Multispecific binding proteins targeting CEA

Abstract

Multispecific binding proteins that bind to the NKG2D receptor, CD16, and a tumor-associated antigen selected from carbonic anhydrase 9 (CAIX), calcium-activated chloride channel protein 1 (ANO1), mesothelin, TROP2, CEA, and tight junction protein-18.2, as well as pharmaceutical compositions and therapeutic methods that can be used to treat cancer are described.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Application Nos. 62 / 464,341, filed February 27, 2017, 62 / 464,344, filed February 27, 2017, 62 / 464,347, filed February 27, 2017, 62 / 467,557, filed March 06, 2017, 62 / 473,652, filed March 20, 2017, and 62 / 473,659, filed March 20, 2017, the entire contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing electronically submitted in ASCII format and incorporated herein by reference in its entirety. The ASCII copy generated on February 27, 2018 is named DFY-012WO_ST25.txt and is 212,480 bytes in size. Technical Field

[0005] The present invention relates to a multispecific binding protein that binds to NKG2D receptor, CD16 and a tumor-associated antigen selected from carbonic anhydrase 9 (CAIX), calcium-activated chloride channel protein 1 (ANO1), mesothelin, trophoblast cell surface antigen (TROP2), carcinoembryonic antigen (CEA) and tight junction protein-18.2. Background Art

[0006] Although a large number of research attempts and scientific progress in treating cancer have been reported in the literature, this disease remains a significant health problem. Some of the most frequently diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Small cell lung cancer remains the most lethal malignancy. Current treatment options for these cancers are not effective for all patients and / or may have a large number of serious side effects. Other types of cancers are also still challenging to treat using existing treatment options.

[0007] 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.

[0008] Natural killer (NK) cells are an integral part of the innate immune system and account for approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and are 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 perforin 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.

[0009] 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., 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.

[0010] Carbonic anhydrase 9 (CAIX) is a zinc metalloenzyme and belongs to a family of enzymes involved in the reversible hydration of carbon dioxide to form bicarbonate and hydrogen ions. CAIX has limited expression in normal tissues, but is overexpressed in a wide variety of different solid tumors and plays an important role in tumor development. Induced by the hypoxic tumor microenvironment, CAIX acts to counteract the deleterious effects of the high rate of glycolytic metabolism in tumor cells by maintaining an intracellular pH that is favorable for tumor cell growth and survival and at the same time generating an acidic extracellular space that promotes tumor cell invasiveness. So far, overexpression of CAIX has been detected in renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, as well as common epithelial cancers such as those of the esophagus, lung, colon, kidney, cervix, and non-small cell lung cancer.

[0011] Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein and is expressed in mesothelial cells of the pleura, peritoneum, and pericardium. In addition to normal expression, mesothelin is overexpressed in a variety of different cancers including mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, bile duct cancer, gastric cancer, uterine serous carcinoma, thymic carcinoma, and acute myeloid leukemia. It has been proposed that mesothelin may play an important role in tumor development including cell adhesion, cell survival / proliferation, and chemotherapy resistance.

[0012] Calcium-activated chloride channel protein 1 (ANO1) is a voltage-sensitive calcium-activated chloride channel. It also has other names, such as TMEM16, TAOS2, ORAOV2, and DOG-1. Calcium-activated chloride channels play a role in many physiological processes, including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth and contraction, and fertilization. ANO1 may be involved in the regulation of epithelial fluid secretion, olfactory and phototransduction, neuronal and cardiac excitability, and vascular tone, including gastrointestinal motility. ANO1 is also highly expressed in certain cancers, where it is thought to increase cell proliferation, cell migration, and metastasis, such as in esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumors (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer, prostate cancer, and sarcomas. It was found that ~85% of HNSCC tumors are ANO1-positive, and HNSCC patients with high-level tumor expression of ANO1 have a reduced overall survival rate; it was also found that overexpression of ANO1 is closely associated with the malignancy of prostate cancer and the poor prognosis of breast cancer.

[0013] TROP2 is a transmembrane glycoprotein and Ca 2+ Signal transducer. In addition to being expressed in certain normal tissues, TROP2 is overexpressed in a variety of different cancers including breast, lung, gastric, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian cancer. It transmits signals to cancer cells for proliferation, invasion, and survival.

[0014] Carcinoembryonic antigen (CEA), also known as CD66e or CEACAM5, is a member of the immunoglobulin superfamily. It is a large cell surface glycoprotein and acts primarily as a cell adhesion molecule that mediates cell-to-cell contact. In addition to its role in cell adhesion and migration, CEA has been found to be overexpressed in a high percentage of human cancers, including 90% of gastrointestinal, colorectal and pancreatic cancers, 70% of non-small cell lung cancer cells and 50% of breast cancers. Overexpression of CEA has been shown to be associated with increased tumorigenicity and tumor invasiveness.

[0015] Claudin-18.2 is one of the two isoforms of Claudin-18, a major component of tight junctions that regulate the permeability, barrier function, and polarity of the epithelial layer. In healthy tissue, Claudin-18.2 is expressed only in short-lived differentiated cells of the gastric mucosa. However, it is overexpressed in up to 80% of gastrointestinal adenocarcinomas and 60% of pancreatic tumors. In addition, its expression has been observed in different subsets of individuals with esophageal cancer, non-small cell lung cancer, ovarian cancer, colon cancer, and several forms of bile duct cancer. Summary of the invention

[0016] The present invention provides multi-specific binding proteins that bind to CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2 on cancer cells and to NKG2D receptors and CD16 receptors on natural killer cells. These proteins can engage more than one NK activation receptor and can block the binding of natural ligands to NKG2D. In certain embodiments, the protein 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.

