Proteins that bind to NKG2D, CD16 and tumor-associated antigens

Abstract

Multispecific binding proteins that bind to the NKG2D receptor, CD16, and tumor associated antigens are described, as well as pharmaceutical compositions and methods of treatment useful for the treatment of cancer.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of September 7, 2018, application number 201880071464.8, and invention name “Protein that binds NKG2D, CD16 and binding protein 4”.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 555,110, filed September 7, 2017, and U.S. Provisional Patent Application No. 62 / 566,824, filed October 2, 2017, the entire disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

[0004] Sequence Listing

[0005] This application contains a sequence listing electronically submitted in ASCII format and incorporated herein by reference in its entirety. The ASCII copy generated on September 6, 2018 is named DFY-038WO_SL.txt and is 321,395 bytes in size. Technical Field

[0006] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16 and tumor-associated antigens. Background Art

[0007] Despite the numerous research attempts and scientific advances in the treatment of cancer reported in the literature, the disease remains a significant health problem. Some of the most frequently diagnosed cancers include prostate cancer, breast cancer, lung cancer, and colorectal cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the 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 effective for all patients and / or may have substantial serious side effects. Other types of cancers also remain challenging to treat using existing treatment options.

[0008] Cancer immunotherapies are ideal 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 tumor cell destruction. Antibodies that bind to certain tumor-associated antigens and to certain immune cells have been described in the literature. See, for example, WO 2016 / 134371 and WO 2015 / 095412.

[0009] Natural killer (NK) cells are an integral part of the innate immune system, accounting for approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues, and were initially characterized by their ability to kill tumor cells without prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells, i.e., by cell lytic granules containing perforins and granzymes and by death receptor pathways. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other leukocytes to target tissues.

[0010] NK cells respond to signals through various activation and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIR). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated through their activation receptors (e.g., natural killer cell surface activation receptor 2 family member D (NKG2D), NCR, DNAM1). NK cells are also activated by the constant regions of certain immunoglobulins through the CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0011] 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. In addition, EpCAM has carcinogenic potential through its ability to upregulate c-myc, e-fabp and cyclins A and E. Since EpCAM is only expressed in epithelial and epithelial-derived neoplasms, EpCAM can be used as a diagnostic marker for a variety of different 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. It appears to play a role in tumorigenesis and metastasis of cancer, so it can also serve as a potential prognostic marker and a potential target for immunotherapy strategies.

[0012] CA125, also known as mucin 16, is a member of the mucin family of glycoproteins. CA-125 has been found to be useful as a tumor marker or biomarker that may be elevated in the blood of certain patients with certain types of cancer, including ovarian cancer, endometrial cancer, and pancreatic cancer. CA-125 has been shown to play a role in promoting tumorigenesis and tumor proliferation through several different mechanisms, including inhibiting the response of natural killer cells, thereby protecting cancer cells from immune responses; and by causing cell growth and promoting cell motility.

[0013] Sodium-dependent phosphate transport protein 2b (NaPi2b) is involved in the active transport of phosphate into cells via Na+ co-transport. For example, it is the major phosphate transporter in the intestinal brush border membrane and plays a role in the synthesis of surfactant in the alveoli. NaPi2b is also an antigen expressed in various different cancers such as lung cancer, ovarian cancer, and thyroid cancer.

[0014] Nectin 4 is a member of the nectin family, a family of proteins involved in the regulation of Ca 2+ A family of cell adhesion molecules that are independent of cell adhesion. Integrin is widely expressed and has an adhesive role in a wide range of tissues, such as adherens junctions of epithelia or chemical synapses of neuronal tissue. It is also a tumor-associated antigen and is expressed in cancers such as bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer and lung cancer.

[0015] Gangliosides have been implicated in many physiological processes, including growth, differentiation, migration, and apoptosis, by regulating both cell-to-cell and cell-to-matrix interactions and cell-to-matrix signaling processes. GM1 is a ganglioside, and fucosyl-GM1 is a ganglioside with a unique structure in which the terminal galactose is α-1,2-fucosylated at the non-reducing end. It is expressed in very few normal tissues, but appears in a variety of different cancers, such as small cell lung cancer, neuroblastoma, and liver cancer. Therefore, fucosyl-GM1 has been considered as a candidate for tumor markers and target antigens in antibody immunotherapy for small cell lung cancer, neuroblastoma, and liver cancer.

[0016] ADAM (disintegrin and metalloproteinase) protein plays a major role in the shedding of the protein extracellular domain of membrane-bound molecules. They have become key regulators of cell signaling in development and homeostasis, and are believed to cause pathological changes, such as cancer, and their regulation is changed. ADAM8 is a member of the ADAM family, overexpressed in pancreatic cancer, breast cancer, lung cancer and renal cancer. ADAM9 has been shown to cut and release many molecules that have an important role in tumorigenesis and angiogenesis, such as EGF, FGFR2iiib, Tie-2, Flk-1, EphB4, CD40, VCAM-1 and VE-cadherin. ADAM9 is overexpressed in renal cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, cervical cancer, prostate cancer, osteosarcoma and brain cancer.

[0017] SLC44A4, also known as CTL4, is a member of a family of solute carrier proteins called choline transporter-like proteins (CTL1-5). SLC44A4 has not been shown to be involved in choline transport, but it has been associated with the synthesis and transport of acetylcholine and the uptake of thiamine pyrophosphate, the phosphorylated form of vitamin B1. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but it is significantly upregulated in a variety of different epithelial tumors, especially pancreatic cancer, prostate cancer, and gastric cancer.

[0018] CA19-9 is a general term for the carbohydrate antigen sialyl Lewis A. It is overexpressed in digestive organ cancers such as pancreatic cancer, colorectal cancer, bile duct cancer, and liver cancer. Therefore, it is the serum tumor marker most commonly used to diagnose these cancers. Summary of the invention

[0019] The present invention provides multispecific binding proteins that bind to tumor-associated antigens (selected from any one of the antigens provided in Table 11) and to NKG2D receptors and CD16 receptors on natural killer cells. These proteins can bind to more than one NK activation receptor and can block the binding of natural ligands to NKG2D. In certain embodiments, the proteins can excite NK cells in humans and other species such as rodents and cynomolgus monkeys. Various aspects and embodiments of the present invention will be described in more detail below.

[0020] Therefore, one aspect of the present invention provides a protein comprising: a first antigen binding site that binds to NKG2D; a second antigen binding site that binds to an antigen selected from EpCAM, cancer antigen 125 (CA125), sodium / phosphate co-transporter 2B (NaPi2b), ligase cell adhesion molecule 4 (ligase 4), fucosyl-GM1 (monosialyltetrahexosylganglioside), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC44A4) and sialyl Lewis a antigen (CA19-9); and an antibody Fc domain, a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds to CD16. The antigen binding sites may each comprise an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged or fused together to form an scFv as in an antibody), or one or more of the antigen binding sites may be a single domain antibody, such as a V H H antibodies such as camel antibodies or V NAR Antibodies as found in cartilaginous fish.

[0021] The present invention provides a multispecific binding protein that binds to the NKG2D receptor, CD16 and an antigen selected from the following: EpCAM, cancer antigen 125 (CA125), sodium / phosphate co-transporter 2B (NaPi2b), integrin cell adhesion molecule 4 (integrin 4), fucosyl-GM1 (monosialyltetrahexosylganglioside), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC44A4) and sialyl Lewis a antigen (CA19-9).

[0022] Certain proteins of the present disclosure include a Fab fragment linked to the antibody Fc domain or a portion thereof sufficient to bind CD16 or the third antigen binding site that binds CD16.

[0023] Certain proteins of the present disclosure include a Fab fragment, wherein the heavy chain portion of the Fab fragment comprises a heavy chain variable domain and a CH1 domain, and wherein the heavy chain variable domain is linked to the CH1 domain.

[0024] Certain proteins of the present disclosure include a Fab fragment linked to the Fc domain of the antibody.

[0025] 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 or a portion thereof sufficient to bind to CD16, 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 a scFv.

[0026] Certain proteins described in the present disclosure include scFv targeting EpCAM, which comprises a heavy chain variable domain and a light chain variable domain, and is connected to an antibody Fc domain or a portion thereof sufficient to bind CD16 or the third antigen binding site that binds CD16 via a hinge comprising Ala-Ser or Gly-Ala-Ser. Certain proteins of the present disclosure include scFv targeting EpCAM connected to an antibody Fc domain. Certain proteins of the present disclosure include a heavy chain variable domain of a scFv targeting EpCAM, which forms a disulfide bridge with the light chain variable domain of the scFv.

[0027] Certain proteins of the present disclosure include a scFv targeting EpCAM, wherein a disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

[0028] Certain proteins disclosed herein include an EpCAM-targeting scFv linked to an antibody Fc domain, wherein 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 (GlyGlyGlyGlySer)4((G4S)4)(SEQ ID NO: 206), and the NKG2D-binding Fab fragment is linked to the antibody Fc domain.

[0029] Certain proteins of the present disclosure include a scFv targeting EpCAM, wherein the heavy chain variable domain is located at the N-terminus or C-terminus of the light chain variable domain of the scFv.

[0030] Certain proteins of the present disclosure include a scFv targeting EpCAM, wherein the light chain variable domain is located at the N-terminus of the heavy chain variable domain of the scFv.

[0031] In one aspect of the present invention, a protein is provided, comprising: (a) a first antigen binding site comprising a single chain variable fragment (scFv) that binds NKG2D; (b) a second antigen binding site that binds EpCAM; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16 or a third antigen binding site that binds CD16. In certain embodiments, the protein of the present disclosure further comprises an additional antigen binding site that binds EpCAM. In certain embodiments, the second antigen binding site of the protein described in the present disclosure is a Fab fragment that binds EpCAM. In certain embodiments, the second and additional antigen binding sites of the protein described in the present disclosure are Fab fragments that bind EpCAM.

[0032] In certain embodiments, the second and additional antigen binding sites of the proteins described in the present disclosure are scFvs that bind EpCAM. In certain embodiments, the heavy chain variable domain of the scFv that binds NKG2D is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. In certain embodiments, the light chain variable domain is located at the N-terminus of the heavy chain variable domain of the scFv that binds NKG2D.

[0033] In certain embodiments, the scFv that binds to NKG2D is linked to the 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 scFv that binds to NKG2D is linked to the antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds to CD16 via a hinge comprising Ala-Ser (e.g., in TriNKETs that comprise additional antigen binding sites that bind to EpCAM, CA125, NaPi2b, Integrin 4, Fucosyl-GM1, ADAM8, ADAM9, SLC44A4, or CA19-9) or Gly-Ala-Ser (e.g., in TriNKETs that do not comprise additional antigen binding sites that bind to EpCAM, CA125, NaPi2b, Integrin 4, Fucosyl-GM1, ADAM8, ADAM9, SLC44A4, or CA19-9). In certain embodiments, the scFv that binds to NKG2D is connected to the C-terminus of the antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds CD16 via a flexible linker comprising G4S. In certain embodiments, the C-terminus of the antibody Fc domain is connected to the N-terminus of the light chain variable domain of the scFv that binds to NKG2D.

[0034] In certain embodiments, within the NKG2D-binding scFv, a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv. In certain embodiments, the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

[0035] Certain proteins of the present disclosure include a sequence selected from SEQ ID NO:210 and SEQ ID NO:211.

[0036] Certain proteins of the present disclosure include a scFv linked to an antibody Fc domain, wherein the scFv linked to an antibody Fc domain is represented by a sequence selected from the group consisting of SEQ ID NO: 208 and SEQ ID NO: 209.

[0037] Certain proteins of the present disclosure include the sequences of SEQ ID NO:205 and SEQ ID NO:213.

[0038] Certain proteins of the present disclosure include a sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211.

[0039] Certain proteins of the present disclosure include a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211.

[0040] Certain proteins of the present disclosure include a sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NO:210 and SEQ ID NO:211.

[0041] Certain proteins of the present disclosure include the amino acid sequence of SEQ ID NO:203.

[0042] Certain proteins of the present disclosure include the amino acid sequences of SEQ ID NO:203 and SEQ ID NO:204.

[0043] Certain 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. Certain 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. Certain 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.

[0044] In certain embodiments, the first antigen binding site that binds to NKG2D may comprise a heavy chain variable domain associated with SEQ ID NO: 1, for example, by 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 / or comprises an amino acid sequence that is identical to the CDR1 (SEQ ID NO: 105), CDR2 (SEQ ID NO: 106) and CDR3 (SEQ ID NO: 107) sequences of SEQ ID NO: 1. The heavy chain variable domain associated with SEQ ID NO: 1 may be coupled to various light chain variable domains to form a NKG2D binding site. For example, the first antigen binding site comprising a heavy chain variable domain associated with SEQ ID NO: 1 may also comprise a light chain variable domain selected from any one of the sequences associated with SEQ ID NO: 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 comprises 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 NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0045] Alternatively, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 41 and a light chain variable domain associated with SEQ ID NO: 42. 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: 41, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44) and CDR3 (SEQ ID NO: 45) sequences of SEQ ID NO: 41. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:42, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:46), CDR2 (SEQ ID NO:47) and CDR3 (SEQ ID NO:48) sequences of SEQ ID NO:42.

[0046] In other embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 49 and a light chain variable domain associated with SEQ ID NO: 50. 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: 49, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 53) sequences of SEQ ID NO: 49. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:50, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:55) and CDR3 (SEQ ID NO:56) sequences of SEQ ID NO:50.

[0047] Alternatively, the first antigen binding site may comprise a heavy chain variable domain related to SEQ ID NO: 57 and a light chain variable domain related to SEQ ID NO: 58, for example by 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: 57 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 58, respectively.

