The TriNKET of the present disclosure comprises a first polypeptide (F4- ​EpCAMFc-AJchainB-NKG2D-binding scFv), and a second polypeptide (anti-EpCAM HC-hinge-Fc) comprising the sequence of SEQ ID NO: 204. The NKG2D-binding F4-TriNKET-EpCAM is composed of. The NKG2D-binding F4-TriNKET-EpCAM EpCAM also includes two light chains targeting EpCAM, each of which comprises the sequence of SEQ ID NO: 214. For example, in the structure of FIG. 36, when the Fab fragment targets EpCAM, the NKG2D-binding F4-TriNKET-EpCAM EpCAM consists of SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, L constant domain and also includes. For example, in the structure of FIG. 36, when the Fab fragment targets EpCAM, the NKG2D-binding F4-TriNKET-EpCAM EpCAM consists of SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM EpCAM contains SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM contains.

[0163] Each arm contains an EpCAM-binding Fab fragment, and the EpCAM-binding Fab fragment is a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, where the heavy chain variable domain and the CH 1 domain are linked, and a light chain portion comprising a light chain variable domain and a light chain constant domain (SEQ ID NO: 214). In the first arm (for example, in F4-EpCAMFc-AJchainB-NKG2D-binding scFv), the CH1 domain is linked to the Fc domain, and the Fc domain is linked to the scFv targeting NKG2D, forming a polypeptide comprising the sequence of SEQ ID NO: 203. In the second arm, the CH1 domain is linked to the Fc domain, forming a polypeptide comprising the sequence of SEQ ID NO: 204. The NKG2D-binding F4-TriNKET-EpCAM EpCAM contains SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM EpCAM contains SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM EpCAM contains SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM EpCAM contains SEQ ID NO: 203 and SEQ ID NO: 214 forming one arm of the TriNKET, and SEQ ID NO: 204 and SEQ ID NO: 214 forming the second arm of the TriNKET. The NKG2D-binding F4-TriNKET-EpCAM

[0164] For example, F4-EpCAMFc-AJchainB-NKG2D-binding scFv (SEQ ID NO: No. 203) is a heavy chain variable domain (V H ) (SEQ ID NO: 139) and and a CH1 domain linked to an Fc domain (hinge-CH2-CH3), which The Fc is linked at the C-terminus to a single chain variable fragment (scFv) that binds to NKG2D. The Fc domain in SEQ ID NO:203 contains an S354C substitution, which is identical to that of another Fc domain. It forms a disulfide bond with the Y349C substitution in the ribosome (SEQ ID NO: 204, below). The Fc domain in column number 203 contains the Q347R substitution, the D399V substitution, and the F405T The scFv that binds to NKG2D is represented by the amino acid sequence of SEQ ID NO: 205. The (G4S)4 linker, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 206) via the heavy chain variable domain (V H ) linked to a light chain variable domain (V L ) V contained in sequence number 205 L and V H V L -(G4S)4-V H Concatenated as And V L and V H is 100V L -44V H Contains S-S bridges (G (Cysteine ​​residues are in bold italics) (The C-terminus of the Fc domain is shown in SEQ ID NO: 203 and is underlined.) At the N-terminus of the scFv (SEQ ID NO: 205), a short SGSGGGGS linker (SEQ ID NO: 20 7). NKG2D binding scFv [ka] F4-EpCAM Fc-AJ chain B-NKG2D-binding scFv

Chem.

[0165] The anti-EpCAM HC-hinge-Fc (SEQ ID NO: 204) contains a CH1 domain linked to a heavy-chain variable domain and an Fc domain (hinge-CH2-CH3) that targets EpCAM. The Fc domain within SEQ ID NO: 204 contains a Y349C substitution, which forms a disulfide bond with the S354C substitution in the CH3 domain of the Fc (SEQ ID NO: 203) linked to the scFv that binds NKG2D. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. chain variable domain and an Fc domain (hinge-CH2-CH3) that targets EpCAM. The Fc domain within SEQ ID NO: 204 contains a Y349C substitution, which forms a disulfide bond with the S354C substitution in the CH3 domain of the Fc (SEQ ID NO: 203) linked to the scFv that binds NKG2D. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. chain variable domain and an Fc domain (hinge-CH2-CH3) that targets EpCAM. The Fc domain within SEQ ID NO: 204 contains a Y349C substitution, which forms a disulfide bond with the S354C substitution in the CH3 domain of the Fc (SEQ ID NO: 203) linked to the scFv that binds NKG2D. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. 2D-binding scFv. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. In SEQ ID NO: 204, the Fc domain also contains K360E and K409W substitutions. Anti-EpCAM V H -CH1-Fc

Chem.

[0166] Anti-EpCAM V L The constant domain (SEQ ID NO: 214) contains a light-chain variable domain and a light-chain constant domain that target EpCAM. constant domain that target EpCAM. Anti-EpCAM V L constant domain

Chem.

[0167] In an exemplary embodiment, the Fc domain linked to the NKG2D-binding scFv fragment contains the mutations K3 60E and K409W, and the Fc domain linked to the EPCAM Fab fragment contains the compatible mutations Q347R, D399V, and F40 5T for heterodimer formation. 5T for heterodimer formation.

[0168] In an exemplary embodiment, the Fc domain linked to the NKG2D-binding scFv contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution in the Fc domain not linked to the NKG2D-binding scFv.

[0169] Another TriNKET of the present disclosure is NKG2D-binding F3’-TriNKET-EPCAM, and its sequence is described below (CDR (Kabat numbering) is underlined).

[0170] Some TriNKETs of the present disclosure are in the form of A49-F3’-TriNKET-EPCAM, and its sequence is provided below (CDR (Kabat numbering) is underlined).

[0171] A49-F3’-TriNKET-EPCAM is a single-chain variable fragment (scFv) that binds to EPCAM, linked to the Fc domain via a hinge containing Gly-Ala-Ser (SEQ ID NOs: 208 and 209 are exemplary sequences of such EPCAM-binding scFv polypeptides) (e.g., SEQ ID NOs: 210 and SEQ ID NOs: 211), and a heavy-chain portion containing a heavy-chain variable domain (SEQ ID NOs: 85) and a CH1 domain, and a light-chain portion containing a light-chain variable domain (SEQ ID NOs: 86) and a light-chain constant domain, an NKG2D-binding Fab fragment (“A49”), wherein the heavy-chain variable domain is linked to the CH1 domain, and the CH1 domain is linked to the Fc domain. The Fc domain linked to the Fab targeting EpCAM contains a K360E substitution, a Q347R substitution for forming a heterodimer with a Fab containing a K409W substitution, a D399V substitution. ​​​​​​​​​​​​​​​ including the substitutions of F405S, F405Y, and F405T (see, for example, SEQ ID NO: 212 below).

[0172] The EPCAM-binding scFv of the present disclosure may include a heavy-chain variable domain linked to a light-chain variable domain by a (G4S)4 linker (V when V L is on the N-terminal side of V H V L (G4S)4V H or represented as LH, and when V H is on the N-terminal side of V L V H (G4S)4V L or represented as HL). SEQ ID NOs: 208 and 209 are exemplary sequences of such EPCAM-binding scFv polypeptides. The V and V V L and V H in scFv (SEQ ID NO: 208 or 209) contain a 100V L -44V H S-S bridge (resulting from G100C substitution and G 44C substitution, respectively) (cysteine residues are in bold italic font and underlined in the following sequences). (G4S)4 is the sequence GGGGSGGGGSGGGGSGGGGS ( SEQ ID NO: 206) that is underlined and in bold in SEQ ID NOs: 208 and 209. EPCAM(MT110LH)scFv

Chemical Structure

Chemical Structure

Chemical Structure

[0173] SEQ ID NOs: 210 and 211 are hinges containing Gly-Ala-Ser (bold , two EPCAM-binding scFvs that can be linked to the Fc domain via a sequence are shown. The Fc domain linked to the scFv contains Q347R, D399V, and F405T substitutions. EPCAM(MT110LH)scFv-Fc

Chemical Structure

Chemical Structure

[0174] SEQ ID NO: 212 represents the heavy chain portion of a Fab fragment containing the heavy chain variable domain (SEQ ID NO: 85) of the NKG2D binding site and the CH1 domain linked to the Fc domain. The Fc domain within SEQ ID NO: 212 contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution in the Fc linked to the scFv that binds to EpCAM (e.g., SEQ ID NO: 210 and SEQ ID NO: 211). In SEQ ID NO: 212 , the Fc domain also contains K360E and K409W substitutions. A49-V H

Chemical Structure

Chemical Structure

[0175] SEQ ID NO: 213 represents the light chain portion of a Fab fragment containing the light chain variable domain (SEQ ID NO: 86) of the NKG2D binding site and the light chain constant domain. A49-V L [Chemistry] A49 LC V L Stationary domain [Chemistry]

[0176] In an exemplary embodiment, the Fc domain linked to the NKG2D-binding Fab fragment contains the mutations Q34 7R, D399V, and F405T, and the F c domain linked to the EPCAM scFv contains the compatible mutations K360E and K409W for forming a heterodimer. In an exemplary embodiment, the Fc domain linked to the NKG2D-binding Fab fragment contains the S354C substitution in the CH3 domain, which forms a disulfide bond with the Y349C substitution in the Fc linked to the EPCAM-binding scFv.

[0177] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain . For example, within human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp26 5~Glu269, Asn297~Thr299, Ala327~Ile332, Leu 234~Ser239, and the carbohydrate residue N-acetyl-D- glucosamine in the CH2 domain (see Sondermann et al., Nature, Vol. 406 (No. 6793) : pages 267~273). Based on known domains, mutations can be selected to enhance or reduce the binding affinity for CD16 by using, for example, phage display libraries or yeast surface display cDNA libraries , or can be designed based on the known three-dimensional structure of the interaction.