[0017] Therefore, one aspect of the present invention provides a protein comprising: a first antigen binding site that binds NKG2D; a second antigen binding site that binds CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2; and an antibody Fc domain sufficient to bind CD16, a portion thereof, or a third antigen binding site that binds 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.

[0018] In one embodiment, the first antigen binding site that binds to NKG2D comprises 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: 51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53) sequences of SEQ ID NO: 1. The heavy chain variable domain associated with SEQ ID NO: 1 can be coupled to various different 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 and 40. For example, the first antigen binding site comprises a heavy chain variable domain having an amino acid sequence 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 at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 40. 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 can 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:54), CDR2 (SEQ ID NO:55) and CDR3 (SEQ ID NO:56) sequences of SEQ ID NO:41.Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 42, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 57), CDR2 (SEQ ID NO: 58) and CDR3 (SEQ ID NO: 59) sequences of SEQ ID NO: 42. In other embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 43 and a light chain variable domain associated with SEQ ID NO: 44. For example, the heavy chain variable domain of the first 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:43, and / or comprise an amino acid sequence identical to CDR1 (SEQ ID NO:60), CDR2 (SEQ ID NO:61), and CDR3 (SEQ ID NO:62) sequences of SEQ ID NO:43. Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 44, 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: 44. In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 45 and a light chain variable domain associated with SEQ ID NO: 46. For example, the heavy chain variable domain of the first 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:45, 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:45.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:46, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:69), CDR2 (SEQ ID NO:70) and CDR3 (SEQ ID NO:71) sequences of SEQ ID NO:46.

[0019] Alternatively, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 47 and a light chain variable domain associated with SEQ ID NO: 48, 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: 47 and SEQ ID NO: 48, respectively. In another embodiment, 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 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: 49 and SEQ ID NO: 50, respectively.

[0020] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 132 and a light chain variable domain associated with SEQ ID NO: 133. 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: 132, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 134), CDR2 (SEQ ID NO: 135), and CDR3 (SEQ ID NO: 136) sequences of SEQ ID NO: 132. 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: 133, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 137), CDR2 (SEQ ID NO: 138) and CDR3 (SEQ ID NO: 139) sequences of SEQ ID NO: 133.

[0021] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 140 and a light chain variable domain associated with SEQ ID NO: 141. 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: 140, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 142), CDR2 (SEQ ID NO: 143), and CDR3 (SEQ ID NO: 144) sequences of SEQ ID NO: 140. 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: 141, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 145), CDR2 (SEQ ID NO: 146) and CDR3 (SEQ ID NO: 147) sequences of SEQ ID NO: 141.

[0022] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 148 and a light chain variable domain associated with SEQ ID NO: 149. 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: 148, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 150), CDR2 (SEQ ID NO: 151), and CDR3 (SEQ ID NO: 152) sequences of SEQ ID NO: 148. 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: 149, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 153), CDR2 (SEQ ID NO: 154) and CDR3 (SEQ ID NO: 155) sequences of SEQ ID NO: 149.

[0023] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 156 and a light chain variable domain associated with SEQ ID NO: 157. 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: 156, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 158), CDR2 (SEQ ID NO: 159), and CDR3 (SEQ ID NO: 160) sequences of SEQ ID NO: 156. 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: 157, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 161), CDR2 (SEQ ID NO: 162) and CDR3 (SEQ ID NO: 163) sequences of SEQ ID NO: 157.

[0024] In certain embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 164 and a light chain variable domain associated with SEQ ID NO: 165. 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: 164, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 166), CDR2 (SEQ ID NO: 167), and CDR3 (SEQ ID NO: 168) sequences of SEQ ID NO: 164. 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: 165, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 169), CDR2 (SEQ ID NO: 170) and CDR3 (SEQ ID NO: 171) sequences of SEQ ID NO: 165.

[0025] The second antigen binding site can bind to CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2. In certain embodiments, the second antigen binding site binds to CAIX and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form a CAIX binding site. For example, the second antigen binding site can comprise a heavy chain variable domain associated with SEQ ID NO: 72 and a light chain variable domain associated with SEQ ID NO: 73. 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: 72, 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: 72. 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:73, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:77), CDR2 (SEQ ID NO:78) and CDR3 (SEQ ID NO:79) sequences of SEQ ID NO:73.

[0026] Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 80 and a light chain variable domain associated with SEQ ID NO: 81. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 80, 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: 80. 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:81, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:85), CDR2 (SEQ ID NO:86) and CDR3 (SEQ ID NO:87) sequences of SEQ ID NO:81.

[0027] Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 88 and a light chain variable domain associated with SEQ ID NO: 89. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 88, 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: 88. 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:89, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:93), CDR2 (SEQ ID NO:94) and CDR3 (SEQ ID NO:95) sequences of SEQ ID NO:89.

[0028] In certain embodiments, the second antigen binding site binds to ANO1 and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form an ANO1 binding site. For example, the second antigen binding site can comprise various heavy chain variable domains associated with SEQ ID NO: 97 and light chain variable domains associated with SEQ ID NO: 98. 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: 97, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 99), CDR2 (SEQ ID NO: 100) and CDR3 (SEQ ID NO: 101) sequences of SEQ ID NO: 97. 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:98, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO:102), CDR2 (SEQ ID NO:103) and CDR3 (SEQ ID NO:104) sequences of SEQ ID NO:98.