[0048] In another embodiment, the first antigen binding site may comprise 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% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 59, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 109), CDR2 (SEQ ID NO: 110) and CDR3 (SEQ ID NO: 111) sequences of SEQ ID NO: 59. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:60, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:112), CDR2 (SEQ ID NO:113) and CDR3 (SEQ ID NO:114) sequences of SEQ ID NO:60.

[0049] In certain embodiments, the first antigen binding site that binds to NKG2D may comprise 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 63), CDR2 (SEQ ID NO: 64) and CDR3 (SEQ ID NO: 65) sequences of SEQ ID NO: 61. Similarly, the light chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:66), CDR2 (SEQ ID NO:67) and CDR3 (SEQ ID NO:68) sequences of SEQ ID NO:62.

[0050] In certain embodiments, the first antigen binding site may comprise 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., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 69, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 71), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73) sequences of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:70, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:74), CDR2 (SEQ ID NO:75) and CDR3 (SEQ ID NO:76) sequences of SEQ ID NO:70.

[0051] In certain embodiments, the first antigen binding site may comprise 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., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 77, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 79), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81) sequences of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:78, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83) and CDR3 (SEQ ID NO:84) sequences of SEQ ID NO:78.

[0052] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 85 and a light chain variable domain associated with SEQ ID NO: 86. 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: 85, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 87), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89) sequences of SEQ ID NO: 85. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:86, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91) and CDR3 (SEQ ID NO:92) sequences of SEQ ID NO:86.

[0053] In certain embodiments, the first antigen binding site may comprise 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 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: 93, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 95), CDR2 (SEQ ID NO: 96) and CDR3 (SEQ ID NO: 97) sequences of SEQ ID NO: 93. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:94, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:98), CDR2 (SEQ ID NO:99) and CDR3 (SEQ ID NO:100) sequences of SEQ ID NO:94.

[0054] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain related to SEQ ID NO: 101 and a light chain variable domain related to SEQ ID NO: 102, for example by 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: 101 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 102, respectively.

[0055] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain related to SEQ ID NO: 103 and a light chain variable domain related to SEQ ID NO: 104, for example by 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: 103 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 104, respectively.

[0056] In certain embodiments, the second antigen binding site can bind to EpCAM and can comprise a heavy chain variable domain associated with SEQ ID NO: 115 and a light chain variable domain associated with SEQ ID NO: 119. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), and CDR3 (SEQ ID NO: 118) sequences of SEQ ID NO: 115. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 119, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 120), CDR2 (SEQ ID NO: 121), and CDR3 (SEQ ID NO: 122) sequences of SEQ ID NO: 119.

[0057] In certain embodiments, the second antigen binding site can bind to EpCAM and can comprise a heavy chain variable domain related to SEQ ID NO: 123 and a light chain variable domain related to SEQ ID NO: 127. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 124), CDR2 (SEQ ID NO: 125), and CDR3 (SEQ ID NO: 126) sequences of SEQ ID NO: 123. Similarly, the light chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 128), CDR2 (SEQ ID NO: 129) and CDR3 (SEQ ID NO: 130) sequences of SEQ ID NO: 127.

[0058] In certain embodiments, the second antigen binding site can bind to EpCAM and can comprise a heavy chain variable domain associated with SEQ ID NO: 131 and a light chain variable domain associated with SEQ ID NO: 135. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), and CDR3 (SEQ ID NO: 134) sequences of SEQ ID NO: 131. Similarly, the light chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 136), CDR2 (SEQ ID NO: 137) and CDR3 (SEQ ID NO: 138) sequences of SEQ ID NO: 135.

[0059] In certain embodiments, the second antigen binding site can bind to EpCAM and can comprise a heavy chain variable domain associated with SEQ ID NO: 139 and a light chain variable domain associated with SEQ ID NO: 143. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO: 141), and CDR3 (SEQ ID NO: 142) sequences of SEQ ID NO: 139. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 143, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 144), CDR2 (SEQ ID NO: 145) and CDR3 (SEQ ID NO: 146) sequences of SEQ ID NO: 143.

[0060] In certain embodiments, the second antigen binding site can bind to CA125 and can comprise a heavy chain variable domain associated with SEQ ID NO: 155 and a light chain variable domain associated with SEQ ID NO: 159. For example, the heavy chain variable domain of the second antigen binding site can 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 / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 156), CDR2 (SEQ ID NO: 157) and CDR3 (SEQ ID NO: 158) sequences of SEQ ID NO: 155. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 159, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 160), CDR2 (SEQ ID NO: 161) and CDR3 (SEQ ID NO: 162) sequences of SEQ ID NO: 159.

[0061] In certain embodiments, the second antigen binding site can bind to CA125 and can comprise a heavy chain variable domain associated with SEQ ID NO: 163 and a light chain variable domain associated with SEQ ID NO: 167. For example, the heavy chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 163, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 164), CDR2 (SEQ ID NO: 165) and CDR3 (SEQ ID NO: 166) sequences of SEQ ID NO: 163. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 167, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 168), CDR2 (SEQ ID NO: 169) and CDR3 (SEQ ID NO: 170) sequences of SEQ ID NO: 167.

[0062] In certain embodiments, the second antigen binding site can bind to NaPi2b and can comprise a heavy chain variable domain associated with SEQ ID NO: 171 and a light chain variable domain associated with SEQ ID NO: 175. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 172), CDR2 (SEQ ID NO: 173) and CDR3 (SEQ ID NO: 174) sequences of SEQ ID NO: 171. Similarly, the light chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 176), CDR2 (SEQ ID NO: 177) and CDR3 (SEQ ID NO: 178) sequences of SEQ ID NO: 175.

[0063] In certain embodiments, the second antigen binding site can bind to bindin 4 and can comprise a heavy chain variable domain associated with SEQ ID NO: 179 and a light chain variable domain associated with SEQ ID NO: 183. For example, the heavy chain variable domain of the second antigen binding site can 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 / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 180), CDR2 (SEQ ID NO: 181), and CDR3 (SEQ ID NO: 182) sequences of SEQ ID NO: 179. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 183, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 184), CDR2 (SEQ ID NO: 185) and CDR3 (SEQ ID NO: 186) sequences of SEQ ID NO: 183.

[0064] In certain embodiments, the second antigen binding site can bind to Fucosyl-GM1 and can comprise a heavy chain variable domain associated with SEQ ID NO: 187 and a light chain variable domain associated with SEQ ID NO: 191. For example, the heavy chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 187, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 188), CDR2 (SEQ ID NO: 189), and CDR3 (SEQ ID NO: 190) sequences of SEQ ID NO: 187. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 191, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 192), CDR2 (SEQ ID NO: 193) and CDR3 (SEQ ID NO: 194) sequences of SEQ ID NO: 191.

[0065] In certain embodiments, the second antigen binding site can bind to SLC44A4 and can comprise a heavy chain variable domain associated with SEQ ID NO: 195 and a light chain variable domain associated with SEQ ID NO: 199. For example, the heavy chain variable domain of the second antigen binding site can 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 comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 196), CDR2 (SEQ ID NO: 197), and CDR3 (SEQ ID NO: 198) sequences of SEQ ID NO: 195. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 199, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 200), CDR2 (SEQ ID NO: 201) and CDR3 (SEQ ID NO: 202) sequences of SEQ ID NO: 199.

[0066] In certain embodiments, the second antigen binding site comprises an amino acid sequence that is identical to the amino acid sequence of the light chain variable domain present in the first antigen binding site.

[0067] In certain embodiments, the protein comprises a portion of an antibody Fc domain sufficient to bind to CD16, wherein the antibody Fc domain comprises a hinge and a CH2 domain, and / or an amino acid sequence that is at least 90% identical to the amino acid sequence 234-332 of a human IgG antibody.

[0068] Certain proteins of the present disclosure have a K of 10 nM D or weaker affinity binding to NKG2D.

[0069] Also provided are dosage forms containing one of these proteins, cells containing one or more nucleic acids expressing these proteins, and methods of using these proteins to increase tumor cell death.

[0070] 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 a multispecific binding protein described herein. Exemplary cancers treated using the multispecific binding proteins 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

[0071] Figure 1 is a diagram of a heterodimeric multispecific antibody (trispecific binding protein (TriNKET)). Each arm can represent a NKG2D binding domain or a tumor-associated antigen binding domain. In certain embodiments, the NKG2D and the tumor-associated antigen binding domain can share a common light chain.

[0072] Figure 2 is a schematic representation of a heterodimeric multispecific antibody. Either the NKG2D binding domain or the tumor associated antigen binding domain may be in a scFv format (right arm).

[0073] Figure 3 Is a line graph showing the binding affinity of the NKG2D binding domains (listed as clones) to human recombinant NKG2D as determined by ELISA.

[0074] Figure 4 Is a line graph showing the binding affinity of the NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D as determined by ELISA.

[0075] Figure 5Is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to mouse recombinant NKG2D as determined by ELISA.

[0076] Figure 6 Is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D by flow cytometry, showing mean fluorescence intensity (MFI) as fold over background (FOB).

[0077] Figure 7 Is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing mouse NKG2D by flow cytometry, showing mean fluorescence intensity (MFI) as fold over background (FOB).

[0078] Figure 8 is a line graph of the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant human NKG2D-Fc shown by competition with the natural ligand ULBP-6.

[0079] Fig. 9 is a line graph of the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant human NKG2D-Fc shown by competition with the natural ligand MICA.

[0080] Fig.10 is a line graph of the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant mouse NKG2D-Fc shown by competition with the natural ligand Rae-1δ.

[0081] Fig.11 Is a bar graph showing activation of human NKG2D by NKG2D binding domains (listed as clones) as shown by quantification of the percentage of TNF-α positive cells expressing human NKG2D-CD3ζ fusion protein.

[0082] Fig.12 Is a bar graph showing activation of mouse NKG2D by NKG2D binding domains (listed as clones) as shown by quantification of the percentage of TNF-α positive cells expressing mouse NKG2D-CD3ζ fusion protein.

[0083] Fig.13 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0084] Fig.14 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0085] Fig.15 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0086] Fig.16 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0087] Fig.17 is a bar graph showing the cytotoxic effects of NKG2D binding domains (listed as clones) on tumor cells.

[0088] Fig.18 is a bar graph showing the melting temperatures of NKG2D binding domains (listed as clones) measured by differential scanning fluorimetry.

[0089] Figures 19A-19C is a bar graph of synergistic activation of NK cells using CD16 and NKG2D binding. Fig.19A Levels of CD107a are shown; Fig.19B Levels of IFNγ are shown; Fig.19C Levels of CD107a and IFNγ are shown. Graphs indicate mean values ​​(n=2)±SD. Data are representative of 5 independent experiments using 5 different healthy donors.

[0090] Fig. 20 is a diagram of a trispecific binding protein (TriNKET) in the form of a bispecific antibody (Triomab), which is a trifunctional, bispecific antibody that maintains an IgG-like shape. The chimera is composed of two half antibodies derived from two parent antibodies, each with one light chain and one heavy chain. The Triomab type can be a heterodimeric construct containing 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0091] Fig.21 is a diagram of TriNKET in KiH common light chain format, which includes knobs-into-holes (KIH) technology. KiH is a heterodimer containing two Fab fragments that bind to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. TriNKET in KiH format can be a heterodimeric construct with two Fab fragments that bind to targets 1 and 2, containing two different heavy chains and a common light chain paired with the two heavy chains.

[0092] Fig. 22 DVD-Ig TM) format, which combines the target binding domains of two monoclonal antibodies through a flexible, naturally occurring linker and produces a tetravalent IgG-like molecule. DVD-Ig TM It is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of the Fab fragment targeting antigen 1. DVD-Ig TM The format contains the normal Fc.

[0093] Fig.23 is a schematic representation of TriNKET in an orthogonal Fab interface (Ortho-Fab) format, which is a heterodimeric construct containing two Fab fragments binding to Target 1 and Target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in Fc.

[0094] Fig.24 This is a diagram of TriNKET in a two-in-one Ig format.

[0095] Fig.25 is a schematic representation of TriNKET in ES format, which is a heterodimeric construct containing two different Fab fragments binding to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in Fc.

[0096] Fig.26 is a diagram of TriNKET in a Fab fragment arm exchange format: the Fab arms are exchanged by exchanging the heavy chain and attached light chain (half-molecule) with the heavy chain-light chain pair from another molecule to generate an antibody of a bispecific antibody. The Fab arm exchange format (cFae) is a heterodimer containing two Fab fragments binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

[0097] Fig. 27 is a schematic representation of TriNKET in the form of a SEEDbody, a heterodimer containing two Fab fragments binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

[0098] Fig.28 is a schematic representation of TriNKET in a LuZ-Y format, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y format is a heterodimer containing two different scFabs binding to targets 1 and 2 fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

[0099] Fig.29 is a schematic representation of TriNKET in the form of a Cov-X body.

[0100] Fig. 30A and 30B is a schematic representation of TriNKET in the form of a Kλbody, which is a heterodimeric construct with 2 different Fab fragments fused to an Fc stabilized by heterodimerization mutations: one Fab fragment targeting antigen 1 contains a κLC, and the second Fab fragment targeting antigen 2 contains a λLC. Fig. 30A is an exemplary illustration of a form of Kλ body; Fig. 30B is an exemplary illustration of another Kλ body.

[0101] Fig.31 is an Oasc-Fab heterodimer construct comprising a Fab fragment binding to target 1 and a scFab binding to target 2, both fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0102] Fig.32 DuetMab is a Fab fragment containing two different Fab fragments that bind to antigens 1 and 2 and a F C Heterodimer construct of Fab fragments 1 and 2. Fab fragments 1 and 2 contain different SS bridges, which ensure the correct pairing of light and heavy chains.