[0178] Construction of heterodimeric antibody heavy chains allows expression of two different antibody heavy chain sequences in the same cell This can be achieved by the construction of homodimers of each antibody heavy chain and the The selective promotion of heterodimer assembly can result in the assembly of heterodimers. 3 / 494870, US16 / 028850, US11 / 533709, US12 / 87 5015, US13 / 289934, US14 / 773418, US12 / 811207 , US13 / 866756, US14 / 647480 and US14 / 830336 As previously reported, different mutations are incorporated into the CH3 domain of each antibody heavy chain constant region. For example, the mutations can be based on human IgG1, a first polypeptide that allows the two chains to selectively heterodimerize with each other; and a CH3 domain incorporating a different pair of amino acid substitutions in a second polypeptide. The positions of the amino acid substitutions exemplified below are as follows, as in Kabat: All numbered according to the EU index.

[0179] In one situation, the amino acid substitution in the first polypeptide replaces the original amino acid with an aryl ginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W) and replacing at least one amino acid in the second polypeptide with a larger amino acid selected from the group consisting of A single amino acid substitution replaces the original amino acid, while a larger amino acid substitution (overhang) replaces a smaller Alanine (A), serine (S), and threo (T) are added to fit the surface of the amino acid substitutions (cavity). The amino acid sequence is replaced with a smaller amino acid selected from the group consisting of tyrosine (T), tyrosine (T), and valine (V). then, one polypeptide can incorporate the T366W substitution, and the other polypeptide can incorporate three substitutions including T366S, L368A, and Y407V .

[0180] The antibody heavy chain variable domain of the present invention can, if necessary, be linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region that has or does not have a CH1 domain and includes a hinge, CH2, and CH3 domains. 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 derived from another mammal, such as a rabbit, dog, cat, mouse, or guinea pig. One or more mutations can be incorporated into the constant region, for example, at Q347, Y349, L3 51, S354, E356, E357, K360, Q362, S364, T366, L3 68, K370, N390, K392, T394, D399, S400, D401, F4 05, Y407, K409, T411, and / or K439 as compared to the human IgG1 constant region. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349 K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L3 51D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364 D, T366V, T366I, T366L, T366M, T366K, T366W, T3 68, K370, N390, K392, T394, D399, S400, D401, F4 05, Y407, K409, T411, and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349 K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L3 K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L3 51D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364 D, T366V, T366I, T366L, T366M, T366K, T366W, T3 66S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394 F, T394W, D399R, D399K, D399V, S400K, S400R, D4 01K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E are included. It is rare.

[0181] In certain embodiments, the mutations that can be incorporated into CH1 of the human IgG1 constant region are , amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, the mutations that can be incorporated into Cκ of the human IgG1 constant region are amino acids E123, F116, S176, V163, S174, and / or T164.

[0182] Alternatively, the amino acid substitutions can be selected from the sets of substitutions set forth in Table 4 below.

Table 4

[0183] Alternatively, the amino acid substitutions can be selected from the sets of substitutions shown in Table 5 below.

Table 5

[0184] Alternatively, the amino acid substitutions can be selected from the sets of substitutions shown in Table 6 below.

Table 6

[0185] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 7 . **Table 7**

[0186] Alternatively, at least one amino acid substitution can be selected from the set of substitutions in Table 8 below, where the position(s) shown in the "First Polypeptide" column can be replaced by any known negatively charged amino acid, and the position(s) shown in the "Second Polypeptide" column can be replaced by any known positively charged amino acid . **Table 8** . **Table 9**

[0187] Alternatively, at least one amino acid substitution can be selected from the set in Table 9 below, where the position(s) shown in the "First Polypeptide" column can be replaced by any known positively charged amino acid, and the position(s) shown in the "Second Polypeptide" column can be replaced by any known negatively charged amino acid . **Table 9** . **Table 10**

[0188] Alternatively, the amino acid substitution can be selected from the set in Table 10 below **Table 10**

[0189] Alternatively or in addition, the structural stability of the heteromultimeric protein is the first or second po Introducing S354C into any of the peptide chains and introducing Y349C into the opposite polypeptide chain can increase it, thereby forming an artificial disulfide cross-link within the interface of the two polypeptides. In some embodiments, the amino acid sequence of one polypeptide chain of the 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 other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366.

[0190] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the 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 other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0191] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the 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 other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0192] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0193] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG The amino acid sequence of the 1 constant region and one or more selected from the group consisting of Y349, E357, S364, L368, K370, T 394, D401, F405 and T411 are different at the positions, where the amino acid sequence of the other polypeptide chain of the antibody constant region is The amino acid sequence of the IgG1 constant region and is different at one or more positions selected from the group consisting of E357, K360, Q362, S364, L36 8, K370, T394, D401, F405, and T411. Selected from the group consisting of One or more positions that are different.

[0194] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG The amino acid sequence of the 1 constant region and is different at one or more positions selected from the group consisting of L351, D399, S400 and Y407, where the other po of the antibody constant region The amino acid sequence of the polypeptide chain is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, N39 0, K392, K409 and T411. Selected from the group consisting of one or more positions That are different.

[0195] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG The amino acid sequence of the 1 constant region and is different at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411, where the antibody constant region Selected from the group consisting of one or more positions The amino acid sequence of the other polypeptide chain of is different from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L35 1, D399, S400 and Y407. Selected from the group consisting of one or more positions

[0196] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG 1 constant region amino acid sequence and one or more positions selected from the group consisting of Q347, Y349, K360 and K409, where the other po lypeptide chain of the antibody constant region is different at one or more positions selected from the group consisting of the amino acid sequence of the IgG1 constant region and Q347, E35 7, D399 and F405 become different.

[0197] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG 1 constant region amino acid sequence and one or more positions selected from the group consisting of Q347, E357, D399 and F405, where the other po lypeptide chain of the antibody constant region is different at one or more positions selected from the group consisting of the amino acid sequence of the IgG1 constant region and Y349, K36 0, Q347 and K409 become different.

[0198] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG 1 constant region amino acid sequence and one or more positions selected from the group consisting of K370, K392, K409 and K439, where the other po lypeptide chain of the antibody constant region is different at one or more positions selected from the group consisting of the amino acid sequence of the IgG1 constant region and D356, E35 7 and D399 become different.

[0199] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is IgG 1 constant region amino acid sequence and is selected from the group consisting of D356, E357 and D399 differ at one or more positions, wherein the other polypeptide chain of the antibody constant region has an amino acid sequence that differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K40 9 and K439.

[0200] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399, wherein the other po lypeptide chain of the antibody constant region has an amino acid sequence that differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Y349, L35 1, L368, K392 and K409. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409, wherein the antibody constant region

[0201] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409, wherein the other polypeptide chain of the antibody constant region has an amino acid sequence that differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L35 1, E356, T366 and D399. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by an S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution. by.

[0202] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by an S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution. by. by.

[0203] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by an S354C substitution. by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by an S354C substitution.

[0204] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V, and F405T substitutions. by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V, and F405T substitutions.

[0205] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by O347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions. by O347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions.

[0206] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG 1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A, and Y407V substitutions. by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S,

[0207] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG ​​​1. The amino acid sequence of the constant region, and the T366S substitution, T368A substitution, and Y407V substitution differ, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region by the T366W substitution.

[0208] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG 1 constant region by the T350V substitution, L351Y substitution, F405A substitution, and Y407V substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region by the T350V substitution, T366L substitution, K392L substitution, and T394W substitution.

[0209] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG 1 constant region by the T350V substitution, T366L substitution, K392L substitution, and T394W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region is different from the amino acid sequence of the IgG1 constant region by the T350V substitution, L351Y substitution, F405A substitution, and Y407V substitution.

[0210] The above multispecific protein is produced using recombinant DNA techniques well known to those skilled in the art and obtained. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vec tor, 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 an immunoglobulin light chain can be cloned into a third expression vector, and the first, second, and third expression vectors can be used to express the proteins encoded by the nucleic acid sequences, and then the expressed proteins can be purified and assembled to obtain the multispecific protein. 3 expression vectors can be used to express the proteins encoded by the nucleic acid sequences, and then the expressed proteins can be purified and assembled to obtain the multispecific protein. Co-transfect the host cell stably with the second and third expression vectors to produce multimeric proteins. It is possible.

[0211] To achieve the highest yield of the multispecific protein, different ratios of the first, second, and third expression vectors can be examined 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. 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. 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. 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.

[0212] Clones can be cultured under conditions suitable for bioreactor scale-up and maintain the expression of the multispecific protein. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography. II. Characteristics of the Multispecific Protein

[0213] The multispecific proteins described herein include an NKG2D binding site, a CD16 binding site, and a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. When the multispecific protein binds to NK cells, the activity of NK cells towards tumor cell destruction can be enhanced. When the multispecific protein binds to NK cells, the activity of NK cells towards tumor cell destruction can be enhanced.

[0214]

Table 11

[0215] In some embodiments, the multispecific protein binds with an affinity similar to that of a monoclonal antibody (i.e., a monoclonal antibody containing the same tumor-associated antigen binding site as that incorporated into the multispecific protein) selected from any one of the antigens presented in Table 11 to a tumor-associated antigen selected from any one of them. In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. and binds to a tumor-associated antigen selected from any one of them. In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. al antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein binds with an affinity similar to that of a monoclonal antibody (i.e., a monoclonal antibody containing the same tumor-associated antigen binding site as that incorporated into the multispecific protein) selected from any one of the antigens presented in Table 11 to a tumor-associated antigen selected from any one of them. In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein binds with an affinity similar to that of a monoclonal antibody (i.e., a monoclonal antibody containing the same tumor-associated antigen binding site as that incorporated into the multispecific protein) selected from any one of the antigens presented in Table 11 to a tumor-associated antigen selected from any one of them. In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In some embodiments, the multispecific protein binds with an affinity similar to that of a monoclonal antibody (i.e., a monoclonal antibody containing the same tumor-associated antigen binding site as that incorporated into the multispecific protein) selected from any one of the antigens presented in Table 11 to a tumor-associated antigen selected from any one of them. In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11.