[0029] In certain embodiments, the second antigen binding site binds to mesothelin and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form a mesothelin binding site. For example, the second antigen binding site can comprise various heavy chain variable domains associated with SEQ ID NO: 106 and light chain variable domains associated with SEQ ID NO: 107. 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: 106, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109) and CDR3 (SEQ ID NO: 110) sequences of SEQ ID NO: 106. Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 107, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 111), CDR2 (SEQ ID NO: 112) and CDR3 (SEQ ID NO: 113) sequences of SEQ ID NO: 107. Alternatively, the second antigen binding site may comprise various heavy chain variable domains associated with SEQ ID NO: 114 and light chain variable domains associated with SEQ ID NO: 115. 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: 114, 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: 115, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO: 120) and CDR3 (SEQ ID NO: 121) sequences of SEQ ID NO: 115.Alternatively, the second antigen binding site may comprise various heavy chain variable domains associated with SEQ ID NO: 122 and light chain variable domains associated with SEQ ID NO: 123. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 122, 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: 122. 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: 123, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 127), CDR2 (SEQ ID NO: 128) and CDR3 (SEQ ID NO: 129) sequences of SEQ ID NO: 123.

[0030] In certain embodiments, the second antigen binding site binds to TROP2 and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form a TROP2 binding site. For example, the second antigen binding site can comprise a heavy chain variable domain associated with SEQ ID NO: 172 and a light chain variable domain associated with SEQ ID NO: 173. 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: 172, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 174), CDR2 (SEQ ID NO: 175) and CDR3 (SEQ ID NO: 176) sequences of SEQ ID NO: 172. Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 173, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 177), CDR2 (SEQ ID NO: 178) and CDR3 (SEQ ID NO: 179) sequences of SEQ ID NO: 173. Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 180 and a light chain variable domain associated with SEQ ID NO: 181. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 180. and / or comprises an amino acid sequence identical to the CDR1 (SEQ ID NO: 182), CDR2 (SEQ ID NO: 183) and CDR3 (SEQ ID NO: 184) sequences of SEQ ID NO: 180. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 181, and / or comprises an amino acid sequence identical to the CDR1 (SEQ ID NO: 185), CDR2 (SEQ ID NO: 186) and CDR3 (SEQ ID NO: 187) sequences of SEQ ID NO: 181.Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 188 and a light chain variable domain associated with SEQ ID NO: 189. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 188, and the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 189. Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 190 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 may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 190, and the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 191. Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 192 and a light chain variable domain associated with SEQ ID NO: 193. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 192, and the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 193.

[0031] In certain embodiments, the second antigen binding site binds to CEA and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form a CEA binding site. For example, the second antigen binding site can comprise a heavy chain variable domain associated with SEQ ID NO: 195 and a light chain variable domain associated with SEQ ID NO: 196. 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: 197), CDR2 (SEQ ID NO: 198) and CDR3 (SEQ ID NO: 199) sequences of SEQ ID NO: 195. Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 196, 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: 196. Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 203 and a light chain variable domain associated with SEQ ID NO: 204. 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: 203, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 205), CDR2 (SEQ ID NO: 206) and CDR3 (SEQ ID NO: 207) sequences of SEQ ID NO: 203. Likewise, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 204, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 208), CDR2 (SEQ ID NO: 209) and CDR3 (SEQ ID NO: 210) sequences of SEQ ID NO: 204. Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO: 211 and a light chain variable domain associated with SEQ ID NO: 212.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: 211, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 213), CDR2 (SEQ ID NO: 214) and CDR3 (SEQ ID NO: 215) sequences of SEQ ID NO: 212. 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: 196, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 216), CDR2 (SEQ ID NO: 217) and CDR3 (SEQ ID NO: 218) sequences of SEQ ID NO: 212.

[0032] In certain embodiments, the second antigen binding site binds to Claudin-18.2 and comprises a heavy chain variable domain and a light chain variable domain that can be coupled to form a Claudin-18.2 binding site. For example, the second antigen binding site can comprise a heavy chain variable domain associated with SEQ ID NO: 220 and a light chain variable domain associated with SEQ ID NO: 221. 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: 220, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 222), CDR2 (SEQ ID NO: 223) and CDR3 (SEQ ID NO: 224) sequences of SEQ ID NO: 220. Likewise, 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: 221, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 225), CDR2 (SEQ ID NO: 226), and CDR3 (SEQ ID NO: 227) sequences of SEQ ID NO: 221. For example, the second antigen binding site can comprise a heavy chain variable domain associated with SEQ ID NO: 228 and a light chain variable domain associated with SEQ ID NO: 229. 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: 228, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 230), CDR2 (SEQ ID NO: 231), and CDR3 (SEQ ID NO: 232) sequences of SEQ ID NO: 228. 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: 229, and / or comprise an amino acid sequence identical to the CDR1 (SEQ ID NO: 233), CDR2 (SEQ ID NO: 234) and CDR3 (SEQ ID NO: 235) sequences of SEQ ID NO: 229.

[0033] In certain embodiments, the second antigen binding site comprises a light chain variable domain having 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.

[0034] 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 at positions 234-332 of a human IgG antibody.

[0035] Also provided are formulations containing one of these proteins, cells containing one or more nucleic acids expressing these proteins, and methods of using these proteins to enhance tumor cell death.