[0103] Fig.33 is a CrossmAb, which is a heterodimeric construct with 2 different Fab fragments binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. The CL and CH1 domains are swapped with the VH and VL domains, e.g., CH1 is fused inline with VL, while CL is fused inline with VH.

[0104] Fig.34 is Fit-Ig, which is a homodimeric construct in which the Fab fragment that binds to Antigen 2 is fused to the N-terminus of the HC of the Fab fragment that binds to Antigen 1. The construct contains a wild-type Fc.

[0105] Fig.35 A trispecific antibody (TriNKET) containing a scFv binding to a tumor-associated antigen, a Fab targeting NKG2D, and a heterodimeric antibody constant region / domain ("CD domain") binding to CD16 (scFv-Fab format) is shown. The antibody format is referred to herein as F3'-TriNKET.

[0106] Fig.36An exemplary trispecific antibody (TriNKET) is shown, which includes a scFv first antigen binding site that binds NKG2D, a second antigen binding site that binds a tumor associated antigen (e.g., EpCAM), an additional tumor associated antigen binding site that binds a tumor associated antigen (e.g., EpCAM), and a heterodimeric antibody constant region that binds CD 16. These antibody formats are referred to herein as F4-TriNKET.

[0107] Fig.37 is a line graph showing the binding of TriNKET and mAb to EpCAM expressed on H747 human colorectal cancer cells.

[0108] Fig.38 is a line graph showing the binding of TriNKET and mAb to EpCAM expressed on HCC827 human lung cancer cells.

[0109] Fig.39 is a line graph showing binding of TriNKET and mAb to EPCAM expressed on HCT116 human colorectal cancer cells.

[0110] Fig.40A and Fig.40B is a line graph showing TriNKET-mediated killing of H747 cells using resting human NK cells from two different donors. The effector cell to target cell ratio was 10:1.

[0111] Fig.41A and Fig.41B is a line graph showing TriNKET-mediated killing of HCC827 cells using resting human NK cells from two different donors. The effector cell to target cell ratio was 10:1.

[0112] Fig.42A and Fig.42B is a line graph showing TriNKET-mediated killing of MCF7 cells using resting human NK cells from two different donors. The effector cell to target cell ratio was 10:1.

[0113] Fig.43A and Fig.43B is a line graph showing TriNKET-mediated killing of HCT116 cells using resting human NK cells from two different donors. The effector cell to target cell ratio was 10:1. Detailed Description

[0115] The present invention provides multispecific binding proteins that bind to EPCAM on cancer cells and NKG2D receptors and CD16 receptors on natural killer cells to activate the natural killer cells, pharmaceutical compositions comprising these multispecific binding proteins, and therapeutic methods using these multispecific proteins and pharmaceutical compositions, including for cancer treatment. Various aspects of the present invention are described in sections below; however, aspects of the present invention described in a particular section should not be limited to any particular section.

[0116] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0117] As used herein, a reference without a specific number means "one or more" and includes the plural referent unless the context is inappropriate.

[0118] When used in this article, the term "antigen binding site" refers to the part of an immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") region of the heavy ("H") chain and the light ("L") chain. The three highly divergent segments in the V region of the heavy and light chains are called "hypervariable regions", which are inserted between the more conservative flanking segments called "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence that is naturally present between and adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are configured relative to each other in three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs". In some animals such as camels and cartilaginous fish, the antigen binding site is formed by a single antibody chain, providing a "single domain antibody". The antigen binding site may be present in an intact antibody, in an antigen binding fragment of an antibody that retains the antigen binding surface, or in a recombinant polypeptide such as a scFv that connects the heavy chain variable domain to the light chain variable domain using a peptide linker in a single polypeptide.

[0119] As used herein, the term "tumor-associated antigen" means any antigen associated with cancer, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, lipids. These antigens may be expressed on malignant cells or in the tumor microenvironment, such as blood vessels, extracellular matrix, mesenchymal matrix, or immune infiltrates associated with the tumor.

[0120] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. These organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0121] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the invention) sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular dosage form or route of administration. As used herein, the term "treatment" includes any effect that results in improvement of a condition, disease, disorder, etc., such as mitigation, reduction, regulation, improvement, or elimination or amelioration of its symptoms.

[0122] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0123] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] .

[0124] When used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or basic salt) of a compound of the present invention, which, after being administered to a subject, can provide a compound of the present invention or its active metabolite or residue. As known to those skilled in the art, the "salt" of a compound of the present invention can be derived from an inorganic or organic acid and base. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids, such as oxalic acid, although not pharmaceutically acceptable per se, can be used to prepare salts that can be used as intermediates in obtaining compounds of the present invention and pharmaceutically acceptable acid addition salts thereof.

[0125] Exemplary bases include, but are not limited to, alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium) hydroxides, ammonia, and compounds of formula NW4 + Compounds (wherein W is C 1-4 Alkyl) etc.

[0126] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, fluoroheptanoate, 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, toluenesulfonate, undecanoate, and the like. Other examples of salts include those with suitable cations such as Na + NH4 + and NW4 + (W is C 1-4 The anion of the compound of the present invention which is combined with an alkyl group or the like.

[0127] For therapeutic use, it is contemplated that the salts of the compounds of the invention are pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0128] Throughout this description, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is contemplated that there are additional compositions of the invention that consist essentially of, or consist of, the recited components, and that there are additional processes and methods consistent with the invention that consist essentially of, or consist of, the recited process steps.

[0129] Generally, compositions specifying percentages are by weight unless otherwise specified. In addition, if a variable is not accompanied by a definition, the preceding definition of the variable will prevail.

[0130] I. Protein

[0131] The present invention provides multispecific binding proteins that bind to NKG2D receptors and CD16 receptors on natural killer cells and tumor-associated antigens selected from any one of the antigens provided in Table 11. The multispecific binding proteins are useful in the pharmaceutical compositions and methods of treatment described herein. The binding of the multispecific binding protein to the NKG2D receptor and the CD16 receptor on natural killer cells enhances the activity of the natural killer cells in destroying tumor cells expressing tumor-associated antigens selected from any one of the antigens provided in Table 11. The binding of the multispecific binding protein to cells expressing tumor-associated antigens brings the cancer cells into the vicinity of the natural killer cells, promoting the direct and indirect destruction of the cancer cells by the natural killer cells. Further descriptions of some exemplary multispecific binding proteins are provided below.

[0132] The first component of the multispecific binding protein binds to cells expressing the NKG2D receptor, which may include but are not limited to NK cells, γδ T cells, and CD8 + αβ T cells. After binding to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 (UL16 binding protein 6) and MICA (class I major histocompatibility complex chain-associated protein A) from binding to NKG2D and activating the NKG2D receptor.

[0133] The second component of the multispecific binding protein binds to a tumor-associated antigen selected from any one of the antigens provided in Table 11. Cells expressing the tumor-associated antigen can be found in leukemias such as acute myeloid leukemia and T-cell leukemia.

[0134] The third component of the multispecific binding protein binds to cells expressing CD 16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0135] The multispecific binding proteins described herein can take a variety of different formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain ( Figure 1). The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and an optional 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 comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and an optional 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 provided in Table 11. The first Fc domain and the second Fc domain together are capable of binding to CD16 ( Figure 1 ). In certain embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0136] Another exemplary format relates to a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain ( Figure 2 ). The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain fused to a single chain variable fragment (scFv) via a connector or an antibody hinge, the scFv consisting of a heavy chain variable domain and a light chain variable domain that are paired and bind to NKG2D or a tumor-associated antigen selected from any one of the antigens provided in Table 11. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and an optional CH1 heavy chain domain. The immunoglobulin light chain includes a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain is paired with the immunoglobulin light chain and binds to NKG2D or a tumor-associated antigen selected from any one of the antigens provided in Table 11. The first Fc domain and the second Fc domain together are capable of binding to CD16 ( Figure 2 ).

[0137] One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the antigen binding motif is a single chain or disulfide-stabilized variable region (scFv), forming a tetravalent or trivalent molecule.

[0138] In certain embodiments, the multispecific binding protein takes the form of a trifunctional antibody, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera is composed of two half antibodies derived from two parent antibodies, each with one light chain and one heavy chain.

[0139] In certain embodiments, the multispecific binding protein is a KiH common light chain (LC) format, which involves knob-in-hole (KIH) technology. The KIH comprises an engineered C H The concept behind the "knob-in-hole (KiH)" Fc technology is to introduce a "knob" in one CH3 domain (CH3A) by replacing a small residue with a bulky residue (e.g. T366W in EU numbering). CH3A To accommodate the "knob", a complementary "hole" surface is created on the other CH3 domain (CH3B) by replacing the residues closest to the knob with smaller residues (e.g. T366S / L368A / Y407V CH3BThe "hole" mutation was optimized by structure-guided phage screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using aphage display library, J Mol Biol (1997) 270 (1): 26-35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction, J Mol Biol (2014) 426(9): 1947–57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K, et al., Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs, Mol Biol (2014) 426(9): 1947–57). Immunol (2014) 58(1):132–8) demonstrated that hydrophobic interactions driven by steric complementarity at the core interface between CH3 domains made heterodimerization thermodynamically favorable, whereas the knob-knob and mortar-mortar interfaces were unfavorable for homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0140] In certain embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig TM ) which combines the target binding domains of two monoclonal antibodies through a flexible, naturally occurring linker and results in a tetravalent IgG-like molecule.

[0141] In certain embodiments, the multispecific binding protein adopts an orthogonal Fab interface (Ortho-Fab) format. In the ortho-Fab IgG method (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface, Nat. Biotechnol. (2014) 32 (2): 191–8), the LC and HC in only one Fab fragment are designed based on the structure of the region. VH-CH1 Complementary mutations were introduced at the interface without making any changes to the other Fab fragment.

[0142] In certain embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In certain embodiments, the multispecific binding protein is in an ES format, which is a heterodimeric construct containing two different Fab fragments that bind to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0143] In certain embodiments, the multispecific binding protein takes the form of a κλbody, which is a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by heterodimerization mutations: Fab fragment 1 targeting Antigen 1 contains a κLC, while the second Fab fragment targeting Antigen 2 contains a λLC. Fig. 30A is an exemplary illustration of a form of Kλ body; Fig. 30B is an exemplary illustration of another Kλ body.

[0144] In certain embodiments, the multispecific binding protein is in the form of a Fab arm exchange (an antibody in which the Fab arms are exchanged by exchanging the heavy chain and attached light chain (half-molecule) with a heavy chain-light chain pair from another molecule to create a bispecific antibody).

[0145] In certain embodiments, the multispecific binding protein takes the form of a SEED body. The chain exchange engineered domain (SEED) platform is designed to produce asymmetric and bispecific antibody-like molecules, which expands the therapeutic application of natural antibodies. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins in the conserved CH3 domain. The SEED design allows efficient production of AG / GA heterodimers, while being unfavorable to the homodimerization of AG and GA SEED CH3 domains. (Muda M. et al., Protein Eng. Des. Sel., 2011, 24 (5): 447-54).

[0146] In certain embodiments, the multispecific binding protein takes the form of LuZ-Y, in which a leucine zipper is used to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0147] In certain embodiments, the multispecific binding protein takes the form of a Cov-X body. In a bispecific CovX body, two different peptides are linked together using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. The antibody scaffold provides a long half-life and Ig-like distribution, while the pharmacophore is responsible for functional activity. The pharmacophore can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies (Doppalapudi VR et al., PNAS (2010), 107 (52); 22611-22616).

[0148] In certain embodiments, the multispecific binding protein takes the form of an Oasc-Fab heterodimer, which includes a Fab fragment that binds to target 1 fused to Fc and a scFab that binds to target 2. Heterodimerization is ensured by mutations in the Fc.

[0149] In certain embodiments, the multispecific binding protein takes the form of a DuetMab, which is a Fab fragment containing two different Fab fragments that bind to antigens 1 and 2 and a Fab fragment that is stabilized by heterodimerization mutations. C Heterodimer construct of Fab fragments 1 and 2. Fab fragments 1 and 2 contain different SS bridges, which ensure correct LC and HC pairing.

[0150] In certain embodiments, the multispecific binding protein takes the form of a CrossmAb, which is a heterodimeric construct with two different Fab fragments binding to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains are swapped with the VH and VL domains, e.g., CH1 is fused inline with VL, and CL is fused inline with VH.

[0151] In certain embodiments, the multispecific binding protein takes the form of Fit-Ig, which is a homodimeric construct in which the Fab fragment that binds to Antigen 2 is fused to the N-terminus of the HC of the Fab fragment that binds to Antigen 1. The construct contains a wild-type Fc.

[0152] Table 1 lists peptide sequences of heavy chain variable domains and light chain variable domains that can bind to NKG2D in combination. The NKG2D binding domains may differ in their binding affinity to NKG2D, however they all activate human NKG2D and NK cells.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 101 can be paired with the light chain variable domain represented by SEQ ID NO: 102 to form an antigen binding site that can bind to NKG2D as shown in US 9,273,136.

[0160] SEQ ID NO: 101

[0161] QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS

[0162] SEQ ID NO: 102

[0163] QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[0164] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 103 can be paired with the light chain variable domain represented by SEQ ID NO: 104 to form an antigen binding site that can bind to NKG2D as shown in US 7,879,985.

[0165] SEQ ID NO: 103

[0166] QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS

[0167] SEQ ID NO: 104

[0168] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0169] Table 2 lists the peptide sequences of the heavy chain variable domain and the light chain variable domain that can bind to EpCAM in combination.

[0170]

[0171]

[0172] Alternatively, novel antigen binding sites that bind to EpCAM can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:147.

[0173] SEQ ID NO: 147

[0174] MAPPQVLAFGLLLAAATATFAAAQEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVIICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPY DSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA

[0175] Antigen binding sites that bind to the tumor-associated antigen CA125 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:148.