[0216] In certain embodiments, the multispecific protein described herein comprising an NKG2D binding site and a binding site for a tumor-associated antigen selected from any one of the antigens presented in Table 11 activates primary human NK cells when co-cultured with cells expressing the tumor-associated antigen. Activation of NK cells is indicated by an increase in the amount of CD107a degranulation and IFN-γ cytokine production. Furthermore, the multispecific protein may exhibit superior activation of human NK cells in the presence of cells expressing the tumor-associated antigen compared to the corresponding monoclonal antibody to a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. onal antibody (selected from any one of the antigens presented in Table 11)). In some embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody with respect to killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens presented in Table 11. In certain embodiments, the multispecific protein described herein comprising an NKG2D binding site and a binding site for a tumor-associated antigen selected from any one of the antigens presented in Table 11 activates primary human NK cells when co-cultured with cells expressing the tumor-associated antigen. Activation of NK cells is indicated by an increase in the amount of CD107a degranulation and IFN-γ cytokine production. Furthermore, the multispecific protein may exhibit superior activation of human NK cells in the presence of cells expressing the tumor-associated antigen compared to the corresponding monoclonal antibody to a tumor-associated antigen selected from any one of the antigens presented in Table 11.

[0217] In certain embodiments, the multispecific protein described herein comprising an NKG2D binding site and a binding site for a tumor-associated antigen selected from any one of the antigens presented in Table 11 activates primary human NK cells when co-cultured with cells expressing the tumor-associated antigen. Activation of NK cells is indicated by an increase in the amount of CD107a degranulation and IFN-γ cytokine production. Furthermore, the multispecific protein may exhibit superior activation of human NK cells in the presence of cells expressing the tumor-associated antigen compared to the corresponding monoclonal antibody to a tumor-associated antigen selected from any one of the antigens presented in Table 11. A multispecific binding protein described herein comprising a binding site for a tumor-associated antigen selected from any one of the following: The heterologous protein enhances the activity of resting human NK cells and IL-2-activated human NK cells when co-cultured with cells expressing the tumor-associated antigen.

[0218] In certain embodiments, the multispecific protein provides an advantage over the corresponding monoclonal antibody that binds to a tumor-associated antigen selected from any one of the antigens presented in Table 11 in targeting tumor cells expressing the tumor-associated antigen. The multispecific binding proteins described herein may be more effective with respect to reducing tumor growth and killing cancer cells.

[0219] In certain embodiments, F4-TriNKET targeting EpCAM (e.g., NKG2D-binding F4-TriNKET-EpCAM) killed target cells more effectively than the parental mAb targeting EpCAM. In certain embodiments, F4-TriNKET also killed target cells more potently than F3’-TriNKET (e.g., NKG2D-binding F3’-TriNKET-EpCAM), which may reflect that F4-TriNKET binds more potently to target cells. III. Therapeutic Use

[0220] The present invention provides a method for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The method comprises administering to a patient in need thereof a therapeutically effective amount of the multispecific binding proteins described herein, and can be used to treat various cancers expressing EPCAM.

[0221] ​​​​​​​​​​​​​This treatment method can be characterized by the cancer to be treated. For example, in certain embodiments in one form, the cancer is acute myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma , thymoma, adenoid cystic carcinoma, gastrointestinal cancer, kidney cancer, breast cancer, glioblastoma, lung cancer, ovarian cancer, brain cancer, prostate cancer, pancreatic cancer, or melanoma.

[0222] In certain other embodiments, the cancer is a solid tumor. In certain other embodiments , the cancer is colon cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, leukemia, liver cancer, rectal cancer, stomach cancer, testicular cancer, or uterine cancer. In yet other embodiments, the cancer is , a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioblastoma, neuroblastoma, sarcoma( e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, biliary tract c ancer, thyroid cancer, lentigo maligna melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myel oid leukemia, adenoid cystic carcinoma, adenoma, adenocarcinoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumors, Bartholin gland adenocarcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma (chondosarcoma), choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma , endothelial cell cancer, epithelioma, epithelial cell cancer, Ewing sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma , glioblastoma, glucagonoma, heart cancer, hemangiblastoma, hemangioendothelioma , angioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma , intaepithelial neoplasia, interepithelial squamous cell neoplasia , intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer , Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, malignant melanoma, lymphoma , male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningioma, mesothelioma , metastatic carcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer , nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer , non-Hodgkin lymphoma, oat cell carcinoma, oligodendroglial cancer , oral cavity cancer, osteosarcoma, serous papillary adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor , plasmacytoma, 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, leiomyoma , soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyoma , subcutaneous cancer, superficial spreading melanoma, T cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer , bladder cancer, urinary system cancer, cervical cancer, uterine body cancer, vitreous melanoma, vaginal cancer, verrucous carcinoma , bipoma, vulvar cancer, well-differentiated carcinoma, or Wilms tumor.

[0223] In certain other embodiments, the cancer is a non- such as B cell lymphoma or T cell lymphoma In certain embodiments, the non-Hodgkin's lymphoma is diffuse large intestine. B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma 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, lymphoplasmocytic lymphoma B-cell lymphoma, such as follicular lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma In certain other embodiments, the non-Hodgkin's lymphoma is precursor T lymphoma. Blastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma lymphoma, extranodal natural killer / T-cell lymphoma, enteropathic T-cell lymphoma, subcutaneous panniculitis T-cell lymphoma, such as myeloblastic T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma It is a pancreatic tumor.

[0224] The cancers to be treated are characterized according to the presence of specific antigens expressed on the surface of the cancer cells. In certain embodiments, the cancer cells express, in addition to EpCAM, the following: CD2, CD 19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ER BB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, TRO P2, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TR AILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD1 One or more may be expressed.

[0225] In some other embodiments, the antibody is treated when the second binding site binds to EpCAM. Cancers include head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, It is selected from liver cancer, esophageal cancer, and lung cancer. In some other embodiments, the second binding site is to an antigen selected from cancer antigen 125 (CA125), sodium / phosphate cotransporter 2B (NaPi2 b), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (monosialotetra rahexosyl ganglioside), disintegrin and metalloprotease domain -containing protein 8 (ADAM8), disintegrin and metalloprotease do main-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SL C44A4), and sialyl Lewis a antigen (CA19-9), the cancer to be treated is selected from ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, thyroid cancer, bladder cancer, breast cancer, colorectal cancer, small cell lung cancer, neuroblastoma, liver cancer, kidney cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma, brain cancer, stomach cancer, cholangiocarcinoma. IV. Combination Therapy Another aspect of the present invention provides a combination therapy. The multispecific binding protein described herein

[0226] can be used in combination with additional therapeutic agents for treating cancer. Exemplary therapeutic agents that can be used as part of a combination therapy in treating cancer include, for example,

[0227] radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carbocon, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole raltitrexed, daunorubicin, fadrozole, fotemustine, timalph ​Asin, Sobuzoxane, Nedaplatin, Cytarabine, Bicalutamide, Vinorelbine, Bes Narinone, Aminoglutethimide, Amsacrine, Progulmid, Elliptinium Acetate, Ke Tanserin, Doxifluridine, Etretinate, Isotretinoin, Streptozocin , Nimustine, Vindesine, Flutamide, Drogenil, Butosine, Carmofur, Lazoki San, Schizophyllan, Carboplatin, Mitolactol, Tegafur, Ifosfamide, Prednimustine, Pibavnil, Levamisole, Teniposide, Inprosulfan, E Nocitabine, Lisuride, Oxymetron, Tamoxifen, Progesterone, Mepithiost Tan, Epithioestanol, Formestane, Interferon-alpha, Interfe Ron-2 alpha, Interferon-beta, Interferon-gamma (IFN-γ ), Colony-Stimulating Factor-1, Colony-Stimulating Factor-2, Denileukin Diftitox, Interleukin-2, Luteinizing Hormone-Releasing Factor, and modified forms of the above-mentioned agents that can exhibit different binding to their homologous receptors and increased or decreased serum half-lives are included .

[0228] A further class of agents that can be used as part of combination therapy in treating cancer are immune check point 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 (P D1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H 4, and (vii) TIM3. Ipilimumab, a CT LA4 inhibitor, has been approved by the US Food and Drug Administration for treating melanoma . ​It is approved.

[0229] Further other agents that can be used as part of a combination therapy when treating cancer are non-check monoclonal antibody agents that target non-checkpoint targets (for example, Herceptin) and non-cytotoxic agents (for example, tyrosine kinase inhibitors).

[0230] Still other categories of anticancer agents include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors + 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 also WEE1 inhibitors selected from; (ii) agonists of OX40, CD137, CD40 , GITR, CD27, HVEM, TNFRSF25, or ICOS; and also (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0231] The protein of the present invention can also be used as an aid in the surgical removal of the primary tumor.

[0232] ​The amounts of the multispecific binding protein and further therapeutic agents and the relative timing of administration may be selected to achieve the desired combination therapy effect. For example, when administering combination therapy to a patient in need thereof, the therapeutic agents to be combined, or one or more pharmaceutical compositions containing the therapeutic agents may be administered in any order, for example, sequentially, together, jointly, simultaneously, etc. Further, for example, the multispecific binding protein may be administered during the time when the further therapeutic agent exerts its prophylactic or therapeutic effect, or vice versa. V. Pharmaceutical Compositions

[0233] The present disclosure also features a pharmaceutical composition containing a therapeutically effective amount of the protein described herein. The composition can be formulated for use in a variety of drug delivery systems. To make suitable formulations, one or more physiologically acceptable excipients or carriers can also be included in the composition. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th Edition, 1985. For a concise overview of methods for drug delivery, see, for example, Langer ( Science, 249:1527 - 1533, 1990).