[0036] Another aspect of the present invention provides a method for treating cancer in a patient. The method comprises administering a therapeutically effective amount of a multispecific binding protein described herein to a patient in need. Cancers treated using a multispecific binding protein targeting CAIX include, for example, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, esophagus, lung, colon, kidney, cervical cancer, and non-small cell lung cancer. Cancers treated using a multispecific binding protein targeting mesothelin include, for example, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, bile duct cancer, gastric cancer, uterine serous carcinoma, thymic carcinoma, and acute myeloid leukemia. Cancers treated using a multispecific binding protein targeting ANO1 include, for example, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumors (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer, prostate cancer, and sarcoma. Cancers treated using a multispecific binding protein targeting TROP2 include, for example, breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian cancer. Cancers treated using multispecific binding proteins targeting CEA include, for example, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Cancers treated using multispecific binding proteins targeting claudin-18.2 include, for example, esophageal cancer, non-small cell lung cancer, ovarian cancer, colon cancer, and several forms of bile duct cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a diagram of a heterodimeric multispecific antibody. Each arm can represent a NKG2D binding domain or a tumor-associated antigen binding domain selected from CAIX, ANO1, mesothelin, TROP2, CEA, and claudin-18.2 binding domains. In certain embodiments, the NKG2D and tumor-associated antigen binding domains can share a common light chain.

[0038] Figure 2 is a schematic representation of a heterodimeric multispecific antibody. The NKG2D binding domain or any one of the tumor associated antigen binding domains selected from the group consisting of CAIX, ANO1, mesothelin, TROP2, CEA and claudin-18.2 binding domains may be in a scFv format (right arm).

[0039] Figure 3Is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

[0040] Figure 4 Is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D in an ELISA assay.

[0041] Figure 5 Is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to mouse recombinant NKG2D in an ELISA assay.

[0042] 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) times background.

[0043] 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) times background.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Fig.11 Is a bar graph showing activation of human NKG2D by NKG2D binding domain (listed as clones) by quantifying the percentage of TNF-α positive cells expressing human NKG2D-CD3ζ fusion protein.

[0048] Fig.12 Is a bar graph showing activation of mouse NKG2D by the NKG2D binding domain (listed as clones) by quantifying the percentage of TNF-α positive cells expressing the human NKG2D-CD3ζ fusion protein.

[0049] Fig.13 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0050] Fig.14 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0051] Fig.15 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0052] Fig.16 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0053] Fig.17 is a bar graph showing the cytotoxic effects of NKG2D binding domains (listed as clones) on tumor cells.

[0054] Fig.18 is a bar graph showing the melting temperatures of NKG2D binding domains (listed as clones) measured by differential scanning fluorimetry.

[0055] Fig.19 A-19C is a bar graph showing synergistic activation of NK cells using CD16 and NKG2D binding. Fig.19 A shows the levels of CD107a; Fig.19 B shows the levels of IFNγ; Fig.19 C shows the levels of CD107a and IFNγ. The graphs indicate mean values ​​(n=2)±SD. The data are representative of 5 independent experiments using 5 different healthy donors.

[0056] Fig. 20 : is a diagram of TriNKET in bispecific antibody (Triomab) form, 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 form can be a heterodimeric construct containing 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0057] Fig.21is a diagram of TriNKET in KiH common light chain (LC) format, which includes knob-in-hole (KIH) technology. KiH is a heterodimer containing two Fabs that bind to targets 1 and 2 and a FC stabilized by heterodimerization mutations. TriNKET in KiH format can be a heterodimeric construct with two Fabs that bind to targets 1 and 2, containing two different heavy chains and one common light chain that pairs with the two heavy chains.

[0058] Fig. 22 DVD-Ig TM ) type TriNKET, 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 targeting Antigen 1. The construct contains a normal Fc.

[0059] Fig.23 TriNKET adopts the orthogonal Fab interface (Ortho-Fab) type, which is a heterodimeric construct containing two Fabs binding to target 1 and target 2 fused to Fc. LC-HC pairing is ensured by the orthogonal interface. Heterodimerization Heterodimerization is ensured by mutations in Fc.

[0060] Fig.24 This is a diagram of TriNKET in a two-in-one Ig format.

[0061] Fig.25 is a schematic representation of TriNKET in ES format, which is a heterodimeric construct containing two different Fabs fused to Fc that bind to target 1 and target 2. Heterodimerization is ensured by electrostatic steering mutations in Fc.

[0062] Fig.26 is a diagram of TriNKET in Fab arm exchange format: 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 generate a bispecific antibody. The Fab arm exchange format (cFae) is a heterodimer containing two Fabs binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

[0063] Fig. 27 is a schematic representation of TriNKET in a SEED format, which is a heterodimer containing two Fabs binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

[0064] Fig.28is a schematic representation of TriNKET in the LuZ-Y format, where 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 Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

[0065] Fig.29 This is an illustration of TriNKET using the Cov-X model.

[0066] Fig.30 A-30B is a schematic representation of TriNKET adopting the Kλ body type, which is a heterodimeric construct with 2 different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains a κ LC, while the second Fab targeting antigen 2 contains a λ LC. Fig.30 A is an exemplary diagram of a type of Kλ body; Fig.30 B is an exemplary illustration of another Kλ body.

[0067] Fig.31 is an Oasc-Fab heterodimer construct comprising a Fab binding to target 1 and a scFab binding to target 2 fused to Fc. Heterodimerization is ensured by mutations in Fc.