[0176] SEQ ID NO: 148

[0177]

[0178] The antigen binding site that can bind to the tumor-associated antigen NaPi2b can be determined by screening for binding to the amino acid sequence defined by SEQ ID NO:149.

[0179] SEQ ID NO: 149

[0180] MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIELLPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGRLILLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPLLGLVIGVLVTVLVQSSSTSTSIVVSMVS SSLLTVRAAIPIIMGANIGTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVKIWCKTFTNKTQINVTVPSTANCTSPSLCWTDGIQNWTMKNVTYKE NIAKCQHIFVNFHLPDLAVGTILLILSLLVLCGCLIMIVKILGSVLKGQVATVIKKTINTDFPFPFAWLTGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGVITIERAYPLTLGSNIGTTTTAILAALASPGNALRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMAKG LGNISAKYRWFAVFYLIIFFFLIPLTVFGLSLAGWRVLVGVGVPVVFIIILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSLKPWDAVVSKFTGCFQMRCCCCCRVCCRACLLCDCPKCCRCSKCCEDLEEAQEGQDVPVKAPETFDNITISREAQGEVPASDSKTECTAL

[0181] The antigen binding site that can bind to tumor-associated antigen binding factor 4 can be determined by screening for binding to the amino acid sequence defined by SEQ ID NO:150.

[0182] SEQ ID NO: 150

[0183] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYEC RVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLED QNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQM TQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV

[0184] Antigen binding sites that can bind to the tumor-associated antigen fucosyl-GM1 can be identified by screening for binding to monosialyltetrahexosylganglioside.

[0185] The antigen binding site that can bind to the tumor-associated antigen ADAM8 can be determined by screening for binding to the amino acid sequence defined by SEQ ID NO:151.

[0186] SEQ ID NO: 151

[0187] LGATGHNFTLHLRKNRDLLGSGYTETYTAANGSEVTEQPRGQDHCFYQGHVEGYPDSAASLSTCAGLRGFFQVGSDLHLIEPLDEGGEGGRHAVYQAEHLLQTAGTCGVSDDSLGSLLGPRTAAVFRPRPGDSLPSRETRYVELYVVVDNAEFQMLGSEAAVRHRVLEVVNHVDKLYQKLNFRVVLVGLEI WNSQDRFHVSPDPSVTLENLLTWQARQRTRRHLHDNVQLITGVDFTGTTVGFARVSAMCSHSSGAVNQDHSKNPVGVACTMAHEMGHNLGMDHDENVQGCRCQERFEAGRCIMAGSIGSSFPRMFSDCSQAYLESFLERPQSVCLANAPDLSHLVGPVCGNLFVERGEQCDCGPPEDCRNRCCNSTTCQL AEGAQCAHGTCCQECKVKPAGELCRPKKDMCDLEEFCDGRHPECPEDAFQENGTPCSGGYCYNGACPTLAQQCQAFWGPGGQAAEESCFSYDILPGCKASRYRADMCGVLQCKGGQQPLGRAICIVDVCHALTTEDGTAYEPVPEGTRCGPEKVCWKGRCQDLHVYRSSNCSAQCHNHGVCNHKQECHCHA GWAPPHCAKLLTEVHAASGSLPVFVVVVLVLLAVVLVTLAGIIVYRKARSRILSRNVAPKTTMGRSNPLFHQAASRVPAKGGAPAPSRGPQELVPTTHPGQPARHPASSVALKRPPPAPPVTVSSPPFPVPVYTRQAPKQVIKPTFAPPVPPVKPGAGAANPGPAEGAVGPKVALKPPIQRKQGAGAPTAP

[0188] The antigen binding site that can bind to the tumor-associated antigen ADAM9 can be determined by screening for binding to the amino acid sequence defined by SEQ ID NO:152.

[0189] SEQ ID NO: 152

[0190] MGSGARFPSGTLRVRWLLLLGLVGPVLGAARPGFQQTSHLSSYEIITPWRLTRERREAPRPYSKQVSYVIQAEGKEHIIHLERNKDLLPEDFVVYTYNKEGT LITDHPNIQNHCHYRGYVEGVHNSSIALSDCFGLRGLLHLENASYGIEPLQNSSHFEHIIYRMDDVYKEPLKCGVSNKDIEKETAKDEEEEPPSMTQLLRRR RAVLPQTRYVELFIVVDKERYDMMGRNQTAVREEMILLANYLDSMYIMLNIRIVLVGLEIWTNGNLINIVGGAGDVLGNFVQWREKFLITRRRHDSAQLVLKKGFGGTAGMAFVGTVCSRSHAGGINVFGQITVETFASIVAHELGHNLGMNHDDGRDCSCGAKSCIMNSGASGSRNFSSCSAEDFEKLTLNKGGNCLLNIPKPD EAYSAPSCGNKLVDAGEECDCGTPKECELDPCCEGSTCKLKSFAECAYGDCCKDCRFLPGGTLCRGKTSECDVPEYCNGSSQFCQPDVFIQNGYPCQNNKAY CYNGMCQYYDAQCQVIFGSKAKAAPKDCFIEVNSKGDRFGNCGFSGNEYKKCATGNALCGKLQCENVQEIPVFGIVPAIIQTPSRGTKCWGVDFQLGSDVPDP GMVNEGTKCGAGKICRNFQCVDASVLNYDCDVQKKCHGHGVCNSNKNCHCENGWAPPNCETKGYGGSVDSGPTYNEMNTALRDGLLVFFFLIVPLIVCAIFI FIKRDQLWRSYFRKKRSQTYESDGKNQANPSRQPGSVPRHVSPVTPPREVPIYANRFAVPTYAAKQPQQFPSRPPPPQPKVSSQGNLIPARPAPAPPLYSSLT

[0191] Antigen binding sites that bind to the tumor-associated antigen SLC44A4 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:153.

[0192] SEQ ID NO: 153

[0193] MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPA LGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTM FYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNP VARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK

[0194] Antigen binding sites that bind 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.

[0195] SEQ ID NO: 154

[0196] MACSRPPSQCEPTSLPPGPPAGRRHLPLSRRRREMSSNKEQRSAVFVILFALITILILYSSNSANEVFHYGSLRGRSRRPVNLKKWSITDGYVPILGNKTLPSRCHQCVIVSSSSHLLGTKLGPEIERAECTIRMNDAPTTGYSADVGNKTTYRVVAHSSVFRVLR RPQEFVNRTPETVFIFWGPPSKMQKPQGSLVRVIQRAGLVFPNMEAYAVSPGRMRQFDDLFRGETGKDREKSHSWLSTGWFTMVIAVELCDHVHVYGMVPPNYCSQRPRLQRMPYHYYEPKGPDECVTYIQNEHSRKGNHHRFITEKRVFSSWAQLYGITFSHPSWT

[0197] Alternatively, Table 3 lists peptide sequences that can be combined to the heavy chain variable domain and light chain variable domain of CA125 (abavuzumab, sofituzumab), NaPi2b (lifastuzumab), enfortumab, fucosyl-GM1 (described in U.S. Patent Application Publication No. 20130142789, the specific sequence is incorporated herein by reference) or SLC44A4 (described in International Application Publication No. WO2010111018, the specific sequence is incorporated herein by reference).

[0198]

[0199]

[0200] Listed below are examples of scFvs linked to antibody constant regions that also include mutations that enable heterodimerization of the two polypeptide chains. H ) and the light chain variable domain (V L ) are used to prepare the multispecific proteins of the present disclosure. Each sequence represents V L -(G4S)4-V H - Hinge (AS or GAS) - Fc containing heterodimerization mutations (underlined). V L and V H Contains 100V L -44V HSS bridge (underlined), and can come from any tumor-targeting or NKG2D-binding antibody. Ala-Ser (AS, bold and underlined) is included in the elbow hinge region sequence to balance between flexibility and optimal geometry. In some embodiments, additional Gly can be added to the N-terminus of the AS sequence, resulting in a hinge with the sequence Gly-Ala-Ser (GAS, bold and underlined). In some embodiments, additional sequences Thr-Lys-Gly can be added to the AS sequence at the hinge. In the sequences listed in the following paragraphs, the (G4S)4 connector is underlined.

[0201] The TriNKET disclosed herein is F4-TriNKET-EpCAM that binds to NKG2D, which comprises a first polypeptide (F4-EpCAMFc-AJ chain B-scFv that binds to NKG2D) containing the sequence of SEQ ID NO: 203, and a second polypeptide (anti-EpCAM antibody HC-hinge-Fc) containing the sequence of SEQ ID NO: 204. The F4-TriNKET-EpCAM that binds to NKG2D further comprises two light chains that target EpCAM, each of which comprises an anti-EpCAM antibody V L - a constant domain comprising the sequence of SEQ ID NO: 214. For example, in Fig.36 In the structure, when the Fab fragment targets EpCAM, the NKG2D-binding F4-TriNKET-EpCAM includes 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.

[0202] Each of the arms comprises a Fab fragment that binds to EpCAM, comprising: a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, wherein the heavy chain variable domain is connected to the CH1 domain; 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 (e.g., in the F4-EpCAMFc-AJ chain B-NKG2D binding scFv), the CH1 domain is connected to the Fc domain, which is connected to the NKG2D targeting scFv, forming a polypeptide comprising the sequence of SEQ ID NO: 203. In the second arm, the CH1 domain is connected to the Fc domain, forming a polypeptide comprising the sequence of SEQ ID NO: 204.

[0203] For example, F4-EpCAMFc-AJ chain B-NKG2D binding scFv (SEQ ID NO: 203) comprises a heavy chain variable domain (V H ) (SEQ ID NO: 139) and a CH1 domain connected to an Fc domain (hinge-CH2-CH3), which is connected to a single chain variable fragment (scFv) that binds NKG2D at the C-terminus of the Fc. The Fc domain in SEQ ID NO: 203 comprises an S354C substitution, which forms a disulfide bond with a Y349C substitution in another Fc domain (SEQ ID NO: 204, described below). The Fc domain in SEQ ID NO: 203 comprises Q347R, D399V and F405T substitutions. The NKG2D-binding scFv is represented by the amino acid sequence of SEQ ID NO: 205, and comprises a heavy chain variable domain (V ) connected to the heavy chain variable domain (V ) via a (G4S)4 linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 206). H )'s light chain variable domain (V L ). The V contained in SEQ ID NO: 205 L and V H Connected to V L -(G4S)4-V H ; V L and V H Contains 100V L -44V H SS bridge (derived from substitutions from G100C and G44C, respectively) (Cysteine ​​residues are bold-italic-underlined). As represented in SEQ ID NO: 203, the C-terminus of the Fc domain is linked to the N-terminus of the scFv (SEQ ID NO: 205) via a short SGSGGGGS linker (SEQ ID NO: 207).

[0204] scFv binding to NKG2D

[0205]

[0206] F4-EpCAMFc-AJ chain B-scFv binding to NKG2D

[0207]

[0208] The anti-EpCAM antibody HC-hinge-Fc (SEQ ID NO: 204) includes a heavy chain variable domain targeting EpCAM and a CH1 domain connected to an Fc domain (hinge-CH2-CH3). The Fc domain in SEQ ID NO: 204 includes a Y349C substitution, which forms a disulfide bond with the S354C substitution in the CH3 domain of the Fc connected to the scFv (SEQ ID NO: 203) that binds to NKG2D. In SEQ ID NO: 204, the Fc domain also includes K360E and K409W substitutions.

[0209] Anti-EpCAM Antibody V H -CH1-Fc

[0210]

[0211] Anti-EpCAM Antibody V L - The constant domain (SEQ ID NO: 214) comprises a light chain portion targeting EpCAM, which comprises a light chain variable domain and a light chain constant domain.

[0212] Anti-EpCAM Antibody V L -Constant domain

[0213] ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEI KGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:214)

[0214] In an exemplary embodiment, the Fc domain linked to the scFv fragment that binds NKG2D comprises mutations of K360E and K409W, and the Fc domain linked to the EPCAM Fab fragment comprises matching mutations Q347R, D399V and F405T for heterodimer formation.

[0215] In an exemplary embodiment, the Fc domain linked to the scFv that binds NKG2D comprises a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution on the Fc domain that is not linked to the scFv that binds NKG2D.

[0216] Another TriNKET of the present disclosure is F3'-TriNKET-EPCAM that binds NKG2D, the sequence of which is described below (CDRs (Kabat numbering) are underlined).

[0217] Certain TriNKETs of the present disclosure take the form of A49-F3'-TriNKET-EPCAM, the sequence of which is provided below (CDRs (Kabat numbering) are underlined).

[0218] A49-F3'-TriNKET-EPCAM comprises: a single-chain variable fragment (scFv) that binds EPCAM (SEQ ID NOs: 208 and 209 are exemplary sequences of such scFv polypeptides that bind EPCAM), which is connected to the Fc domain via a hinge comprising Gly-Ala-Ser (e.g., in SEQ ID NOs: 210 and 211); and a Fab fragment that binds NKG2D ("A49"), which comprises a heavy chain portion comprising a heavy chain variable domain (SEQ ID NO: 85) and a CH1 domain, and a light chain portion comprising a light chain variable domain (SEQ ID NO: 86) and a light chain constant domain, wherein the heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the Fc domain. The Fc domain linked to the Fab targeting EpCAM comprises Q347R, D399V and F405T substitutions for forming heterodimers with a Fab comprising K360E and K409W substitutions (see, e.g., SEQ ID NO: 212 described below).