[0234] The pharmaceutical composition may contain a therapeutically effective amount of a multispecific binding protein comprising antigenic (listed in Table 11) sites.

[0235] The intravenous drug delivery formulations of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag is connected to a channel that includes a tube and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried and may be contained in about 12 to 6 0 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of freeze-dried formulation may be contained in one vial. In certain embodiments, the freeze-dried formulation from 12, 27, or 45 vials may be combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 2 50 mg / vial.

[0236]

[0237] The protein may be present in a liquid aqueous pharmaceutical formulation that contains a therapeutically effective amount of the protein in a buffer solution that forms the formulation.

[0237] These compositions may be sterilized by conventional sterilization techniques or may be filter-sterilized. The resulting aqueous solution may be packaged for use as is or may be freeze-dried, and the freeze-dried preparation is combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8. is between 7 and 8, most preferably between 7 and 8, such as 7 to 7.5. The resulting solid composition may be packaged in multiple single-dose units, each containing a fixed amount of one or more of the above-mentioned drugs. The solid composition may also be packaged in a container for a flexible amount as well.

[0238] In certain embodiments, the present disclosure provides a formulation having an extended shelf life, comprising mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and the protein of the present disclosure in combination with sodium oxide. In certain embodiments, an aqueous formulation containing the protein of the present disclosure is prepared in a pH buffer solution. The buffer solution of the present invention may have a pH in the range of about 4 to about 8, such as about 4.5 to about 6.0, or about 4. 8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Intermediate ranges of the pH values listed above are also intended to be part of the present disclosure. For example, ranges of values using any combination of the values listed above as upper and / or lower limits are intended to be included. Examples of buffer solutions for controlling the pH within this range include acetate

[0239] (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffer solutions. In certain embodiments, the formulation comprises a buffer system containing citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range is about In certain embodiments, the pH range is about For example, ranges of values using any combination of the values listed above as upper and / or lower limits are intended to be included. Examples of buffer solutions for controlling the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffer solutions.

[0240] In certain embodiments, the formulation comprises a buffer system containing citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range is about In certain embodiments, the pH range is about A pH range of 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about 5.0 to about 5.2 may be used. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / mL of citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL of sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL of disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL of sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg / mL of sodium chloride (e.g., 6.165 mg / mL). 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 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. In certain embodiments, the pH of the formulation is adjusted using sodium hydroxide. A polyol that acts as a tonicifier and can stabilize the antibody can also be included in the formulation. The polyol is added to the formulation in an amount that can vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added can also vary with respect to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) is added compared to a disaccharide (such as trehalose).

[0241] ​​​​​​​​​​​​​​​​It is also possible. In certain embodiments, the polyol that can be used in the formulation as an isotonic agent is man nitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / ml It may be. In certain embodiments, the concentration of mannitol is about 7.5 to 15 mg / ml may be. In certain embodiments, the concentration of mannitol is about 10 to 14 mg / ml may be. In certain embodiments, the concentration of mannitol is about 12 mg / m l may be. In certain embodiments, the polyol sorbitol can be included in the formulation It can be.

[0242] Detergents or surfactants may also be added to the formulation. Exemplary detergents include polyso rbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., polo xamer 188), etc. non-ionic detergents. The amount of detergent added is 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 may contain a surfactant that is polysorb ate. In certain embodiments, the formulation may contain polysorbate 80 or Tween 80 of the detergent 80. Tween 80 is a term used to represent polyoxyeth ylene (20) sorbitan monooleate (see Fiedler, L exikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th Edition, 1996 for reference). In certain embodiments, the formulation contains from about 0.1 mg / mL to about 10 mg / mL of polysorbate 80, or between about 0.5 mg / mL and about 5 mg / mL It may contain. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation. It may be added.

[0243] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation is provided in a USP / Ph Eur Type I 50R vial, closed with a rubber stopper and sealed with an aluminum crimp seal closure, at a concentration of 10 mg / mL. The stopper may be made of an elastomer compliant with USP and Ph Eur. In certain embodiments, the vial may be filled with 61.2 mL of the protein product solution to allow for a withdrawal volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline. In certain embodiments, the liquid formulation of the present disclosure may be prepared as a 10 mg / mL concentration solution combined with sugar at a stabilizing level. In certain embodiments, the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount below that which would result in a viscosity that is undesirable or inappropriate for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also contain one or more of a buffer, a surfactant, and a preservative. In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0244] In certain embodiments, the liquid formulation of the present disclosure may be prepared as a 10 mg / mL concentration solution combined with sugar at a stabilizing level. In certain embodiments, the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount below that which would result in a viscosity that is undesirable or inappropriate for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also contain one or more of a buffer, a surfactant, and a preservative.

[0245] In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0246] In addition to aggregation, deamidation is a common product barrier for peptides and proteins that can occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage 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 Dalton mass decrease of the parent peptide. Subsequent hydrolysis results in an 18 Dalton mass increase. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is typically detectable as a 1 Dalton mass increase. Deamidation of asparagine results in either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser following Asn in the protein sequence result in higher susceptibility to deamidation .

[0247] 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

[0248] The aqueous carriers for the purposes herein are pharmaceutically acceptable (safe and non-toxic for human administration ), and are 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, Ringer's solution or dextrose solution

[0249] ​​​​​​​​​The preservative can be added to the formulations herein, if necessary, to reduce bacterial action. This can be done, for example, to facilitate the manufacture of multi-use (multiple-dose) formulations.

[0250] An intravenous (IV) formulation can be a preferred route of administration in certain cases, such as when the patient is hospitalized after transplantation and receiving all medications via the IV route. In certain embodiments, the liquid formulation is diluted with a 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0251] In certain embodiments, salts or buffering components can be added in an amount of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) using "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can function as counterions.

[0252] The preservative can be added to the formulations herein, if necessary, to reduce bacterial action. This can be done, for example, to facilitate the production of multi-use (multiple-dose) formulations.

[0253] The aqueous carriers for the purposes herein are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for the preparation of liquid formulations. Exemplary carriers include sterile water for injection ( ​​​​​​​​​​​​SWFI), bacteriostatic water for injection (BWFI), a pH buffer solution (e.g., phosphate buffered saline ), sterile saline, Ringer's solution or dextrose solution is included.

[0254] The protein of the present disclosure can also exist as a lyophilized preparation containing the protein and a lyoprotectant. The lyoprotectant can be a sugar, for example a disaccharide. In certain embodiments the lyoprotectant can be sucrose or maltose. The lyophilized preparation can also contain one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0255] The amount of sucrose or maltose useful for stabilizing the lyophilized drug product can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

[0256] In certain embodiments, the pH of the formulation before lyophilization can be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

[0257] Before lyophilization, the pH of the solution containing the protein of the present disclosure can be adjusted to be between 6 and 8. In certain embodiments, the pH range for the lyophilized drug product can be from 7 to 8.

[0258] In certain embodiments, the salt or buffer component is added in an amount of 10 mM to 200 mM. It is possible. The salt and / or buffer is pharmaceutically acceptable and is derived from various known acids (inorganic and organic) using "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can function as counterions. In certain embodiments, "bulking agents" can 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., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents. In certain embodiments, a preservative can be added to the formulations herein, if desired, to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multiple dose) formulations. In certain embodiments, the lyophilized drug product can be composed of an aqueous carrier. The aqueous carriers for the purposes herein are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solutions.

[0259] In certain embodiments, it is possible to add a "bulking agent". A "bulking agent" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (for example, facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such a bulking agent.

[0260] Preservatives can be added to the formulations herein, if desired, to reduce bacterial action. The addition of preservatives can, for example, facilitate the production of multi-use (multiple dose) formulations.

[0261] In certain embodiments, the lyophilized drug product can be composed of an aqueous carrier. The aqueous carriers for the purposes herein are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solutions.

[0262] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either Sterile Water for Injection, USP (SW FI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into a solution.

[0263] In certain embodiments, the lyophilized protein product of the present disclosure is constituted with about 4.5 mL of water for injection and diluted with a 0.9% aqueous physiological saline solution (sodium chloride solution).

[0264] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is an amount of the active ingredient that does not cause toxicity to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. This may vary to obtain the

[0265] Specific dosages may be uniform dosages for each patient, e.g., 50 - 5000 mg of protein. Alternatively, the dosage for a patient may be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is customarily made by one of ordinary skill in the art, particularly in view of the dosage information and assays disclosed in the present specification. The dosage can also be determined by use of known assays for determining the dosage to be used in conjunction with appropriate dose - response data. The dosage for an individual patient may be adjusted as the progression of the disease is monitored. The blood level of a targetable construct or complex in a patient is adjusted such that the effective concentration is reached or maintained. ​​​​​​​​​It can be measured to determine whether it needs to be truncated. Which targetable constructs and / or complexes, and their dosages, can be determined using pharmacogenomics to determine if they are likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta, Volume 308: pages 43 - 53, 2001; Steimer et al., Clinica Chimica Acta Volume 308: pages 33 - 41, 2001).

[0266] Generally, dosages based on body weight are 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 5 0 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 / k g 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, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 10 0 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 10 0 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight 、about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 m g to about 100 mg / kg body weight.

[0267] The dosage can be administered once or multiple times a day, once or multiple times a week, once or multiple times a month or once or multiple times a year, or even once every 2 to 20 years. One of ordinary skill in the art can readily estimate the repetition rate for dosing based on the measured residence time and concentration of the targetable construct or complex in body fluids or tissues. Administration of the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, and may also be by perfusion through a catheter, or by direct intralesional injection . This can be administered once or multiple times a day, once or multiple times a week, once or multiple times a month, and once or multiple times a year.

[0268] The above description describes multiple aspects and embodiments of the present invention. This application specifically contemplates all combinations and permutations of aspects and embodiments.