[0068] Fig.32 DuetMab is a Fab containing two different Fabs that bind to antigens 1 and 2 and a F C Heterodimer construct of Fab 1 and 2. Fab 1 and 2 contain different SS bridges, which ensure the correct light chain (LC) and heavy chain (HC) pairing.

[0069] Fig.33 is a CrossmAb, which is a heterodimeric construct with 2 different Fabs 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, while CL is fused inline with VH.

[0070] Fig.34 is Fit-Ig, which is a homodimeric construct in which a Fab that binds to Antigen 2 is fused to the N-terminus of the HC of a Fab that binds to Antigen 1. The construct contains a wild-type Fc.

[0071] Fig.35 Binding of TriNKET comprising a CAIX binding domain and a NKG2D binding domain to NKG2D expressed on EL4 cells is shown.

[0072] Fig.36Binding of TriNKET comprising a CAIX binding domain and a NKG2D binding domain to CAIX expressed on human renal cell carcinoma cells A498 is shown.

[0073] Fig.37 A shows TriNKET-mediated cytotoxicity of KHYG-1 effector cells against CAIX-positive renal cell carcinoma A498 cells. The dashed line indicates background killing of A498 cells during co-culture with quiescent KHYG-1 cells without the addition of TriNKET. Fig.37 B shows TriNKET-mediated cytotoxicity of resting NK cells against CAIX-positive renal cell carcinoma A498 cells.

[0074] Detailed Description

[0075] The present invention provides multispecific binding proteins that bind to CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2 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 therapeutic methods for treating cancer. 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.

[0076] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0077] When used in this article, no specific number of references means "one or more", and includes plural references unless the context is inappropriate. 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 amino acid residues in 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 can be present in an intact antibody, an antigen binding fragment of an antibody that retains the antigen binding surface, or a recombinant polypeptide such as a scFv, which uses a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0078] 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.

[0079] 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.

[0080] 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 formulation 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.

[0081] 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.

[0082] 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] .

[0083] 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.

[0084] 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.

[0085] 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 1-4The anion of the compound of the present invention which is combined with an alkyl group or the like.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] I. Protein

[0090] The present invention provides a multispecific binding protein that binds to CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2 on cancer cells and NKG2D receptors and CD16 receptors on natural killer cells to activate the natural killer cells. The multispecific binding protein is useful in the pharmaceutical compositions and treatment methods described herein. The binding of the multispecific binding protein to the NKG2D receptor and CD16 receptor on natural killer cells enhances the activity of the natural killer cells in destroying cancer cells. The binding of the multispecific binding protein to CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2 on cancer cells brings the cancer cells to 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 exemplary multispecific binding proteins are provided below.

[0091] 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 and MICA from binding to NKG2D and activating the NKG2D receptor.

[0092] The second component of the multispecific binding protein binds to cells expressing CAIX, ANO1, mesothelin, TROP2, CEA or tight junction protein-18.2. Cells expressing CAIX include, but are not limited to, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, esophagus, lung, colon, kidney, cervical cancer and non-small cell lung cancer. Cells expressing mesothelin include, but are not limited to, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, bile duct cancer, gastric cancer, uterine serous carcinoma, thymic carcinoma and acute myeloid leukemia. Cells expressing ANO1 include, but are not limited to, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumors (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer, prostate cancer and sarcoma. Cells expressing TROP2 include, but are not limited to, breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma and ovarian cancer. Cells expressing CEA include, but are not limited to, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Cells expressing claudin-18.2 include, but are not limited to, esophageal cancer, non-small cell lung cancer, ovarian cancer, colon cancer, and several forms of bile duct cancer.

[0093] 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.

[0094] 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 CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2. 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.

[0095] 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 to one of the tumor-associated antigens selected from CAIX, ANO1, mesothelin, TROP2, CEA, and tight junction protein-18.2. 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 one of the tumor-associated antigens selected from CAIX, ANO1, mesothelin, TROP2, CEA, and tight junction protein-18.2. The first Fc domain and the second Fc domain together are capable of binding to CD16 ( Figure 2 ).

[0096] One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the antigen binding site may be a single chain or disulfide stabilized variable region (scFv) or may form a tetravalent or trivalent molecule.

[0097] 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.

[0098] In certain embodiments, the multispecific binding protein is a KiH common light chain (LC) type, which involves a 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" (e.g., T366W in EU numbering) into one CH3 domain (CH3A) by replacing a small residue with a bulky residue. CH3ATo 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 CH3B The "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 a phage display library, J Mol Biol (1997) 270 (1): 26-35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction, J Mol Biol (2014) 426(9): 1947–57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K, et al., Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for Fc gammaRs, 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 make heterodimerization thermodynamically favorable, while the knob-knob and hole-hole interfaces are unfavorable for homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0099] In certain embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig TM) type, which combines the target binding domains of two monoclonal antibodies through a flexible, naturally occurring linker and results in a tetravalent IgG-like molecule.

[0100] 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 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.

[0101] 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 type, which is a heterodimeric construct containing two different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0102] In certain embodiments, the multispecific binding protein adopts a Kλ body form, which is a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1 targeting Antigen 1 contains a κ LC, while the second Fab targeting Antigen 2 contains a λ LC. Fig.30 A is an exemplary illustration of a type of Kλ body; Fig.30 B is an exemplary illustration of another Kλ body.

[0103] 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).

[0104] In certain embodiments, the multispecific binding protein adopts a SEED body shape (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 within the conserved CH3 domain. The SEED design allows the efficient generation 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).

[0105] In certain embodiments, the multispecific binding protein is of the LuZ-Y type, 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).