[0219] The EPCAM-binding scFv of the present disclosure may include a heavy chain variable domain (denoted as V L (G4S)4V H or LH, where V L In V H N-terminal, and is represented by V H (G4S)4V L or HL, where V H In V L SEQ ID NO: 208 and 209 are exemplary sequences of such scFv polypeptides that bind to EPCAM. The V contained in the scFv (SEQ ID NO: 208 or 209) L and V H Contains 100V L -44V HSS bridge (derived from substitutions of G100C and G44C, respectively) (the cysteine ​​residues are bold-italic-underlined in the following sequences). In SEQ ID NO:208 and SEQ ID NO:209 (G4S)4 is the bold-underlined sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:206).

[0220] EPCAM(MT110LH)scFv

[0221]

[0222] EPCAM(MT100HL)scFv

[0223]

[0224] SEQ ID NO: 210 and SEQ ID NO: 211 represent two sequences of scFv binding to EPCAM, which can be linked to the Fc domain via a hinge (bold-underlined) comprising Gly-Ala-Ser. The Fc domain linked to the scFv includes Q347R, D399V and F405T substitutions.

[0225] EPCAM(MT110LH)scFv-Fc

[0226]

[0227]

[0228] EPCAM(MT110HL)scFv-Fc

[0229]

[0230] SEQ ID NO: 212 represents the heavy chain portion of the Fab fragment, which comprises the heavy chain variable domain of the NKG2D binding site (SEQ ID NO: 85) and the CH1 domain, and is connected to the Fc domain. The Fc domain in SEQ ID NO: 212 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc connected to the EpCAM-binding scFv (e.g., SEQ ID NO: 210 and SEQ ID NO: 211). In SEQ ID NO: 212, the Fc domain also includes K360E and K409W substitutions.

[0231] A49-V H

[0232] EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS SISSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR GAPMGAAAGWFDP WGQGTLVTVSS (SEQ ID NO: 85)

[0233] A49 V H -CH1-Fc

[0234]

[0235] SEQ ID NO: 213 represents the light chain portion of the Fab fragment, which comprises the light chain variable domain of the NKG2D binding site (SEQ ID NO: 86) and the light chain constant domain.

[0236] A49–V L

[0237] DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGVSFPRTF GGGTKVEIK (SEQ ID NO: 86)

[0238] A49 LC V L -Constant domain

[0239] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK RTVAAPSPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:213)

[0240] In an exemplary embodiment, the Fc domain of the Fab fragment that binds NKG2D includes mutations of Q347R, D399V, and F405T, and the Fc domain that is connected to the EPCAM scFv contains matching mutations K360E and K409W for forming heterodimers. In an exemplary embodiment, the Fc domain of the Fab fragment that binds NKG2D includes a S354C substitution in the CH3 domain, which forms a disulfide bond with the Y349C substitution on the Fc of the scFv that is connected to EPCAM.

[0241] In the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala327-Ile 332, Leu 234-Ser 239 and the sugar residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406 (6793): 267-273). Based on the known domain, mutations can be selected to increase or decrease the binding affinity with CD16, for example, by using a phage display library or a yeast surface display cDNA library, or mutations can be designed based on the known three-dimensional structure of the interaction.

[0242] The assembling of heterodimeric antibody heavy chains can be realized by expressing two different antibody heavy chain sequences in the same cell, which may result in the assembling of homodimers of every kind of antibody heavy chains and the assembling of heterodimers. The preference assembling of promoting heterodimers can be realized by incorporating different mutations into the CH3 domain of every kind of antibody heavy chain constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480 and US14 / 830336. For example, mutations can be made in the CH3 domain on the basis of human IgG1, and different pairs of amino acid replacements can be incorporated into the first polypeptide and the second polypeptide to allow these two chains to selectively heterodimerize each other. The position of the amino acid replacement shown below is all numbered according to the EU index in Kabat.

[0243] In one case, the amino acid substitution in the first polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W), and at least one amino acid substitution in the second polypeptide replaces the original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T) or valine (V), so that the larger amino acid substitution (protrusion) fits into the surface of the smaller amino acid substitution (hole). For example, one polypeptide can include a T366W substitution, and another polypeptide can include three substitutions, including T366S, L368A and Y407V.

[0244] The antibody heavy chain variable domain of the present invention can be optionally coupled to an amino acid sequence with at least 90% identity to an antibody constant region, such as an IgG constant region including hinge, CH2 and CH3 domains and containing or not containing a CH1 domain. In certain 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, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In certain other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region compared to a human IgG1 constant region, for example at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366 S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K , D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[0245] In certain embodiments, mutations that can be incorporated into the CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into the CK of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0246] Alternatively, amino acid substitutions may be selected from the group of substitutions shown in Table 4 below.

[0247] Table 4 The first polypeptide The second polypeptide Group 1 S364E / F405A Y349K / T394F Group 2 S364H / D401K Y349T / T411E Group 3 S364H / T394F Y349T / F405A Group 4 S364E / T394F Y349K / F405A Group 5 S364E / T411E Y349K / D401K Group 6 S364D / T394F Y349K / F405A Group 7 S364H / F405A Y349T / T394F Group 8 S364K / E357Q L368D / K370S Group 9 L368D / K370S S364K Group 10 L368E / K370S S364K Group 11 K360E / Q362E D401K Group 12 L368D / K370S S364K / E357L Group 13 K370S S364K / E357Q Group 14 F405L K409R Group 15 K409R F405L

[0248] Alternatively, amino acid substitutions may be selected from the groups of substitutions shown in Table 5 below.

[0249]

[0250]

[0251] Alternatively, amino acid substitutions may be selected from the group of substitutions shown in Table 6 below.

[0252] Table 6 The first polypeptide The second polypeptide Group 1 T366K / L351K L351D / L368E Group 2 T366K / L351K L351D / Y349E Group 3 T366K / L351K L351D / Y349D Group 4 T366K / L351K L351D / Y349E / L368E Group 5 T366K / L351K L351D / Y349D / L368E Group 6 E356K / D399K K392D / K409D

[0253] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 7.

[0254]

[0255] Alternatively, at least one amino acid substitution may be selected from the group of substitutions in Table 8 below, wherein the position noted in the first polypeptide column is replaced by any known negatively charged amino acid, and the position noted in the second polypeptide column is replaced by any known positively charged amino acid.

[0256] Table 8 The first polypeptide The second polypeptide K392, K370, K409 or K439 D399, E356 or E357

[0257] Alternatively, at least one amino acid substitution may be selected from the group of substitutions in Table 9 below, wherein the positions noted in the first polypeptide column are replaced by any known positively charged amino acid, and the positions noted in the second polypeptide column are replaced by any known negatively charged amino acid.

[0258] Table 9 The first polypeptide The second polypeptide D399, E356 or E357 K409, K439, K370 or K392

[0259] Alternatively, amino acid substitutions may be selected from the group of substitutions in Table 10 below.

[0260] Table 10 The first polypeptide The second polypeptide T350V, L351Y, F405A and Y407V T350V, T366L, K392L and T394W

[0261] Alternatively or additionally, the structural stability of the heteromultimeric protein may be improved by introducing S354C on either the first or second polypeptide chain and Y349C on the opposing polypeptide chain, said mutations forming an artificial disulfide bridge within the interface of the two polypeptides.

[0262] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and wherein 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 at one or more positions selected from T366, L368 and Y407.

[0263] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368 and Y407, and wherein 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 at position T366.

[0264] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an 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 wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411.

[0265] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an 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.

[0266] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400 and Y407, and wherein 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 at one or more positions selected from T366, N390, K392, K409 and T411.

[0267] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409 and T411, and wherein 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 at one or more positions selected from L351, D399, S400 and Y407.

[0268] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, Y349, K360 and K409, and wherein 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 at one or more positions selected from Q347, E357, D399 and F405.

[0269] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399 and F405, and wherein 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 at one or more positions selected from Y349, K360, Q347 and K409.

[0270] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409 and K439, and wherein 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 at one or more positions selected from D356, E357 and D399.

[0271] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357 and D399, and wherein 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 at one or more positions selected from K370, K392, K409 and K439.

[0272] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366 and D399, and wherein 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 at one or more positions selected from Y349, L351, L368, K392 and K409.

[0273] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392 and K409, and wherein 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 at one or more positions selected from L351, E356, T366 and D399.

[0274] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by an S354C substitution, and wherein 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.

[0275] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a Y349C substitution, and wherein 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 S354C substitution.

[0276] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region in the substitutions of K360E and K409W, and wherein 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 in the substitutions of O347R, D399V and F405T.

[0277] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of O347R, D399V and F405T, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of K360E and K409W.

[0278] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region in the substitution of T366W, and wherein 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 in the substitutions of T366S, T368A and Y407V.

[0279] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region in the substitutions of T366S, T368A and Y407V, and wherein 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 in the substitution of T366W.

[0280] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of T350V, L351Y, F405A and Y407V, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of T350V, T366L, K392L and T394W.

[0281] In certain embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of T350V, T366L, K392L, and T394W, and wherein the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by substitutions of T350V, L351Y, F405A, and Y407V.

[0282] The above-mentioned multispecific proteins can be produced using recombinant DNA techniques known to those skilled in the art. For example, a first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding the second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding the immunoglobulin light chain can be cloned into a third expression vector; and the first, second and third expression vectors can be stably transfected into a host cell together to produce the multimeric protein.

[0283] In order to obtain the highest yield of the multispecific protein, different ratios of the first, second and third expression vectors can be explored to determine the optimal ratio for transfection into the host cell. After transfection, single clones can be isolated using methods known in the art such as limiting dilution, ELISA, FACS, microscopy or Clonepix for cell bank production.

[0284] The clones can be cultured under conditions suitable for bioreactor scale-up and maintaining 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.

[0285] II. Characteristics of multispecific proteins

[0286] The multispecific proteins described herein include a NKG2D binding site, a CD16 binding site, and a binding site for a tumor-associated antigen selected from any one of the antigens provided in Table 11. In certain embodiments, the multispecific protein simultaneously binds to cells expressing NKG2D and / or CD16, such as NK cells, and binds to tumor cells expressing a tumor-associated antigen selected from any one of the antigens provided in Table 11. The binding of the multispecific protein to NK cells can increase the activity of the NK cells in destroying the tumor cells.

[0287] Table 11

[0288]

[0289] In certain embodiments, the multispecific protein binds to a tumor-associated antigen selected from any one of the antigens provided in Table 11 with an affinity similar to that of a corresponding monoclonal antibody (i.e., a monoclonal antibody that contains a binding site for the same tumor-associated antigen (selected from any one of the antigens provided in Table 11) as contained in the multispecific protein). In certain embodiments, the multispecific protein is more effective than the corresponding monoclonal antibody in killing tumor cells expressing a tumor-associated antigen selected from any one of the antigens provided in Table 11.

[0290] In certain embodiments, the multispecific proteins described herein comprising a NKG2D binding site and a binding site for a tumor-associated antigen selected from any one of the antigens provided in Table 11 activate primary human NK cells when co-cultured with cells expressing the tumor-associated antigen. NK cell activation is characterized by increased CD107a degranulation and IFN-γ cytokine production. In addition, the multispecific proteins may show higher activation of human NK cells in the presence of cells expressing the tumor-associated antigens compared to the corresponding monoclonal antibodies for tumor-associated antigens selected from any one of the antigens provided in Table 11.

[0291] In certain embodiments, the multispecific proteins described herein comprising a binding site for NKG2D and a binding site for a tumor-associated antigen selected from any one of the antigens provided in Table 11 enhance the activity of resting and IL-2-activated human NK cells co-cultured with cells expressing the tumor-associated antigen.

[0292] In certain embodiments, the multispecific protein provides advantages in targeting tumor cells expressing a tumor-associated antigen compared to a corresponding monoclonal antibody that binds to a tumor-associated antigen selected from any one of the antigens provided in Table 11. The multispecific binding proteins described herein may be more effective in reducing tumor growth and killing cancer cells.

[0293] In certain embodiments, F4-TriNKET targeting EpCAM (e.g., F4-TriNKET-EpCAM binding to NKG2D) kills target cells more efficiently than the parent mAb targeting EpCAM. In certain embodiments, the F4-TriNKET also kills target cells more efficiently than F3'-TriNKET (e.g., F3'-TriNKET-EpCAM binding to NKG2D), which may be a reflection of the stronger binding of F4-TriNKET to target cells.

[0294] III. Therapeutic Applications

[0295] The present invention provides methods for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat various cancers expressing EPCAM by administering a therapeutically effective amount of the multispecific binding proteins described herein to a patient in need thereof.

[0296] The treatment method can be characterized according to the cancer to be treated. For example, in certain embodiments, 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.

[0297] 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 other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, bile duct cancer, thyroid cancer, acral lentigo melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytoma, Bartholin's gland carcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland cancer, carcinoid tumor, bile duct cancer, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic Lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, islet cell tumor, Intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct carcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kapos sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo maligna, melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal cancer, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial gland carcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, serous papillary gland cancer, penile cancer, glossopharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumors, spinal cancer, squamous cell carcinoma, rhabdomyocarcinoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, urethral bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0298] In certain other embodiments, the cancer is a non-Hodgkin's lymphoma such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, e.g., pre-T lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer cell / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0299] The cancer to be treated can be characterized by the presence of specific antigens expressed on the surface of cancer cells. In certain embodiments, in addition to EpCAM, the cancer cells may express one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, TROP2, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1.

[0300] In certain other embodiments, when the second binding site binds EpCAM, the cancer to be treated is selected from the group consisting of head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer. In certain other embodiments, when the second binding site binds to an antigen selected from cancer antigen 125 (CA125), sodium / phosphate co-transporter 2B (NaPi2b), nexin cell adhesion molecule 4 (nexin 4), fucosyl-GM1 (monosialyltetrahexosylganglioside), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC44A4) 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, gastric cancer, bile duct cancer.

[0301] IV. Combination Therapy

[0302] Another aspect of the invention provides combination therapies.The multispecific binding proteins described herein can be used in combination with other therapeutic agents to treat cancer.