Example

[0269] ​ The invention generally described herein will be more readily understood by reference to the following examples, which are included for purposes of illustration only of certain aspects and embodiments (Example 1) The NKG2D binding domain binds to NKG2D The NKG2D binding domain binds to purified recombinant NKG2D

[0270] The nucleic acid sequence of the extracellular domain of human, mouse, or cynomolgus monkey NKG2D was fused with the nucleic acid sequence encoding the human IgG1 Fc domain and introduced into mammalian cells for expression. After purification, the NKG2D-Fc protein was adsorbed to the wells of a microplate. The wells were blocked with bovine serum albumin to prevent non-specific binding, and then the NKG2D binding domain was titrated and added to the wells pre-adsorbed with the NKG2D-Fc fusion protein. Primary antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and specifically recognizing human kappa light chain to avoid Fc cross-reactivity. The substrate for horseradish peroxidase, 3,3’,5,5’-tetramethylbenzidine (TMB), was added to the wells to visualize the binding signal, and its

[0271] The isotype control showed only weak binding to the recombinant NKG2D-Fc protein. However, the positive control bound most strongly to the recombinant antigen. Although the affinity differed for each clone, the NKG2D binding domains produced by all clones showed binding to all of the recombinant NKG2D-Fc proteins of human, mouse, and cynomolgus monkey. Generally, each anti-NKG2D clone bound to the recombinant NKG2 D-Fc of human (Figure 3) and cynomolgus monkey (Figure 4) with a similar affinity, but the affinity for the recombinant NKG2D-Fc of mouse (Figure 5) was relatively low. The NKG2D binding domain binds to cells expressing NKG2D.

[0272] The EL4 mouse lymphoma cell line was engineered to express a chimeric antigen receptor of the NKG2D-CD3 zeta signaling domain of human or mouse. The NKG2D binding clone, isotype control, or positive control was used at a concentration of 100 nM to stain the extracellular NKG2D expressed in EL4 cells. Antibody binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells was used to calculate the fold over background (FOB).

[0273] The NKG2D binding domains produced by all clones bound to EL4 cells expressing NKG2D of human and mouse. The positive control antibodies (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or including the anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) had the best FOB. resulted in a binding signal. The NKG2D binding affinity of each clone was similar between cells expressing human NKG2D (Figure 6) and cells expressing mouse NKG2D (Figure 7). It was the same between cells expressing human NKG2D (Figure 6) and cells expressing mouse NKG2D (Figure 7). (Example 2) The NKG2D binding domain blocks the binding of natural ligands to NKG2D Competition with ULBP-6

[0274] Recombinant human NKG2D-Fc protein was adsorbed to the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. ULBP-6-His-biotin at saturation concentration was added to the wells, followed by addition of NKG2D binding domain clones. After a 2-hour incubation, the wells were washed, and the ULBP-6-His-biotin remaining bound to the wells coated with NKG2D-Fc was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 It was blocked with bovine serum albumin to reduce non-specific binding. ULBP-6-His-biotin at saturation concentration was added to the wells, followed by addition of NKG2D binding domain clones. After a 2-hour incubation, the wells were washed, and the ULBP-6-His-biotin remaining bound to the wells coated with NKG2D-Fc was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 It was added to the wells, followed by addition of NKG2D binding domain clones. After a 2-hour incubation, the wells were washed, and the ULBP-6-His-biotin remaining bound to the wells coated with NKG2D-Fc was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 After a 2-hour incubation, the wells were washed, and the ULBP-6-His-biotin remaining bound to the wells coated with NKG2D-Fc was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 The ULBP-6-His-biotin remaining bound to the wells coated with NKG2D-Fc was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 It was detected with streptavidin conjugated to horseradish peroxidase and a TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 Absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 After subtracting the background, specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 Specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin whose binding to the NKG2D-Fc protein in the wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101 to 104) and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 and various NKG2D binding domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 domains blocked the binding of ULBP-6 to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8). The ULBP-6 sequence is represented by SEQ ID NO: 108 The ULBP-6 sequence is represented by SEQ ID NO: 108 is represented by SEQ ID NO: 108

Chemical formula

[0275] The recombinant human MICA-Fc protein was adsorbed onto the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. NKG2D-Fc-biotin was added to the wells, followed by the addition of the NKG2D binding domain. After incubation and washing, the NKG2D-Fc-biotin that remained bound to the wells coated with MICA-Fc was detected using streptavidin-HRP and a TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the wells coated with MICA-Fc was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked the binding of MICA to NKG2D, while the isotype control showed little competition with MICA (Figure 9). Competition with Rae-1 delta Recombinant mouse Rae-1 delta-Fc (purchased from R&D Systems) was adsorbed onto the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the addition of the NKG2D binding domain. After incubation and washing, the NKG2D-Fc-biotin that remained bound to the wells coated with Rae-1 delta-Fc was detected using streptavidin-HRP and a TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the wells coated with Rae-1 delta-Fc was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked the binding of MICA to NKG2D, while the isotype control showed little competition with MICA (Figure 9). domains blocked the binding of MICA to NKG2D, while the isotype control showed little competition with MICA (Figure 9). The absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the wells coated with Rae-1 delta-Fc was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked the binding of MICA to NKG2D, while the isotype control showed little competition with MICA (Figure 9).

[0276] Recombinant mouse Rae-1 delta-Fc (purchased from R&D Systems) was adsorbed onto the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the addition of the NKG2D binding domain. After incubation and washing, the NKG2D-Fc-biotin that remained bound to the wells coated with Rae-1 delta-Fc was detected using streptavidin-HRP and a TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the wells coated with Rae-1 delta-Fc was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked the binding of MICA to NKG2D, while the isotype control showed little competition with MICA (Figure 9). After incubation and washing, the NKG2D-Fc-biotin that remained bound to the wells coated with Rae-1 delta-Fc was detected using streptavidin-HRP and a TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After subtracting the background, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the wells coated with Rae-1 delta-Fc was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding Measured at 450 nM and corrected at 540 nM. After subtracting the background, N The specific binding of the NKG2D binding domain to the NKG2D-Fc protein was determined by the percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). The percentage of NKG2D-Fc-biotin that was blocked from binding to wells coated with Rae-1 delta-Fc was calculated. Positive controls (heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) and various NKG2D binding domain clones blocked the binding of Rae-1 delta to mouse NKG2D, but isotype control antibodies showed little competition with Rae-1 delta (Figure 10). (Example 3) The NKG2D binding domain clones activate NKG2D

[0277] The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. The nucleic acid sequence encoding the CD3 zeta signaling domain was fused with the nucleic acid sequences of human and mouse NKG2D to obtain a chimeric antigen receptor (CAR) construct. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retrovirus production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8 μg / mL of polybrene. Twenty-four hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected. To determine whether the NKG2D binding domain activates NKG2D,

[0278] To determine whether the NKG2D binding domain activates NKG2D, These were adsorbed onto the wells of a microplate and, in the wells coated with the antibody fragment, NKG2D-CAR EL4 cells were cultured for 4 hours in the presence of brefeldin-A and monensin. Intracellular TNFα production, which is an indicator of NKG2D activation, was assayed by flow cytometry. The percentage of TNFα-positive cells was normalized relative to the cells treated with the positive control. All NKG2D binding domains activated both human NKG2D (Figure 11) and mouse NKG2D (Figure 12). These were adsorbed onto the wells of a microplate and, in the wells coated with the antibody fragment, NKG2D-CAR EL4 cells were cultured for 4 hours in the presence of brefeldin-A and monensin. Intracellular TNFα production, which is an indicator of NKG2D activation, was assayed by flow cytometry. The percentage of TNFα-positive cells was normalized relative to the cells treated with the positive control. All NKG2D binding domains activated both human NKG2D (Figure 11) and mouse NKG2D (Figure 12). (Example 4) The NKG2D binding domain activates NK cells Primary human NK cells

[0279] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 CD56 ) were isolated from PBMCs using negative selection with magnetic beads, and the purity of the isolated NK cells was typically >95%. - CD56 + ) were isolated from PBMCs using negative selection with magnetic beads, and the purity of the isolated NK cells was typically >95%. Next, the isolated NK cells were cultured in a medium containing 100 ng / mL of IL-2 for 24 to 48 hours and then transferred to the wells of a microplate adsorbed with the NKG2D binding domain and cultured in a medium containing a fluorophore-conjugated anti-CD107a antibody, brefeldin-A, and monensin. After culturing, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-γ. NK cell activation was evaluated by analyzing the staining of CD 107a and IFN-γ in CD3 CD56 cells. The activation of NK cells was evaluated by analyzing the staining of CD107 - CD56 + cells for CD 107a and IFN-γ. The activation of NK cells was evaluated by analyzing the staining of CD107 ​Increase in a / IFN-γ double-positive cells is due to association of two activating receptors rather than one receptor The results show that the NKG2D binding domain and the positive control ( For example, the heavy chain variable domain is represented by SEQ ID NO: 101 or SEQ ID NO: 103, and the light chain The variable domain is represented by SEQ ID NO: 102 or SEQ ID NO: 104. A higher percentage of NK cells expressing CD107a than in control mice + and IFN-γ + Nina (Figures 13 and 14 show that PBMCs from different donors were used for the preparation of NK cells.) Data from two independent experiments, each using MC, are presented. Primary mouse NK cells