[0106] In certain embodiments, the multispecific binding protein adopts the Cov-X body type (in the bispecific CovX body, two different peptides are linked together using a branched azetidinone linker and fused to the scaffold antibody in a site-specific manner under mild conditions). The antibody scaffold provides 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).

[0107] In certain embodiments, the multispecific binding protein is a deOasc-Fab heterodimer that includes a Fab that binds to target 1 and a scFab that binds to target 2 fused to Fc. Heterodimerization is ensured by mutations in the Fc.

[0108] In certain embodiments, the multispecific binding protein is of the DuetMab type, which is a protein containing two different Fabs that bind to antigens 1 and 2 and a Fab that is stabilized by heterodimerization mutations. C Heterodimer construct of Fab 1 and 2. Fab 1 and 2 contain different SS bridges, which ensure correct LC and HC pairing.

[0109] In certain embodiments, the multispecific binding protein is of the CrossmAb type, which is a heterodimeric construct with two different Fabs 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.

[0110] In certain embodiments, the multispecific binding protein is in the form of Fit-Ig, which is a homodimeric construct in which a Fab that binds to Antigen 2 is fused to the N-terminus of the HC of a Fab that binds to Antigen 1. The construct contains a wild-type Fc.

[0111] 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.

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 47 can be paired with the light chain variable domain defined by SEQ ID NO: 48 to form an antigen binding site that can bind to NKG2D as shown in US 9,273,136.

[0125] SEQ ID NO: 47

[0126] QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS

[0127] SEQ ID NO: 48

[0128] QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[0129] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 49 can be paired with the light chain variable domain defined by SEQ ID NO: 50 to form an antigen binding site that can bind to NKG2D as shown in US 7,879,985.

[0130] SEQ ID NO: 49

[0131] QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS

[0132] SEQ ID NO: 50

[0133] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0134] Table 2 lists peptide sequences that, in combination, can bind to the heavy chain variable domain and light chain variable domain of CAIX.

[0135]

[0136]

[0137] Alternatively, novel antigen binding sites that bind to CAIX can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:96.

[0138] SEQ ID NO: 96

[0139] MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLVPVHPQRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLP TVEAPGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHW GAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTV FNQTVMLSAKQLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA

[0140] Table 3 lists peptide sequences that can bind to the heavy chain variable domain and light chain variable domain of ANO1 in combination.

[0141]

[0142] Alternatively, novel antigen binding sites that bind to ANO1 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:105.

[0143] SEQ ID NO: 105

[0144] MRVNEKYSTLPAEDRSVHIINICAIEDIGYLPSEGTLLNSLSVDPDAECKYGLYFRDGRRKVDYILVYHHKRPSGNRTLVRRVQHSDTPSGARSVKQDHPLPGKGASLDAGSGEPPMDYHEDDKRFRREEYEGNLLEAGLELERDEDTKIHGVGFVKIHAPWNVLCREAEFLKLKMPTKKMYHINETRGLLKKINSVLQKITDPIQPKVAEHRPQTMKRLSYPFSREKQHLFDLSDKDSFFDSKTRSTIVYEILKRTTCTKAKYSMGITSLLANGVYAAAYPLHDGDYNGENVEFNDRKLLYEEWARYGVFYKYQPIDLVRKYFGEKIGLYFAWLGVYTQMLIPASIVGIIVFLYGCATMDENIPSMEMCDQRHNITMCPLCDKTCSYWKMSSACATARASHLFDNPATVFFSVFMALWAATFMEHWKRKQMRLNYRWDLTGFEEEEEAVKDHPRAEYEARVLEKSLKESRNKEKRRIPEESTNKWKQRVK TAMAGVKLTDKVKLTWRDRFPAYLTNLVSIIFMIAVTFAIVLGVIIYRISMAAALAMNSSPSVRSNIRVTTVATAVIINLVVIILLDEVYGCIARWLTKIEVPKTEXFEERLIFKAFLLKFVNSYTPIFYVAFFKGRFVGRPGDYVYIFRSFRMEECAPGGCLMELCIQLSIIMLGKQLIQNNLFEIGIPKMKKLIRYLKQQSPPDHEECVKRKQRYEVDYNLEPFAGLTPEYMEMIIQFGFV TLFVASFPLAPLFALLNNIIEIRLDAKFVTELRRPVAVRAKDIGIWYNILRGIGKLAVIINAFVISFTSDFIPRLVYLYMYSKNGTMHGFVNHTLSSFVNSDFQNGTAPNDPLDLGYEVQICRYKDYREPPWSENKYDISKDFWAVLAARLAFVIVFQNLVMFMSDFVDWVIPDIPKDISQQIHKEKVLMVELFMREEQDKQQLLETWMEKERQKDEPPCNHHNTKACPDSLGSPAPSHAYHGGVL

[0145] Table 4 lists peptide sequences that, in combination, can bind to the heavy chain variable domain and the light chain variable domain of mesothelin.

[0146]

[0147]

[0148]

[0149] Alternatively, novel antigen binding sites that bind to mesothelin can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:130.

[0150] SEQ ID NO: 130

[0151] MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAP ERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESL IFYKKWELEEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQD LDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

[0152] Table 5 lists peptide sequences that can bind to the heavy chain variable domain and light chain variable domain of TROP2 in combination.

[0153]

[0154]

[0155]

[0156] Alternatively, novel antigen binding sites that bind to TROP2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:194.