[0303] Exemplary therapeutic agents that can be used as part of a combination therapy in treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, dibromomannitol, methotrexate, doxorubicin, carboquinone, pentostatin, nitrocorin, zenastatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzosane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesanaline, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozotocin, nimustine, vindesine, flutamide Flutamide, drogenil, oxytocin, carmofur, razoxane, siroxan, carboplatin, dibromodulan, tegafur, ifosfamide, prednimustine, bixibanib, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, melastidine, cyclothiodide, formestran, interferon-α, interferon-2α, interferon-β, interferon-γ (IFN-γ), colony stimulating factor-1, colony stimulating factor-2, denileukin, interleukin-2, luteinizing hormone releasing factor and the above agents may exhibit differential binding to cognate receptors and variations in increased or decreased serum half-life.

[0304] Another class of agents that can be used as part of a combination therapy in treating cancer is an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of the following: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor Ipilimumab has been approved by the United States Food and Drug Administration for the treatment of melanoma.

[0305] Another class of agents that can be used as part of a combination therapy in treating cancer are monoclonal antibodies directed against non-checkpoint targets (eg, Herceptin) and non-cytotoxic agents (eg, tyrosine kinase inhibitors).

[0306] Another class of anticancer agents includes, for example: (i) an ALK inhibitor, an ATR inhibitor, an A2A antagonist, a base excision repair inhibitor, a Bcr-Abl tyrosine kinase inhibitor, a Bruton's tyrosine kinase inhibitor, a CDC7 inhibitor, a CHK1 inhibitor, a cyclin-dependent kinase inhibitor, a DNA-PK inhibitor, an inhibitor of both DNA-PK and mTOR, a DNMT1 inhibitor, a DNMT1 inhibitor plus 2-chloro-deoxyadenosine, an HDAC inhibitor, a Hedgehog signaling pathway inhibitor, an IDO inhibitor, a JAK inhibitor, an mTO inhibitor, R inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphatidylinositol 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25 or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF and G-CSF.

[0307] The proteins of the invention may also be used as an adjunct to surgical removal of primary lesions.

[0308] The amounts of the multispecific binding protein and other therapeutic agents and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the combined therapeutic agents or one or more pharmaceutical compositions comprising the therapeutic agents can be administered in any order, such as sequentially, concurrently, together, simultaneously, etc. In addition, for example, the multispecific binding protein can be administered at a time when the other therapeutic agent exerts its prophylactic or therapeutic effect, and vice versa.

[0309] V. Pharmaceutical Compositions

[0310] The present invention also describes pharmaceutical compositions containing therapeutically effective amounts of proteins described herein. The compositions can be formulated for use in a variety of different drug delivery systems. For suitable dosage forms, one or more physiologically acceptable excipients or carriers can also be included in the compositions. For dosage forms suitable for use in the present disclosure, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249: 1527-1533, 1990).

[0311] The pharmaceutical composition may contain a therapeutically effective amount of a multispecific binding protein comprising sites for the antigens listed in Table 11.

[0312] The intravenous drug delivery dosage form of the present disclosure can be contained in a bag, a pen or a syringe. In certain embodiments, the bag can be connected to a channel comprising a tube and / or a needle. In certain embodiments, the dosage form can be a freeze-dried dosage form or a liquid dosage form. In certain embodiments, the dosage form can be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the dosage form can be freeze-dried, and the freeze-dried dosage form of 45mg can be contained in a vial. In certain embodiments, the freeze-dried dosage form of about 40mg-about 100mg can be contained in a vial. In certain embodiments, the freeze-dried dosage form from 12, 27 or 45 vials is merged to obtain the therapeutic dose of the protein in the intravenous drug dosage form. In certain embodiments, the dosage form can be a liquid dosage form and is stored at about 250mg / vial to about 1000mg / vial. In certain embodiments, the dosage form can be a liquid dosage form and is stored at about 600mg / vial. In certain embodiments, the dosage form may be a liquid dosage form and stored at about 250 mg per vial.

[0313] The protein may be in the form of an aqueous liquid pharmaceutical dosage form comprising a therapeutically effective amount of the protein in a buffered solution forming the dosage form.

[0314] These compositions can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The aqueous solution obtained can be packaged as is for use, or can be freeze-dried, and the freeze-dried preparation is merged with a sterile aqueous carrier before administration. The pH of the preparation is generally between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, for example 7 to 7.5. The composition in the solid form obtained can be packaged in multiple single dose units, each containing a fixed amount of one or more of the above-mentioned medicaments. The composition in the solid form can also be packaged in a container with a flexible amount.

[0315] In certain embodiments, the present disclosure provides dosage forms with extended shelf life comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0316] In certain embodiments, an aqueous dosage form is prepared, which comprises a protein of the present disclosure in a pH buffered solution. The buffer of the present invention can 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 can have a pH of about 5.0 to about 5.2. Ranges between the above pHs are also intended to be part of the present disclosure. For example, it is intended to include a range of values ​​using a combination of any of the above values ​​as an upper and / or lower limit. Examples of buffers that control pH within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, histidine, citrates, and other organic acid buffers.

[0317] In certain embodiments, the dosage form includes a buffer system containing citrate and phosphate to maintain pH in the range of about 4 to about 8. In certain embodiments, the pH range can be about 4.5 to about 6.0 or about pH 4.8 to about 5.5, or in the pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg / mL citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL disodium hydrogen phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g., 0.86) and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system comprises 1-1.5 mg / mL citric acid, 0.25 to 0.5 mg / mL sodium citrate, 1.25 to 1.75 mg / mL disodium hydrogen phosphate dihydrate, 0.7 to 1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / mL sodium chloride. In certain embodiments, the pH of the dosage form is adjusted with sodium hydroxide.

[0318] A polyol that acts as an isotonic agent and can stabilize an antibody may also be included in the dosage form. The polyol is added to the dosage form in an amount that may vary according to the desired isotonicity of the dosage form. In certain embodiments, the aqueous dosage form may be isotonic. The amount of the added polyol may also vary according to the molecular weight of the polyol. For example, a lower amount of monosaccharides (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used as an isotonic agent in the dosage form is mannitol. In certain embodiments, the concentration of mannitol may be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol may be about 7.5 to 15 mg / mL. In certain embodiments, the concentration of mannitol may be about 10-14 mg / mL. In certain embodiments, the concentration of mannitol may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the dosage form.

[0319] Detergents or surfactants may also be added to the dosage form. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulate matter in the dosage form and / or reduces adsorption. In certain embodiments, the dosage form may include a polysorbate as a surfactant. In certain embodiments, the dosage form may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, EditioCantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the dosage form may contain between about 0.1 mg / mL to about 10 mg / mL or between about 0.5 mg / mL to about 5 mg / mL of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the dosage form.

[0320] In embodiments, the protein product of the present disclosure is formulated into a liquid dosage form. The liquid dosage form can be present in a USP / Ph Eur I type 50R vial at a concentration of 10 mg / mL, closed with a rubber stopper and sealed with an aluminum press-fit seal cap. The rubber stopper can be made of an elastomer that complies with USP and Ph Eur. In certain embodiments, the vial can be filled with 61.2 mL of the protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid dosage form can be diluted with a 0.9% saline solution.

[0321] In certain embodiments, liquid dosage forms of the present disclosure can be prepared into solutions with a concentration of 10 mg / mL and combined with the sugar at the level of stabilizer. In certain embodiments, the liquid dosage form can be prepared in an aqueous carrier. In certain embodiments, stabilizers can be added in an amount that does not exceed the viscosity that may be undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar can be a disaccharide such as sucrose. In certain embodiments, the liquid dosage form can also include one or more buffers, surfactants, and preservatives.

[0322] In certain embodiments, the pH of the liquid dosage form can be set by adding a pharmaceutically acceptable acid and / or a base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0323] In addition to aggregation, deamidation is a common product variation that may occur in peptides and proteins during fermentation, harvest / cell clarification, purification, storage of drug substances / drug products, and sample analysis. Deamidation is the loss of NH3 from a protein to form a succinimide intermediate, which may undergo hydrolysis. The succinimide intermediate causes a mass reduction of 17 Daltons in the parent peptide. Subsequent hydrolysis results in a mass increase of 18 Daltons. Separation of the succinimide intermediate is difficult due to instability under aqueous conditions. Therefore, deamidation is usually detectable as a mass increase of 1 Dalton. Deamidation of asparagine produces 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 rate of deamidation. Gly and Ser after Asn in the protein sequence lead to higher sensitivity to deamidation.

[0324] In certain embodiments, liquid dosage forms of the present disclosure may be stored under pH and humidity conditions that prevent deamidation of the protein product.

[0325] Aqueous carriers of interest herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and aqueous carriers that can be used to prepare liquid dosage forms. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0326] Preservatives may optionally be added to the dosage forms of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multiple-dose) dosage forms.

[0327] In certain circumstances, such as when a patient receives all medications by IV route in a hospital after a transplant, an intravenous (IV) dosage form may be the preferred route of administration. In certain embodiments, the liquid dosage form is diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted injectable drug product is isotonic and suitable for administration by intravenous infusion.

[0328] In certain embodiments, salt or buffer components can be added in an amount of 10mM-200mM. The salt and / or buffer is pharmaceutically acceptable and is derived from a variety of known acids (inorganic and organic acids) and "alkali-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycine salt, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can serve as counter ions.

[0329] Preservatives may optionally be added to the dosage forms of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multiple-dose) dosage forms.

[0330] Aqueous carriers of interest herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and aqueous carriers that can be used to prepare liquid dosage forms. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0331] The protein of the present disclosure may be in the form of a freeze-dried dosage form, which includes the protein and a lyoprotectant. The lyoprotectant may be a sugar such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The freeze-dried dosage form may also include one or more buffers, surfactants, bulking agents and / or preservatives.

[0332] The amount of sucrose or maltose that can be used to stabilize the freeze-dried pharmaceutical product can be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be 1:2 to 1:5.

[0333] In certain embodiments, the pH of the dosage form can be set by adding a pharmaceutically acceptable acid and / or base prior to freeze drying. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

[0334] Prior to freeze drying, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range of the freeze-dried pharmaceutical preparation may be 7 to 8.

[0335] In certain embodiments, salt or buffer components can be added in an amount of 10mM-200mM. The salt and / or buffer is pharmaceutically acceptable and is derived from a variety of known acids (inorganic and organic acids) and "alkali-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycine salt, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can serve as counter ions.

[0336] In certain embodiments, "bulking agents" may be added. "Bulking agents" are compounds that add mass to the lyophilized mixture and contribute to the physical structure of the lyophilized cake (e.g., facilitate the production of a substantially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized dosage forms of the present invention may contain these bulking agents.

[0337] Preservatives may optionally be added to the dosage forms of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multiple-dose) dosage forms.

[0338] In certain embodiments, the freeze-dried pharmaceutical product can be constructed with an aqueous carrier. The aqueous carrier of interest herein is a pharmaceutically acceptable (e.g., safe and nontoxic for human administration) and aqueous carrier that can be used to prepare a liquid dosage form after freeze drying. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0339] In certain embodiments, the lyophilized pharmaceutical products of the present disclosure are reconstituted with USP Grade Sterile Water for Injection (SWFI) or USP Grade 0.9% Sodium Chloride Injection.During reconstitution, the lyophilized powder is dissolved in solution.

[0340] In certain embodiments, the freeze-dried protein product of the present disclosure is made up to about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0341] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, and which is not toxic to the patient.

[0342] The specific dosage can be a uniform dosage for each patient, such as 50-5000 mg protein. Alternatively, the dosage of the patient can be customized according to the approximate weight or surface area of ​​the patient. Other factors determining a suitable dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine a suitable dosage for treatment is routinely performed by those skilled in the art, particularly according to the dosage information and assay disclosed herein. The dosage can also be determined by using a known assay for determining the dosage used and in combination with suitable dose response data. When disease progression is monitored, the dosage of an individual patient can be adjusted. The blood level of a targetable construct or complex can be measured in a patient to observe whether the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0343] Generally, a dosage based on body weight is from about 0.01 μg to about 100 mg per 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, kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 1 00 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 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 100 mg / kg body weight. To about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0344] The medicament can be administered once or more daily, weekly, monthly or annually, or even once every 2 to 20 years, and one of ordinary skill in the art can easily estimate the repetition rate of administration based on the measured residence time and concentration of the targetable construct or complex in the body fluid or tissue. The administration of the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion or by direct intralesional injection. This can be administered once or more daily, once or more weekly, once or more monthly or once or more annually.

[0345] The above description describes various aspects and embodiments of the present invention. This application specifically contemplates all combinations and permutations of the described aspects and embodiments. Example

[0346] The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0347] Example 1 - NKG2D binding domain binds to NKG2D

[0348] NKG2D binding domain binds to purified recombinant NKG2D

[0349] The nucleic acid sequence of the human, mouse or cynomolgus monkey NKG2D extracellular domain (ectodomains) is fused with the nucleic acid sequence encoding the human IgG1 Fc domain and introduced into mammalian cells for expression. After purification, the NKG2D-Fc fusion protein is adsorbed to the wells of the microplate. After blocking the wells with bovine serum albumin to prevent nonspecific binding, the NKG2D binding domain is titrated and added to the wells pre-adsorbed with the NKG2D-Fc fusion protein. The first antibody is combined with a second antibody coupled to horseradish peroxidase and specifically recognizes human kappa light chains to avoid Fc cross-reactivity for detection. The substrate 3,3',5,5'-tetramethylbenzidine (TMB) of horseradish peroxidase is added to the wells to visualize the binding signal, which is measured at 450nM and corrected at 540nM. To each well, a NKG2D binding domain clone, isotype control or positive control (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D antibody clones MI-6 and CX-5 available at eBioscience) was added.