[0280] Spleens were obtained from C57Bl / 6 mice and crushed through a 70 μm cell strainer. A single cell suspension was obtained by pelleting the cells and lysing them in ACK lysis buffer (Thermo F Purchased from Isher Scientific, #A1049201; 155 mM chloride 10 mM potassium bicarbonate, 0.01 mM EDTA) The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours. The cells were then cultured and harvested to prepare for NK cell isolation. Using negative depletion techniques, splenic tissue was extracted, typically at a purity of >90%. NK cells (CD3 - NK1.1 + ) were isolated. Purified NK cells were After 48 hours of culture in medium containing 0 ng / mL mIL-15, NKG The 2D binding domains were transferred to the wells of a microplate and fluorophore conjugates were added. In a medium containing a ugate anti-CD107a antibody, brefeldin-A, and monensin After culturing in wells coated with the NKG2D binding domain, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1 and IFN-γ CD3 - NK1.1 + cells The staining of CD107a and IFN-γ in the cells was analyzed to evaluate NK cell activation . An increase in CD107a / IFN-γ double-positive cells indicates better NK cell activation due to the association of two activating receptors rather than one receptor. The NKG2D binding domain and also the positive control (selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) showed a higher percentage of NK cells becoming CD107a and IFN-γ + than the isotype control (Figures 15 and 1 + 6 represent data from two independent experiments using different mice for the preparation of NK cells, respectively). 6 represent data from two independent experiments using different mice for the preparation of NK cells, respectively). (Example 5) (Example 5) The NKG2D binding domain enables cytotoxicity of target tumor cells

[0281] Human and mouse primary NK cell activation assays demonstrated an increase in cytotoxicity markers in NK cells after incubation with the NKG2D binding domain. To address whether this leads to an increase in tumor cell lysis, a cell-based assay was utilized in which each NKG2D binding domain was developed into a single-specificity antibody. The Fc region was used as one targeting arm while the Fab fragment region (NKG2D binding domain) activates NK cells To address whether this leads to an increase in tumor cell lysis, a cell-based assay was utilized in which each NKG2D binding domain was developed into a single-specificity antibody. The Fc region was used as one targeting arm while the Fab fragment region (NKG2D binding domain) activates NK cells while the Fab fragment region (NKG2D binding domain) activates NK cells served as another targeting arm for. It is of human origin and expresses high levels of Fc receptors The THP-1 cells that do so were used as tumor targets, and the Perkin Elmer DELFIA cytotoxicity kit was used. The THP-1 cells were labeled with the BATDA reagent and resuspended in the culture medium at 10 5 / mL Next, the labeled THP-1 cells were combined with the NKG2D antibody and incubated at 37 °C for 3 hours in the wells of a microtiter plate to isolate mouse NK cells After incubation, 20 μL of the culture supernatant was taken out, mixed with 200 μL of the europium solution, and incubated with shaking in the dark for 15 minutes. The fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (excitation 337 nM, emission 620 nM), and the specific lysis was calculated according to the instructions of the kit (Fig. 17). The positive control ULBP-6 conjugated to Fc (a natural ligand for NKG2D

[0282] showed an increase in the specific lysis of THP-1 target cells by mouse NK cells. The N KG2D antibody also increased the specific lysis of THP-1 target cells, while the isotype control antibody showed a decrease in specific lysis. The dotted line indicates the specific lysis of THP-1 cells by mouse NK cells without the addition of antibody (Fig. 17). (Example 6) (Example 6) The NKG2D antibody exhibits high thermal stability

[0283] The melting temperature of the NKG2D binding domain was assayed using differential scanning fluorimetry . The extrapolated apparent melting temperature is high compared to that of a typical IgG1 antibody (Fig. 18). (Example 7) Synergistic activation of human NK cells by cross-linking of NKG2D and CD16 Primary Human NK Cell Activation Assay

[0284] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation NK cells were purified from PB MCs using negative magnetic beads (StemCell #17955). NK cells were determined by flow cytometry to be >9 0% CD3 - CD56 + Next, the cells were expanded in medium containing 10 0 ng / mL of hIL-2 (Peprotech #200-02) for 48 hours prior to use in the activation assay Antibodies were coated onto 96-well flat-bottom plates at 4°C overnight at concentrations of 2 μg / mL in 100 μL of sterile PBS (anti-CD16, Bio legend #302013) and 5 μg / mL (anti-NKG2D, R&D #MA B139), followed by washing the wells thoroughly to remove excess antibody. For the evaluation of degranulation, IL-2-activated NK cells were resuspended in culture medium supplemented with 100 ng / mL of human IL-2 (hIL2) and 1 μg / mL of APC conjugated anti-CD107a mAb (Biolegend #328619) at 5 × 10 cells / mL. Next, 1 × 10 5 cells / 5 well were added onto the antibody-coated plates. The protein transport inhibitors brefeldin A (BFA, Biolegend #420601) and monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively (Biolegend #420701). The seeded cells were incubated at 37°C for 4 hours in 5% CO2 . For intracellular staining of IFN-γ, NK cells were stained with anti-CD3 (Bio legend #420601) and anti-CD107a mAb (Biolegend #328619) at 5 × 10 labeled with legend #300452) and anti-CD56 mAb (Biolegend #3 18328), followed by fixation, permeabilization, and labeling with anti-IFN-γ mAb (Biole gend #506507). NK cells were gated on live CD56 + CD3 - cells and then analyzed for CD107a and IFN-γ expression by flow cytometry.

[0285] To investigate the relative potency of receptor combinations, crosslinking of NKG2D or CD16 and co-crosslinking of both receptors were performed by plate-bound stimulation. As shown in Fig. 19 (Figs. 19A - 19C), combined stimulation of CD16 and NKG2D resulted in a large increase in the level of CD107a ( degranulation) (Fig. 19A) and / or IFN-γ production (Fig. 19B). The dotted lines represent the additive effect of individual stimulation of each receptor. After 4-hour plate-bound stimulation with anti-CD16, anti-NKG2D, or a combination of both monoclonal antibodies, the CD107a level and intracellular IFN N-γ production of IL-2-activated NK cells were analyzed. The graphs show mean (n = 2) ± Sd. Fig. 19A shows the level of CD107a, Fig. 19B shows the level of IFN-γ, and Fig. 19C shows the levels of CD107a and IFN-γ. The data shown in Figs. 19A - 19C are representative of five (Example 8) Enhancement of cytotoxicity of target cells mediated by trispecific binding protein (TriNKET) Evaluation of the binding of TriNKET or mAb to cells expressing human cancer antigens

[0286] Using human cancer cell lines expressing EPCAM, the tumor antigen binding of TriNKETs derived from clone MT110 targeting EPCAM in the F4 format and F3' format was evaluated. Using human cell lines H747, HCC827, and HCT116, the binding of TriNKETs and mAbs to cells expressing EPCAM was evaluated. TriNKETs or mAbs were diluted and incubated with the respective cells. Binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the binding MFI to cells expressing EPCAM was normalized to a control stained with human recombinant IgG1 to obtain the fold over background value. TriNKET or mAb was diluted and incubated with each cell. Binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the binding MFI to cells expressing EPCAM was normalized to a control stained with human recombinant IgG1 to obtain the fold over background value. by flow cytometry, and the binding MFI to cells expressing EPCAM was normalized to a control stained with human recombinant IgG1 to obtain the fold over background value. by flow cytometry, and the binding MFI to cells expressing EPCAM was normalized to a control stained with human recombinant IgG1 to obtain the fold over background value.

[0287] Figure 37 shows the binding of the trispecific binding proteins (TriNKETs) (A49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) of the present disclosure and the parental monoclonal antibody (mAb) to H747 human colorectal cancer cells expressing EpCAM. Figure 38 shows the binding of the trispecific binding proteins (TriNKETs) (A 49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) of the present disclosure and the parental monoclonal antibody (mAb) to HCC827 human lung cancer cells expressing EpCAM. Figure 39 shows the binding of the trispecific binding proteins (TriNKETs) (A 49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) of the present disclosure and the parental monoclonal antibody (mAb) to HCT116 human colorectal cancer cells expressing EpCAM. The overall binding is F4-TriNKET Figure 37 shows the binding of the trispecific binding proteins (TriNKETs) (A49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) of the present disclosure and the parental monoclonal antibody (mAb) to H747 human colorectal cancer cells expressing EpCAM. Figure 38 shows the binding of the trispecific binding proteins (TriNKETs) (A 49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) of the present disclosure and the parental monoclonal antibody (mAb) to HCC827 human lung cancer cells expressing EpCAM. Figure 39 shows the binding of the trispecific binding proteins (TriNKETs) (A In T, compared with F3’-TriNKET incorporating the MT110-EPCAM conjugate it was more potent. Cytotoxicity assay of primary human NK cells

[0288] PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using negative selection technology with magnetic beads. The purity of the achieved isolated NK cells was typically higher than 90% CD3 - CD56 + . The isolated NK cells were incubated overnight without cytokines and used for the cytotoxicity assay the next day. DELFIA cytotoxicity assay Human cancer cell lines expressing the target of interest were harvested from cultures, washed with HBS, and resuspended

[0289] in growth medium at 10 cells / mL for labeling with BATD 6 A reagent (Perkin Elmer, AD0116). The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with HBS and resuspended in culture medium at 0.5×10 5 cells / mL. To prepare the background wells, an aliquot of the labeled cells was taken and the cells were spun out of the medium. 100 μL of medium was carefully added in triplicate to the wells without disturbing the pelleted cells. 100 μL of BATDA-labeled cells were added to each well of a 96-well plate. The wells were reserved for the natural release from the target cells and prepared for the lysis of the target cells by the addition of 1% T riton-X. Against the tumor target of interest Dilute the monoclonal antibody or TriNKET in the culture medium and add 50 μL of the diluted mA b or TriNKET to each well. Harvest the resting NK cells from the culture, wash them, and resuspend them in the culture medium at 1.0×10 5 ~2.0 ×10 6 cells / mL according to the desired effector cell to target cell ratio. Add 50 μL of the NK cells to each well of the plate to make a total culture volume of 200 μL. Incubate the plate at 37 °C in 5% CO2 for 2 - 4 hours before the assay is developed.