[0157] SEQ ID NO: 194

[0158] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHR PTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL

[0159] Table 6 lists peptide sequences that, in combination, can bind to the heavy chain variable domain and light chain variable domain of CEA.

[0160]

[0161]

[0162] Alternatively, novel antigen binding sites that bind to CEA can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:219.

[0163] SEQ ID NO: 219

[0164] MESPSAPPHRWCIPWQRLLLTASLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDAT YLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQN TTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPE AQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI

[0165] Table 7 lists peptide sequences that can bind to the heavy chain variable domain and light chain variable domain of Claudin-18.2 in combination.

[0166]

[0167]

[0168] Alternatively, novel antigen binding sites that bind to Claudin-18.2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:236.

[0169] SEQ ID NO: 236

[0170] MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGL CAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[0171] 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 cDNA library, or mutations can be designed based on the known three-dimensional structure of the interaction.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] Alternatively, amino acid substitutions may be selected from the groups of substitutions shown in Table 8 below.

[0177] Table 8 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

[0178] Alternatively, amino acid substitutions may be selected from the groups of substitutions shown in Table 9 below.

[0179] Table 9 The first polypeptide The second polypeptide Group 1 K409W D399V / F405T Group 2 Y349S E357W Group 3 K360E Q347R Group 4 K360E / K409W Q347R / D399V / F405T Group 5 Q347E / K360E / K409W Q347R / D399V / F405T Group 6 Y349S / K409W E357W / D399V / F405T

[0180] Alternatively, amino acid substitutions may be selected from the groups of substitutions shown in Table 10 below.

[0181] Table 10 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

[0182] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 11.

[0183]

[0184] Alternatively, at least one amino acid substitution can be selected from the group of substitutions in Table 12 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.

[0185] Table 12 The first polypeptide The second polypeptide K392, K370, K409 or K439 D399, E356 or E357

[0186] Alternatively, at least one amino acid substitution can be selected from the group of substitutions in Table 13 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.

[0187] Table 13 The first polypeptide The second polypeptide D399, E356 or E357 K409, K439, K370 or K392

[0188] Alternatively, amino acid substitutions may be selected from the group of substitutions in Table 14 below.

[0189] Table 14 The first polypeptide The second polypeptide T350V, L351Y, F405A and Y407V T350V, T366L, K392L and T394W

[0190] 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.

[0191] The above-mentioned multispecific protein can be produced using recombinant DNA techniques known to those skilled in the art. For example, the first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector; the second nucleic acid sequence encoding the second immunoglobulin heavy chain can be cloned into a second expression vector; the third nucleic acid sequence encoding the immunoglobulin light chain can be cloned into a third expression vector; the first, second and third expression vectors can be stably transfected into a host cell together to produce the multimeric protein.

[0192] 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.

[0193] 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.

[0194] II. Characteristics of multispecific proteins

[0195] In certain embodiments, the multispecific binding proteins described herein comprising a NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 bind to cells expressing human NKG2D. In certain embodiments, the multispecific binding proteins bind to the tumor-associated antigens CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 at a level comparable to a monoclonal antibody having the same corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 binding domain. However, the multispecific proteins described herein may be more effective in reducing tumor growth and killing cancer cells expressing CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 than the corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 monoclonal antibodies.

[0196] In certain embodiments, the multispecific binding proteins described herein comprising a NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2 can activate primary human NK cells when cultured with tumor cells expressing the antigen CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2. NK cell activation is marked by increased CD107a degranulation and IFNγ cytokine production. In addition, compared to a monoclonal antibody comprising the same CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2 binding domain, the multispecific binding protein shows higher activation of human NK cells in the presence of tumor cells expressing the antigen. For example, compared to the monoclonal antibody trastuzumab, the multispecific binding protein shows higher activation of human NK cells in the presence of tumor cells expressing the antigen CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2.

[0197] In certain embodiments, the multispecific binding proteins described herein comprising a NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 can enhance the activity of resting and IL-2-activated human NK cells in the presence of tumor cells expressing the antigen CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2.

[0198] In certain embodiments, the multispecific binding proteins described herein comprising a NKG2D binding domain and a binding domain for a tumor-associated antigen CAIX, ANO1, mesothelin, TROP2, CEA, or claudin-18.2 can enhance the cytotoxic activity of resting and IL-2 activated human NK cells in the presence of tumor cells expressing the antigen CAIX, ANO1, mesothelin, TROP2, CEA, or claudin-18.2. In certain embodiments, the multispecific proteins can provide advantages against tumor cells expressing moderate and low CAIX, ANO1, mesothelin, TROP2, CEA, or claudin-18.2 compared to the corresponding monoclonal antibodies.

[0199] In certain embodiments, the multispecific proteins described herein may be advantageous in the treatment of cancers with high expression of Fc receptors (FcRs) or cancers residing in a tumor microenvironment with high levels of FcRs, compared to corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or tight junction protein-18.2 monoclonal antibodies. Monoclonal antibodies exert their effects on tumor growth through a variety of mechanisms including ADCC, CDC, phagocytosis, and signal blocking. Among FcγRs, CD16 has the lowest affinity for IgG Fc; FcγRI (CD64) is a high-affinity FcR that binds to IgG Fc about 1000 times more strongly than CD16. CD64 is typically expressed on many hematopoietic lineages, such as myeloid lineages, and may be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML) cells. Immune cells such as MDSCs and monocytes that infiltrate the tumor also express CD64 and are known to infiltrate the tumor microenvironment. The expression of CD64 by the tumor or in the tumor microenvironment may have a deleterious effect on monoclonal antibody therapy. The expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to engage CD16 on the surface of NK cells because the antibody preferentially engages the high-affinity receptor. The multispecific protein can overcome the deleterious effects of CD64 expression (on tumors or tumor microenvironments) on monoclonal antibody therapy by targeting two activating receptors on the surface of NK cells. Regardless of whether CD64 is expressed on the tumor cells, the multispecific protein can mediate human NK cell responses for all tumor cells because the dual targeting of the two activating receptors on NK cells provides a stronger specific binding to NK cells.