[0350] The isotype control showed minimal binding to the recombinant NKG2D-Fc protein, while the positive control bound most strongly to the recombinant antigen. The NKG2D binding domains produced by all clones showed binding across human, mouse, and cynomolgus monkey recombinant NKG2D-Fc proteins, albeit with varying affinities between clones. Overall, each anti-NKG2D clone bound to human ( Figure 3 ) and cynomolgus monkeys ( Figure 4 ) recombinant NKG2D-Fc, but bound to mouse ( Figure 5 )The affinity of recombinant NKG2D-Fc is low.

[0351] NKG2D binding domain binds to cells expressing NKG2D

[0352] The EL4 mouse lymphoma cell line was engineered to express human or mouse NKG2D-CD3ζ signaling domain chimeric antigen receptors. NKG2D binding clones, isotype controls or positive controls were used at a concentration of 100 nM to stain extracellular NKG2D expressed on the EL4 cells. Antibody binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and the folds above background (FOB) were calculated using a comparison of the mean fluorescence intensity (MFI) of cells expressing NKG2D to that of parent EL4 cells.

[0353] The NKG2D binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. The positive control antibody (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D antibody clones MI-6 and CX-5 available at eBioscience) gave the best FOB binding signal. Figure 6 ) and mice ( Figure 7 )NKG2D-binding affinity of each clone was similar.

[0354] Example 2 - NKG2D binding domain blocks natural ligand binding to NKG2D

[0355] Competition with ULBP-6

[0356] Recombinant human NKG2D-Fc protein was adsorbed to the wells of the microplate, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by the NKG2D binding domain clone. After incubation for 2 hours, the wells were washed, and the ULBP-6-His-biotin still bound to the wells coated with NKG2D-Fc was detected by streptavidin-coupled horseradish peroxidase and TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin that was blocked from binding to the NKG2D-Fc protein in the wells. The positive control antibody (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked ULBP-6 binding to NKG2D, while the isotype control showed little competition with ULBP-6 ( Figure 8 ).

[0357] The ULBP-6 sequence is represented by SEQ ID NO: 108

[0358] MAAAAIPALLLCLPLLFLLFGWSRARRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARM SCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSGTTQLRATATTLILCCLLIILPCFILPGI(SEQ ID NO: 108) Competition with MICA

[0359] Recombinant human MICA-Fc protein was adsorbed to the wells of a microplate and the wells were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells followed by the NKG2D binding domain. After incubation and washing, NKG2D-Fc-biotin still bound to the MICA-Fc coated wells was detected using streptavidin-HRP and TMB substrate. The absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin blocked from binding to the MICA-Fc coated wells. The positive control antibody (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked MICA binding to NKG2D, while the isotype control showed little competition with MICA ( Fig. 9 ).

[0360] Competition with Rae-1δ

[0361] Recombinant mouse Rae-1δ-Fc (purchased from R&D Systems) was adsorbed to the wells of the microplate, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the NKG2D binding domain. After incubation and washing, streptavidin-HRP and TMB substrates were used to detect NKG2D-Fc-biotin still bound to the wells coated with Rae-1δ-Fc. The absorbance was measured at 450nM and corrected at 540nM. 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 blocked from binding to the wells coated with Rae-1δ-Fc. The positive control (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D antibody clones MI-6 and CX-5 available at eBioscience) and various NKG2D binding domain clones blocked Rae-1δ binding to mouse NKG2D, while the isotype control antibody showed little competition with Rae-1δ ( Fig.10 ).

[0362] Example 3-NKG2D binding domain cloning activates NKG2D

[0363] The nucleic acid sequences of human and mouse NKG2D are fused to the nucleic acid sequences encoding the CD3ζ signaling domain to obtain a chimeric antigen receptor (CAR) construct. The NKG2D-CAR construct is then cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells are infected with a virus containing NKG2D-CAR together with 8 μg / mL polybrene. 24 hours after infection, the expression level of NKG2D-CAR in the EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0364] To determine whether the NKG2D binding domains activated NKG2D, they were adsorbed to wells of a microtiter plate and NKG2D-CAR EL4 cells were cultured on antibody fragment-coated wells in the presence of Brefeldin-A and Monensin for 4 hours. Intracellular TNF-α production, an indication of NKG2D activation, was determined by flow cytometry. TNF-α positive cells were normalized to cells treated with a positive control. All NKG2D binding domains activated human NKG2D ( Fig.11 ) and mouse NKG2D ( Fig.12 )Both.

[0365] Example 4-NKG2D binding domain activates NK cells

[0366] Primary human NK cells

[0367] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ), the purity of the isolated NK cells is usually >95%. The isolated NK cells are then cultured in medium containing 100ng / mL IL-2 for 24-48 hours, and then they are transferred to microtiter wells adsorbed with the NKG2D binding domain and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin-A and monensin. After culture, NK cells are measured by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56 and IFN-γ. In CD3 - CD56 +CD107a and IFN-γ staining were analyzed in cells to assess NK cell activation. An increase in CD107a / IFN-γ double positive cells indicates better NK cell activation by binding to two activation receptors rather than one receptor. The NKG2D binding domain and positive controls (e.g., heavy chain variable domains represented by SEQ ID NO: 101 or SEQ ID NO: 103, and light chain variable domains represented by SEQ ID NO: 102 or SEQ ID NO: 104) showed changes to CD107a compared to isotype controls. + and IFN-γ + The percentage of NK cells is higher ( Fig.13 and Fig.14 Represents data from two independent experiments, each using PBMCs from different donors for NK cell preparation).

[0368] Primary mouse NK cells

[0369] Spleens were obtained from C57Bl / 6 mice and crushed through a 70 μm cell sieve to obtain a single cell suspension. The cells were centrifuged and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific #A1049201; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove red blood cells. The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours and then harvested and prepared for NK cell isolation. NK cells (CD3 - NK1.1 + ), typically with a purity of >90%. Purified NK cells were cultured in medium containing 100 ng / mL mIL-15 for 48 hours, and then they were transferred to microtiter wells adsorbed with the NKG2D binding domain and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin-A, and monensin. After culture in wells coated with the NKG2D binding domain, NK cells were measured by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-γ. - NK1.1 + CD107a and IFN-γ staining were analyzed in the cells to assess NK cell activation. An increase in CD107a / IFN-γ double positive cells indicates better NK cell activation by binding to two activating receptors rather than one receptor. The NKG2D binding domain and positive control (selected from anti-mouse NKG2D antibody clones MI-6 and CX-5 available at eBioscience) showed changes to CD107a compared to the isotype control + and IFN-γ+ The percentage of NK cells is higher ( Fig.15 and Fig.16 Represents data from two independent experiments, each using different mice for NK cell preparation).

[0370] Example 5 - NKG2D binding domain can induce cytotoxicity to target tumor cells

[0371] Human and mouse primary NK cell activation assays showed an increase in cytotoxic markers on NK cells after incubation with the NKG2D binding domain. To investigate whether this translates into an increase in tumor cell lysis, a cell-based assay was utilized in which each NKG2D binding domain was developed into a monospecific antibody. The Fc region was used as one targeting arm, while the Fab fragment region (NKG2D binding domain) served as another targeting arm to activate NK cells. THP-1 cells of human origin and expressing high levels of Fc receptors were used as tumor targets, and the Perkin Elmer DELFIA cytotoxicity kit was used. THP-1 cells were labeled with BATDA reagent and incubated at 10 5 The NKG2D antibody and the isolated mouse NK cells were then combined in the wells of a microplate and incubated for 3 hours at 37°C. After incubation, 20 μL of culture supernatant was taken out, mixed with 200 μL of europium solution, and incubated in the dark for 15 minutes with shaking. The fluorescence was measured over time by a PheraStar plate reader equipped with a time-resolved fluorescence module (excitation 337 nm, emission 620 nm), and specific lysis was calculated according to the kit instructions.

[0372] The positive control ULBP-6 coupled to Fc, the natural ligand of NKG2D, showed an increase in the specific lysis of THP-1 target cells by mouse NK cells. The NKG2D 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 ).

[0373] Example 6 - NKG2D antibodies show high thermal stability

[0374] The melting temperature of the NKG2D binding domain was determined using differential scanning fluorimetry. The extrapolated apparent melting temperature is higher than that of a typical IgG1 antibody ( Fig.18 ).

[0375] Example 7 - Synergistic activation of human NK cells by cross-linked NKG2D and CD16

[0376] Primary Human NK Cell Activation Assay

[0377] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells were purified from PBMCs using negative magnetic beads (StemCell #17955). NK cells were determined to be >90% CD3 by flow cytometry. - CD56 + . The isolated NK cells were then expanded in medium containing 100 ng / mL hIL-2 (Peprotech #200-02) for 48 hours before being used in activation assays. Antibodies were coated in 100 μL sterile PBS at a concentration of 2 μg / mL (anti-CD16 antibody, Biolegend #302013) and 5 μg / mL (anti-NKG2D antibody, R&D #MAB139) in 96-well flat-bottom plates at 4°C overnight, and the wells were then washed extensively to remove excess antibody. To assess degranulation, IL-2 activated NK cells were plated at 5×10 5 1×10 cells / mL were resuspended in culture medium supplemented with 100 ng / mL hIL2 and 1 μg / mL APC-conjugated anti-CD107a mAb (Biolegend #328619). 5 Cells / well were added to the antibody coated plates. Protein transport inhibitors Brefeldin A (BFA, Biolegend #420601) and Monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively. The plated wells were incubated at 37°C for 4 hours in 5% CO2. For intracellular staining of IFN-γ, NK cells were labeled with anti-CD3 antibody (Biolegend #300452) and anti-CD56 mAb (Biolegend #318328), then fixed, permeabilized, and labeled with anti-IFN-γ mAb (Biolegend #506507). In the presence of live CD56 + CD3 - After cell gating, the expression of CD107a and IFN-γ in NK cells was analyzed by flow cytometry.

[0378] To investigate the relative potency of receptor combinations, cross-linking of NKG2D or CD16 and co-cross-linking of both receptors were performed by plate-bound stimulation. Figures 19A-19C ), combined stimulation of CD16 and NKG2D resulted in greatly increased CD107a levels (degranulation) ( Fig.19A ) and / or IFN-γ production ( Fig.19B ). Dashed lines indicate the additive effect of individual stimulation of each receptor.

[0379] CD107a levels and intracellular IFN-γ production of IL-2 activated NK cells were analyzed after 4 hours of plate-bound stimulation with anti-CD16 antibody, anti-NKG2D antibody or a combination of both monoclonal antibodies. Graphs indicate mean values ​​(n=2)±Sd. Fig.19A Levels of CD107a are shown; Fig.19B Levels of IFNγ are shown; Fig.19C Levels of CD107a and IFNγ are shown. Figures 19A-19C The data shown are representative of 5 independent experiments using 5 different healthy donors.

[0380] Example 8 - Trispecific Binding Protein (TriNKET) Mediated Enhanced Cytotoxicity of Target Cells Evaluation of TriNKET or mAb Binding to Cells Expressing Human Cancer Antigens

[0381] Human cancer cell lines expressing EPCAM were used to evaluate tumor antigen binding of TriNKET derived from clone MT110 targeting EPCAM in F4 and F3' formats. Human cell lines H747, HCC827 and HCT116 were used to evaluate the binding of TriNKET and mAbs to cells expressing EPCAM. TriNKET or mAbs were diluted and incubated with the corresponding cells. Detection was performed using a fluorophore-coupled anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the MFI bound to cells expressing EPCAM was normalized to the control stained with human recombinant IgG1 to obtain a multiple compared to the background value.

[0382] Fig.37 Binding of trispecific binding proteins (TriNKET) of the present disclosure (A49-F4-TriNKET-MT110 and A49-F3′-TriNKET-MT110) and the parent monoclonal antibody (mAb) to H747 human colorectal cancer cells expressing EpCAM is shown. Fig.38 Binding of trispecific binding proteins (TriNKET) of the present disclosure (A49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) and the parent monoclonal antibody (mAb) to HCC827 human lung cancer cells expressing EpCAM is shown. Fig.39Binding of the trispecific binding proteins (TriNKETs) of the present disclosure (A49-F4-TriNKET-MT110 and A49-F3'-TriNKET-MT110) and the parent monoclonal antibody (mAb) to HCT116 human colorectal cancer cells expressing EpCAM is shown. F4-TriNKET containing the MT110 EPCAM conjugate has stronger overall binding than F3'-TriNKET.

[0383] Primary Human NK Cell Cytotoxicity Assay

[0384] PBMCs are isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs are washed and prepared for NK cell isolation. NK cells are isolated by negative selection technique using magnetic beads. The purity of isolated NK cells obtained is usually higher than 90% CD3 - CD56 + The isolated NK cells were incubated overnight without cytokines and used in cytotoxicity assays the next day.

[0385] DELFIA Cytotoxicity Assay

[0386] Human cancer cell lines expressing the target of interest were collected from culture, washed with HBS, and incubated at 10 6 The cells were resuspended in growth medium at a density of 10 cells / mL in preparation for labeling with BATDA 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 incubated at 0.5 × 10 5 Resuspend in culture medium at a density of 10 cells / mL. To prepare background wells, set aside an aliquot of the labeled cells and centrifuge the cells from the culture medium. Carefully add 100 μL of culture medium to the wells in triplicate to avoid disturbing the settled cells. Add 100 μL of BATDA-labeled cells to each well of the 96-well plate. Reserve wells for measuring spontaneous release from target cells and prepare wells for lysing target cells by adding 1% Triton-X. Dilute a monoclonal antibody or TriNKET against the tumor target of interest in culture medium and add 50 μL of the diluted mAb or TriNKET to each well. Harvest resting NK cells from the culture medium, wash, and plate at 1.0 × 10 5 -2.0×10 6 NK cells were resuspended in culture medium at a density of 10 cells / mL. 50 μL of NK cells were added to each well of the plate to provide a total culture volume of 200 μL. The plates were incubated at 37° C. and 5% CO 2 for 2-4 hours prior to assay development.