[0290] After culturing for 2 - 3 hours, remove the plate from the incubator and pellet the cells by centrifugation at 200×g for 5 minutes. Transfer 20 μL of the culture supernatant to a clean microplate provided by the manufacturer and add 200 μL of room temperature europium solution to each well . Protect the plate from light and incubate it on a plate shaker at 250 rpm for 15 minutes. Read the plate using a SpectraMax® i3 X instrument (Molecular Devices) and calculate the percent specific lysis (Percent specific lysis = (Experimental release - Spontaneous release) / (Maximum release - Spontaneous release)) × 100).

[0291] Figures 40A and 40B show the TriNKET - mediated cytotoxicity of resting human NK cells from two different healthy donors against H747 human cancer cells. Figures 41A and 4 1B show the TriNKET - mediated cytotoxicity of resting human NK cells from two different healthy donors against HCC827 human cancer cells. Figures 42A and 42B show the TriNKET - mediated cytotoxicity of resting human NK cells from two different healthy donors against MCF7 cells. NK cells. Figures 42A and 42B show the TriNKET - mediated cytotoxicity of resting human NK cells from two different healthy donors against MCF7 TriNK of resting human NK cells from two different healthy donors against human cancer cells is ET-mediated cytotoxicity. Figures 43A and 43B show the TriNK ET-mediated cytotoxicity of resting human NK cells from two different healthy donors against HCT116 human cancer cells is ET-mediated cytotoxicity. F4-TriNKET targeting EPCAM killed target cells more effectively than the parental mAb targeting EPCAM is ET-mediated cytotoxicity. F4-TriNKET also killed target cells more potently than F3’-TriNKET, which may reflect stronger binding of F4-TriNKET to target cells than that of the parental mAb targeting EPCAM. F4-TriNKET also killed target cells more potently than F3’-TriNKET, which may reflect stronger binding of F4-TriNKET to target cells than that of the parental mAb targeting EPCAM. F4-TriNKET also killed target cells more potently than F3’-TriNKET, which may reflect stronger binding of F4-TriNKET to target cells than that of the parental mAb targeting EPCAM. F4-TriNKET also killed target cells more potently than F3’-TriNKET, which may reflect stronger binding of F4-TriNKET to target cells Incorporation by reference

[0292] The entire disclosure of each patent document and scientific paper referenced herein is incorporated by reference for all purposes The entire disclosure of each patent document and scientific paper referenced herein is incorporated by reference for all purposes Equivalents

[0293] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalents of the claims are intended to be embraced therein

Claims

1. (a) a first antigen binding to natural killer group 2 member D (NKG2D); A joining portion, (b) EpCAM, cancer antigen 125 (CA125), sodium / phosphate cotransporter 2B (NaPi2b), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (Mo nosialotetrahexosylganglioside), a disintegrin and metalloproteinase ADAM8, a disintegrin and metalloproteinase domain-containing protein ADAM9, a member of the solute carrier family 44 Barr 4 (SLC44A4), and sialylated Lewis a antigen (CA19-9) and a second antigen-binding site that binds to an antigen selected from (c) an antibody Fc domain sufficient to bind cluster of differentiation 16 (CD16); or a third antigen-binding site that binds to a portion thereof or to CD16; Proteins including:

2. (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to EpCAM; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

3. (a) a first antigen-binding site that binds to NKG2D; (b) Cancer antigen 125 (CA125), sodium / phosphate cotransporter 2B (NaPi2 b), nectin cell adhesion molecule 4 (Nectin4), fucosyl-GM1 (monosialyltransferase A disintegrin and metalloproteinase domain ADAM8, a disintegrin and metalloproteinase-containing protein ADAM9, solute carrier family 44 member 4 (SL C44A4), and sialylated Lewis a antigen (CA19-9). a second antigen-binding site that binds to an antigen; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

4. (a) a first antigen-binding site that binds to NKG2D; (b) a tumor selected from sodium-dependent phosphate transport protein 2b (NaPi2b) a second antigen-binding site that binds to a tumor-associated antigen; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

5. (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to the tumor-associated antigen Nectin4; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

6. (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to the multiple myeloma associated antigen Fucosyl-GM1; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

7. (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding portion that binds to a T cell-associated tumor antigen selected from SLC44A4; Rank and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

8. The first antigen-binding site is an antigen binding site that binds to NKG2 in humans, non-human primates, and rodents. A protein according to any one of claims 1 to 7, which binds to D.

9. The first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

9. A protein described in any one of 1 to 8.

10. The heavy chain variable domain and the light chain variable domain are present on the same polypeptide.

10. The protein according to claim 9.

11. The second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

11. The protein according to claim 9 or 10.

12. the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are the same The protein of claim 11 , which is present on a polypeptide.

13. The light chain variable domain of the first antigen-binding site is a light chain variable domain of the second antigen-binding site.

13. The method according to claim 11 or 12, wherein the amino acid sequence of the variable domain of the The protein described.

14. (a) a first antigen-binding site comprising a Fab fragment that binds to NKG2D; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds to EpCAM; 、 (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

15. The scFv comprises an Fc domain of the antibody or a portion thereof sufficient to bind to CD16. Ala-Ser or Gly in the third antigen-binding site that binds to the portion or CD16 -Ala-Ser, and the scFv is linked via a hinge comprising a heavy chain variable domain. The protein of claim 14, comprising a light chain variable domain and a light chain variable domain.

16. The protein of claim 15, wherein the scFv is linked to the antibody Fc domain. quality.

17. The heavy chain variable domain of the scFv is disulfide bonded to the light chain variable domain of the scFv.

16. A protein according to claim 14 or 15, which forms a phosphoryl bridge.

18. The disulfide bridge is formed by binding C44 of the heavy chain variable domain and C 18. The protein of claim 17, wherein the amino acid sequence is between 1 and 100.

19. The scFv is linked to the antibody Fc domain, and the light chain variable domain of the scFv a domain located at the N-terminus of the heavy chain variable domain of the scFv, and The heavy chain variable domain of Fv is provided with a flexible linker (GlyGlyGlyGlySer). ４ ((G4S) ４ ) linked to the antibody Fc domain, The protein of claim 18.

20. The heavy chain variable domain of the scFv may be linked to the light chain variable domain of the scFv.

20. The protein of claim 15, wherein the protein is linked via a flexible linker. Quality.

21. The flexible linker is (GlyGlyGlyGlySer) ４ ((G4S) ４ ) The protein of claim 20.

22. The heavy chain variable domain of the scFv is N-terminal to the light chain variable domain of the scFv.

22. The protein according to any one of claims 15 to 21, wherein the protein is located at the C-terminus or the C-terminus. 。

23. The light chain variable domain of the scFv is N-terminal to the heavy chain variable domain of the scFv. The protein of claim 22, which is located at the end.

24. The Fab fragment comprises a sufficient Fc domain of the antibody or an Fc domain thereof to bind to CD16.

15. The method of claim 14, wherein the third antigen-binding site is linked to a portion or the third antigen-binding site that binds to CD16.

24. A protein according to any one of claims 23 to 24.

25. The heavy chain portion of the Fab fragment comprises a heavy chain variable domain and a CH1 domain.

25. The protein of claim 24, wherein the chain variable domain is linked to the CH1 domain. 。

26. 26. The method of claim 24 or 25, wherein the Fab fragment is linked to the antibody Fc domain. Proteins listed.

27. 27. The method according to claim 14, comprising a sequence selected from SEQ ID NO: 208 and SEQ ID NO:

209. The protein according to any one of claims 1 to 4.

28. comprising an scFv linked to an antibody Fc domain, cFv is represented by a sequence selected from SEQ ID NO: 210 and SEQ ID NO: 211; 28. A protein according to any one of claims 15 to 27.

29. 27. The method according to claim 15, comprising a sequence selected from SEQ ID NO: 212 and SEQ ID NO:

213. The protein according to any one of claims 1 to 4.

30. At least 90% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211 27. A protein according to any one of claims 15 to 26, comprising an identical sequence.

31. At least 95% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211 27. A protein according to any one of claims 15 to 26, comprising an identical sequence.

32. At least 99% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211 27. A protein according to any one of claims 15 to 26, comprising an identical sequence.

33. At least 90% identical to an amino acid sequence selected from SEQ ID NO:212 and SEQ ID NO:213 33. A protein according to any one of claims 15 to 32, comprising an identical sequence.

34. At least 95% identical to an amino acid sequence selected from SEQ ID NO:212 and SEQ ID NO:213 33. A protein according to any one of claims 15 to 32, comprising an identical sequence.

35. At least 99% of an amino acid sequence selected from SEQ ID NO:212 and SEQ ID NO:213 33. A protein according to any one of claims 15 to 32, comprising an identical sequence.

36. (a) a first antigen-binding site comprising a single chain variable fragment (scFv) that binds to NKG2D; 、 (b) a second antigen-binding site that binds to EpCAM; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16 or a CD a third antigen-binding site that binds to 16; and Proteins including:

37. 37. The protein of claim 36, further comprising an additional antigen binding site that binds to EpCAM. Quality.

38. 36. The method of claim 35, wherein the second antigen binding site that binds to EpCAM is a Fab fragment.

37. The protein according to claim 37.

39. The second antigen-binding site that binds to EpCAM and the further antigen-binding site are 39. The protein of claim 37 or 38, which is an ab fragment.

40. The second antigen-binding site that binds to EpCAM and the further antigen-binding site are 38. The protein of claim 36 or 37, which is a cFv.

41. The heavy chain variable domain of the scFv that binds to NKG2D is 41. Any one of claims 36 to 40, which is located at the N-terminus or C-terminus of the chain variable domain. The protein according to claim 3.

42. The light chain variable domain of the scFv that binds to NKG2D. The protein of claim 41, which is located at the N-terminus of

43. The scFv that binds to NKG2D is provided with an antibody Fc domain sufficient to bind to CD16. or a third antigen-binding site that binds to CD16.

43. A protein according to any one of claims 36 to 42.

44. The scFv that binds to NKG2D is provided with an antibody Fc domain sufficient to bind to CD16. Ala-S in the main or a part thereof or in the third antigen-binding site that binds to CD16 The method according to claim 43, wherein the amino acid sequence is linked via a hinge comprising Gly-Ala-Ser or Gly-Ala-Ser. Proteins listed.

45. The scFv that binds to NKG2D is provided with an antibody Fc domain sufficient to bind to CD16. The C-terminus of the main or a part thereof or the third antigen-binding site that binds to CD16 is and linked via a flexible linker comprising GSGGGGS (SEQ ID NO: 207).

43. The protein according to claim 43.

46. The C-terminus of the antibody Fc domain is the light chain variable domain of the scFv that binds to NKG2D. The protein of claim 45, wherein the domain is linked to the N-terminus.

47. Within the scFv that binds to NKG2D, the heavy chain variable domain of the scFv and A disulfide bridge is formed between the light chain variable domain of the scFv.

47. A protein according to any one of claims 6 to 46.

48. The disulfide bridge is formed by binding C44 of the heavy chain variable domain and C 48. The protein of claim 47, wherein the amino acid sequence is between 1 and 100.

49. In the scFv that binds to NKG2D, the heavy chain variable domain is 49. The method according to any one of claims 36 to 48, wherein the Proteins listed.

50. The flexible linker is (GlyGlyGlyGlySer) ４ (G4S) ４ ) 50. The protein of claim 49.

51. The second antigen-binding site scFv and the further antigen-binding site scFv are or a portion thereof sufficient to bind to CD16. and the third antigen-binding site is linked to the third antigen-binding site via a hinge comprising Ala-Ser.

51. The protein according to any one of claims 40 to 50.

52. The second antigen-binding site scFv and the further antigen-binding site scFv are The Fc domain is linked to the IgG1 domain via a hinge comprising Ala-Ser.

51. A protein according to any one of claims 51.

53. The heavy chain variable domain of the second antigen-binding site and / or the further antigen-binding site 51. A disulfide bridge is formed between the amino acid sequence of claim 50 and the light chain variable domain. or 52.

54. The disulfide bridge is formed by binding C44 of the heavy chain variable domain and C 54. The protein of claim 53, wherein the amino acid sequence is between 1 and 100.

55. A light chain in which the scFv that binds to NKG2D is located at the N-terminus of the heavy chain variable domain. wherein the light chain variable domain is linked to the heavy chain variable domain of the scFv. , flexible linker (GlyGlyGlyGlySer) ４ (G4S) ４ ) The scFv that binds to NKG2D has an Ala-Se residue in the antibody Fc domain. r or Gly-Ala-Ser, 5. A protein according to any one of claims 4.

56. A protein comprising the amino acid sequence of SEQ ID NO:

203.

57. A protein comprising the amino acid sequence of SEQ ID NO:203 and SEQ ID NO:

204.

58. A protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

203. quality.

59. A protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

203. quality.

60. A protein comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

203. quality.

61. The first antigen-binding site is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57 , SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:85, and A heavy chain variable domain that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:

93.

4. A protein according to any one of the preceding claims.

62. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:41 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

42.

1. A protein according to any one of claims 1 to 0.

63. The first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:

49. and a light chain variable domain that is at least 90% identical to SEQ ID NO:

50.

1. A protein according to any one of claims 1 to 0.

64. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:57 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

58.

1. A protein according to any one of claims 1 to 0.

65. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:59 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

60.

1. A protein according to any one of claims 1 to 0.

66. The first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:

61. and a light chain variable domain that is at least 90% identical to SEQ ID NO:

62.

1. A protein according to any one of claims 1 to 0.

67. The first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:

69. and a light chain variable domain that is at least 90% identical to SEQ ID NO:

70.

1. A protein according to any one of claims 1 to 0.

68. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:77 and a light chain variable domain at least 90% identical to SEQ ID NO:

78.

1. A protein according to any one of claims 1 to 0.

69. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:85 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

86.

1. A protein according to any one of claims 1 to 0.

70. the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:93 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

94.

1. A protein according to any one of claims 1 to 0.

71. The first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:

101. and a light chain variable domain that is at least 90% identical to SEQ ID NO:

102.

61. A protein described in any one of claims 60 to 60.

72. The first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:

103. and a light chain variable domain that is at least 90% identical to SEQ ID NO:

104.

61. A protein described in any one of claims 60 to 60.

73. 11. The method of claim 1 , wherein the first antigen-binding site is a single domain antibody. The protein according to claim 3.

74. The single domain antibody is Ｈ H fragment or V ＮＡＲ 74. The method of claim 73, protein.

75. The second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

75. A protein according to any one of claims 1 to 10 or 73 to 74.

76. the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are the same 76. The protein of claim 75, which is present on a polypeptide.

77. The second antigen-binding site binds to EpCAM, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 115, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

119.

73. A protein according to any one of claims 1, 2, or 61 to 72, comprising a amino acid sequence.

78. The heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 116; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 117, and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO:118 78. The protein of claim 77, comprising an amino acid sequence comprising:

79. the light chain variable domain of the second antigen-binding site comprising: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 120; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 121; and The light chain CDR3 sequence is identical to the amino acid sequence of SEQ ID NO:

122.

79. The protein of claim 78, comprising an amino acid sequence comprising:

80. The second antigen-binding site binds to EpCAM, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 123, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

127.

73. The protein of any one of claims 1, 2, or 61 to 72, comprising a amino acid sequence.

81. The heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 124; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 125; and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 126 81. The protein of claim 80, comprising an amino acid sequence comprising:

82. the light chain variable domain of the second antigen-binding site comprising: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 128; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 129, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 130 82. The protein of claim 81 , comprising an amino acid sequence comprising:

83. The second antigen-binding site binds to EpCAM, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 131, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

135.

73. The protein of any one of claims 1, 2, or 61 to 72, comprising a amino acid sequence.

84. The heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 132; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 133, and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 134 84. The protein of claim 83, comprising an amino acid sequence comprising:

85. the light chain variable domain of the second antigen-binding site comprising: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 136; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 137, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 138 85. The protein of claim 84, comprising an amino acid sequence comprising:

86. The second antigen-binding site binds to EpCAM, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 139, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

143.

73. The protein of any one of claims 1, 2, or 61 to 72, comprising a amino acid sequence.

87. The heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 140; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 141; and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 142 87. The protein of claim 86, comprising an amino acid sequence comprising:

88. the light chain variable domain of the second antigen-binding site comprising: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 144; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 145, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 146 88. The protein of claim 87, comprising an amino acid sequence comprising:

89. The second antigen-binding site binds to CA125, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 155, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

159.

73. The protein of any one of claims 1, 3, or 61-72, comprising a amino acid sequence.

90. The second antigen-binding site binds to CA125, and the heavy chain of the second antigen-binding site the variant domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 163, The light chain variable domain of the antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO:

167.

73. The protein of any one of claims 1, 3, or 61-72, comprising a cDNA sequence.

91. the second antigen-binding site binds to NaPi2b, and the heavy chain of the second antigen-binding site the variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 171, The light chain variable domain of the antigen-binding site of SEQ ID NO: 175 is an amino acid sequence that is at least 90% identical to SEQ ID NO:

175.

73. The protein of any one of claims 1, 4, or 61 to 72, comprising the amino acid sequence 。

92. The second antigen-binding site binds to Nectin4, and the overlap of the second antigen-binding site the chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 179, The light chain variable domain of the second antigen-binding site is an antigen having at least 90% identity to SEQ ID NO:

183.

73. The protein of any one of claims 1, 5, or 61 to 72, comprising the amino acid sequence quality.

93. The second antigen-binding site binds to Fucosyl-GM1, and the second antigen-binding site the heavy chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 187, the light chain variable domain of the second antigen-binding site is at least 90% identical to SEQ ID NO: 191 73. The protein of any one of claims 1, 6, or 61 to 72, comprising the amino acid sequence Quality.

94. the second antigen-binding site binds to SLC44A4, and the overlap of the second antigen-binding site the chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195, The light chain variable domain of the second antigen-binding site is an antigen having at least 90% identity to SEQ ID NO:

199.

73. The protein of any one of claims 1, 7, or 61 to 72, comprising the amino acid sequence quality.

95. The antibody Fc domain comprises a hinge and a CH2 domain of a human IgG1 antibody.

95. The protein according to any one of claims 1 to 94.

96. The Fc domain has a structure identical to that of amino acids 234 to 332 of a human IgG1 antibody at least 90% 96. The protein of claim 95, comprising an identical amino acid sequence.

97. The Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1. The sequence includes Q347, Y349, L351, S354, E356, E357, K360 , Q362, S364, T366, L368, K370, N390, K392, T394 , D399, S400, D401, F405, Y407, K409, T411, K439 The tag according to claim 96, which differs at one or more positions selected from the group consisting of: Compact quality.

98. K of 10 nM for NKG2D Ｄ or weaker affinity than that of claims 1 to 9.

7. A protein according to any one of claims 6 to 6.

99. A method for the preparation of a pharmaceutical composition comprising the protein of any one of the preceding claims and a pharma- ceutically acceptable carrier. A preparation that contains.

100. 99. One or more nucleic acids expressing a protein according to any one of claims 1 to 98. Cells containing

101. The protein of any one of claims 1 to 98 is used to treat tumor and natural killer cells. A method of directly and / or indirectly enhancing tumor cell death comprising exposing tumor cells to a cytoplasmic medium.

102. A protein according to any one of claims 1 to 98 or a formulation according to claim 99.

23. A method of treating cancer comprising administering to a patient

103. If the second binding site binds to EpCAM, the cancer is head and neck cancer, ovarian cancer, , bladder cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, and lung cancer.

103. The method of claim 102, wherein the cancer is selected from the group consisting of:

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