[0387] After 2-3 hours of incubation, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 x g for 5 minutes. 20 μL of culture supernatant was transferred to a clean microplate provided by the manufacturer and 200 μL of room temperature europium solution was added to each well. The plate was protected from light and incubated on a shaker at 250 rpm for 15 minutes. The plate was read on an i3X instrument (Molecular Devices) and the % of specific lysis was calculated (% of specific lysis = ((experimental release - spontaneous release) / (maximal release - spontaneous release)) x 100).

[0388] Fig.40A and Fig.40B Shown is TriNKET-mediated cytotoxicity of resting human NK cells from two different healthy donors against H747 human cancer cells. Fig.41A and Fig.41B Shown is TriNKET-mediated cytotoxicity of resting human NK cells from two different healthy donors against HCC827 human cancer cells. Fig.42A and Fig.42B Shown is TriNKET-mediated cytotoxicity of resting human NK cells from two different healthy donors against MCF7 human cancer cells. Fig.43A and Fig.43B TriNKET-mediated cytotoxicity of resting human NK cells from two different healthy donors against HCT116 human cancer cells is shown. F4-TriNKET targeting EPCAM kills target cells more efficiently than the parent mAb targeting EPCAM. F4-TriNKET also kills target cells more efficiently than F3'-TriNKET, which may be a reflection of the stronger binding of F4-TriNKET to target cells.

[0389] Incorporated by Reference

[0390] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0391] Equivalence

[0392] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments should be considered in all cases as illustrative rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than the above description, and it is intended that all changes within the meaning and equivalent scope of the claims are included therein.

Claims

1. A protein comprising: (a) binds to the first antigen binding site of natural killer cell surface activating receptor group 2 member D (NKG2D); (b) a second antigen binding site that binds an antigen selected from the group consisting of: EpCAM, cancer antigen 125 (CA125), sodium / phosphate co-transporter 2B (NaPi2b), integrin cell adhesion molecule 4 (integrin 4), fucosyl-GM1 (monosialyltetrahexosylganglioside), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC44A4), and sialyl Lewis a antigen (CA19-9); and (c) an antibody Fc domain or a portion thereof sufficient to bind to cluster of differentiation antigen 16 (CD16), or a third antigen binding site that binds to CD16.

2. A protein comprising: (a) binds to the first antigen binding site of NKG2D; (b) a second antigen binding site that binds to EpCAM; and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

3. A protein comprising: (a) binds to the first antigen binding site of NKG2D; (b) a second antigen binding site that binds to a tumor-associated antigen selected from the group consisting of cancer antigen 125 (CA125), sodium / phosphate co-transporter 2B (NaPi2b), integrin cell adhesion molecule 4 (integrin 4), fucosyl-GM1 (monosialyltetrahexosylganglioside), disintegrin and metalloproteinase domain-containing protein 8 (ADAM8), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), solute carrier family 44 member 4 (SLC44A4), and sialyl Lewis a antigen (CA19-9); and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

4. A protein comprising: (a) binds to the first antigen binding site of NKG2D; (b) a second antigen binding site that binds a tumor-associated antigen selected from sodium-dependent phosphate transporter 2b (NaPi2b); and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

5. A protein comprising: (a) binds to the first antigen binding site of NKG2D; (b) a second antigen binding site that binds to tumor-associated antigen binding protein 4; and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

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

7. A protein comprising: (a) binds to the first antigen binding site of NKG2D; (b) a second antigen binding site that binds a T-cell associated tumor antigen selected from SLC44A4; and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

8. The protein of any one of claims 1-7, wherein the first antigen binding site binds to NKG2D in humans, non-human primates, and rodents.

9. The protein of any one of claims 1-8, wherein the first antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

10. The protein of claim 9, wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide.

11. The protein of claim 9 or 10, wherein the second antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

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

13. The protein of claim 11 or 12, wherein the light chain variable domain of the first antigen binding site has an amino acid sequence that is identical to the amino acid sequence of the light chain variable domain of the second antigen binding site.

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

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

17. The protein of claim 14 or 15, wherein the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv.

18. The protein of claim 17, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

19. The protein of claim 18, wherein the scFv is linked to the antibody Fc domain, wherein 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 (GlyGlyGlyGlySer)4((G4S)4), and the Fab is linked to the antibody Fc domain.

20. The protein of any one of claims 15-19, wherein the heavy chain variable domain of the scFv is connected to the light chain variable domain of the scFv via a flexible linker.

21. The protein of claim 20, wherein the flexible linker comprises (GlyGlyGlyGlySer)4((G4S)4).

22. The protein of any one of claims 15-21, wherein the heavy chain variable domain of the scFv is located at the N-terminus or C-terminus of the light chain variable domain of the scFv.

23. The protein of claim 22, wherein the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv.

24. The protein according to any one of claims 14 to 23, wherein the Fab fragment is linked to the antibody Fc domain or a portion thereof sufficient to bind to CD16 or the third antigen binding site that binds CD16.

25. The protein of claim 24, wherein the heavy chain portion of the Fab fragment comprises a heavy chain variable domain and a CH1 domain, and wherein the heavy chain variable domain is linked to the CH1 domain.

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

27. The protein of any one of claims 14 to 26, comprising a sequence selected from SEQ ID NO: 208 and SEQ ID NO:

209.

28. The protein of any one of claims 15-27, comprising a scFv linked to an antibody Fc domain, wherein the scFv linked to an antibody Fc domain is represented by a sequence selected from the group consisting of SEQ ID NO: 210 and SEQ ID NO:

211.

29. The protein of any one of claims 15-27, comprising a sequence selected from the group consisting of SEQ ID NO: 212 and SEQ ID NO:

213.

30. The protein of any one of claims 15-26, comprising a sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 210 and SEQ ID NO:

211.

31. The protein of any one of claims 15-26, comprising a sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 210 and SEQ ID NO:

211.

32. The protein of any one of claims 15-26, comprising a sequence that is at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 210 and SEQ ID NO:

211.

33. The protein of any one of claims 15-32, comprising a sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 212 and SEQ ID NO:

213.

34. The protein of any one of claims 15-32, comprising a sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 212 and SEQ ID NO:

213.

35. The protein of any one of claims 15-32, comprising a sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 212 and SEQ ID NO:

213.

36. A protein comprising: (a) comprising a first antigen binding site of a single chain variable fragment (scFv) that binds NKG2D; (b) a second antigen binding site that binds to EpCAM; and (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen binding site that binds to CD16.

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

38. The protein of claim 36 or 37, wherein the second antigen binding site that binds EpCAM is a Fab fragment.

39. The protein of claim 37 or 38, wherein the second and additional antigen binding site that binds EpCAM is a Fab fragment.

40. The protein of claim 36 or 37, wherein the second and additional antigen binding site that binds EpCAM is a scFv.

41. The protein of any one of claims 36-40, wherein the heavy chain variable domain of the NKG2D binding scFv is located at the N-terminus or C-terminus of the light chain variable domain of the scFv.

42. The protein of claim 41, wherein the light chain variable domain is located at the N-terminus of the heavy chain variable domain of the NKG2D binding scFv.

43. The protein of any one of claims 36-42, wherein the scFv that binds to NKG2D is linked to the antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds CD16.

44. The protein of claim 43, wherein the scFv that binds to NKG2D is linked to the antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds to CD16 via a hinge comprising Ala-Ser or Gly-Ala-Ser.

45. The protein of claim 43, wherein the scFv that binds to NKG2D is linked to the C-terminus of the antibody Fc domain or a portion thereof sufficient to bind to CD16 or a third antigen binding site that binds CD16 via a flexible linker comprising SGSGGGGS (SEQ ID NO: 207).

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

47. The protein of any one of claims 36-46, wherein within the NKG2D binding scFv, a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv.

48. The protein of claim 47, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

49. The protein of any one of claims 36-48, wherein within the NKG2D binding scFv, the heavy chain variable domain is connected to the light chain variable domain by a flexible linker.

50. The protein of claim 49, wherein the flexible linker comprises (GlyGlyGlyGlySer)4((G4S)4).

51. The protein of any one of claims 40 to 50, wherein the second and additional antigen binding site scFv is linked to the antibody Fc domain or a portion thereof sufficient to bind CD16 or the third antigen binding site that binds CD16 via a hinge comprising Ala-Ser.

52. The protein of any one of claims 40 to 51, wherein the second and additional antigen binding site scFv is linked to the antibody Fc domain via a hinge comprising Ala-Ser.

53. The protein of claim 51 or 52, wherein a disulfide bridge is formed between the heavy chain variable domain and the light chain variable domain of the second antigen binding site and / or the additional antigen binding site.

54. The protein of claim 53, wherein the disulfide bridge is formed between C44 from the heavy chain variable domain and C100 from the light chain variable domain.

55. The protein of any one of claims 36 to 54, wherein the NKG2D-binding scFv comprises a light chain variable domain at the N-terminus of the heavy chain variable domain, wherein the light chain variable domain is connected to the heavy chain variable domain of the scFv via a flexible linker (GlyGlyGlyGlySer)4((G4S)4), and the NKG2D-binding scFv is connected to the antibody Fc domain via a hinge comprising Ala-Ser or Gly-Ala-Ser.

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

203.

57. A protein comprising the amino acid sequences 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.

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

203.

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

203.

61. A protein according to any of the preceding claims, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to an amino acid sequence 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 SEQ ID NO:

93.

62. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:41 and a light chain variable domain at least 90% identical to SEQ ID NO:

42.

63. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:49 and a light chain variable domain at least 90% identical to SEQ ID NO:

50.

64. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:57 and a light chain variable domain at least 90% identical to SEQ ID NO:

58.

65. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO:59 and a light chain variable domain at least 90% identical to SEQ ID NO:

60.

66. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 61 and a light chain variable domain at least 90% identical to SEQ ID NO:

62.

67. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 69 and a light chain variable domain at least 90% identical to SEQ ID NO:

70.

68. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 77 and a light chain variable domain at least 90% identical to SEQ ID NO:

78.

69. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 85 and a light chain variable domain at least 90% identical to SEQ ID NO:

86.

70. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 93 and a light chain variable domain at least 90% identical to SEQ ID NO:

94.

71. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 101 and a light chain variable domain at least 90% identical to SEQ ID NO:

102.

72. The protein of any one of claims 1-60, wherein the first antigen binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 103 and a light chain variable domain at least 90% identical to SEQ ID NO:

104.

73. The protein of any one of claims 1-10, wherein the first antigen binding site is a single domain antibody.

74. The protein of claim 73, wherein the single domain antibody is a V H H fragment or V NAR Fragment.

75. The protein of any of claims 1-10 or 73-74, wherein the second antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

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

77. The protein of any of claims 1, 2, or 61-72, wherein the second antigen binding site binds EpCAM, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 115, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

119.

78. The protein of claim 77, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence comprising: 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.

79. The protein of claim 78, wherein the light chain variable domain of the second antigen binding site comprises an amino acid sequence 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 A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO:

122.

80. The protein of any of claims 1, 2, or 61-72, wherein the second antigen binding site binds EpCAM, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 123, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

127.

81. The protein of claim 80, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence comprising: 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.

82. The protein of claim 81, wherein the light chain variable domain of the second antigen binding site comprises an amino acid sequence 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.

83. The protein of any of claims 1, 2, or 61-72, wherein the second antigen binding site binds EpCAM, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 131, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

135.

84. The protein of claim 83, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence comprising: 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.

85. The protein of claim 84, wherein the light chain variable domain of the second antigen binding site comprises an amino acid sequence 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.

86. The protein of any of claims 1, 2, or 61-72, wherein the second antigen binding site binds EpCAM, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 139, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

143.

87. The protein of claim 86, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence comprising: 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.

88. The protein of claim 87, wherein the light chain variable domain of the second antigen binding site comprises an amino acid sequence 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.

89. The protein of any one of claims 1, 3, or 61-72, wherein the second antigen binding site binds CA125, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 155, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

159.

90. The protein of any one of claims 1, 3, or 61-72, wherein the second antigen binding site binds CA125, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 163, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

167.

91. The protein of any one of claims 1, 4, or 61-72, wherein the second antigen binding site binds NaPi2b, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 171, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

175.

92. The protein of any of claims 1, 5, or 61-72, wherein the second antigen binding site binds to binding protein 4, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 179, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

183.

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

191.

94. The protein of any of claims 1, 7, or 61-72, wherein the second antigen binding site binds SLC44A4, the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195, and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

199.

95. The protein of any one of claims 1-94, wherein the antibody Fc domain comprises the hinge and CH2 domains of a human IgG1 antibody.

96. The protein of claim 95, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgGl antibody.

97. The protein of claim 96, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgGl and differs at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, K439.

98. The protein of any one of claims 1-96, wherein the protein has a K of 10 nM. D or weaker affinity binding to NKG2D.

99. A dosage form comprising the protein of any preceding claim and a pharmaceutically acceptable carrier.

100. A cell comprising one or more nucleic acids expressing a protein according to any one of claims 1-98.

101. A method of directly and / or indirectly increasing tumor cell death, the method comprising exposing tumor and natural killer cells to a protein according to any one of claims 1-98.

102. A method of treating cancer, wherein the method comprises administering to a patient a protein according to any one of claims 1-98 or a dosage form according to claim 99.

103. The method of claim 102, wherein when the second binding site binds EpCAM, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, esophageal cancer, and lung cancer.

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