[0200] In certain embodiments, the multispecific proteins described herein can provide better safety by reducing on-target off-tumor side effects. Both natural killer cells and CD8 T cells can directly lyse tumor cells, although the mechanisms by which NK cells and CD8 T cells recognize normal self cells and tumor cells are different. The activity of NK cells is regulated by the balance of signals from activation (NCR, NKG2D, CD16, etc.) and inhibition (KIR, NKG2A, etc.) receptors. The balance of these activation and inhibition signals allows NK cells to determine whether they are healthy self cells or stressed, virally infected or transformed self cells. This "built-in" mechanism of self-tolerance will help protect normal healthy tissues from NK cell responses. To expand this principle, self-tolerance of NK cells will allow TriNKET to target antigens expressed on both autologous and tumors without deviating from tumor side effects, or with an increased therapeutic window. Unlike natural killer cells, T cells need to recognize specific peptides presented by MHC molecules to activate and exert effector functions. T cells have been used as the main target of immunotherapy, and many strategies have been developed to redirect T cell responses against tumors. T cell bispecific substances, checkpoint inhibitors, and CAR-T cells have all been approved by the FDA, but they are often suffering from dose-limiting toxicity. T cell bispecific substances and CAR-T cells work around the TCR-MHC recognition system by using binding domains to target antigens on the surface of tumor cells and using engineered signaling domains to conduct activation signals into effector cells. Although effective in eliciting anti-tumor immune responses, these therapies are often accompanied by cytokine release syndrome (CRS) and tumor side effects that deviate from the target. In this context, the multispecific proteins are unique because they do not "rewrite" the natural system of NK cell activation and inhibition. Instead, the multispecific proteins are designed to shake the balance and provide additional activation signals to NK cells while maintaining NK tolerance to healthy autologous cells.

[0201] In certain embodiments, the multispecific proteins described herein are more effective in delaying tumor progression than corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 monoclonal antibodies comprising the same antigen binding domain. In certain embodiments, the multispecific proteins described herein are more effective in combating cancer metastasis than corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 monoclonal antibodies comprising the same antigen binding domain.

[0202] III. Therapeutic Applications

[0203] 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 different cancers expressing CAIX, ANO1, mesothelin, TROP2, CEA or claudin-18.2, comprising administering to a patient in need thereof a therapeutically effective amount of the multispecific binding protein described herein.

[0204] The treatment method can be characterized according to the cancer to be treated. Cancers treated using a multispecific binding protein targeting CAIX include, for example, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, esophagus, lung, colon, kidney, cervical cancer, and non-small cell lung cancer. Cancers treated using a multispecific binding protein targeting mesothelin include, for example, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, bile duct cancer, gastric cancer, uterine serous carcinoma, thymic carcinoma, and acute myeloid leukemia. Cancers treated using a multispecific binding protein targeting ANO1 include, for example, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumors (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer, prostate cancer, and sarcoma. Cancers treated using a multispecific binding protein targeting TROP2 include, for example, breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian cancer. Cancers treated using multispecific binding proteins targeting CEA include, for example, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Cancers treated using multispecific binding proteins targeting claudin-18.2 include, for example, esophageal cancer, non-small cell lung cancer, ovarian cancer, colon cancer, and several forms of bile duct cancer.

[0205] In certain other embodiments, the cancer is brain cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer. In other embodiments, the cancer is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, 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, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, islet cell tumor, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, 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 cancer, oral cancer, osteosarcoma, serous papillary adenocarcinoma, vaginal Stem 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, urethrobladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0206] 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.

[0207] The cancer to be treated can be characterized by the presence of specific antigens expressed on the surface of the cancer cells. In certain embodiments, the cancer cells can express one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1.

[0208] IV. Combination Therapy

[0209] Another aspect of the invention provides combination therapies. The multispecific binding proteins described herein are used in combination with other therapeutic agents to treat cancer.

[0210] 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, sobuzosine, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesanaline, aminoglutethimide, amsacrine, proglumide, elixirsium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozotocin, nimustine, vindesine, , 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, colony stimulating factor-1, colony stimulating factor-2, denileukin, interleukin-2, luteinizing hormone releasing factor and combinations of the foregoing agents may exhibit differential binding to cognate receptors and variations in increased or decreased serum half-lives.

[0211] 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.

[0212] 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).

[0213] 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.

[0214] The proteins of the invention may also be used as an adjunct to surgical removal of primary lesions.

[0215] 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, or vice versa.

[0216] V. Pharmaceutical Compositions

[0217] 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 drug delivery systems. For suitable formulations, one or more physiologically acceptable excipients or carriers can also be included in the compositions. Formulations suitable for use in the present disclosure are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249: 1527-1533, 1990).

[0218] The intravenous drug delivery preparation 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 preparation can be a freeze-dried preparation or a liquid preparation. In certain embodiments, the preparation can be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the preparation can be freeze-dried, and the freeze-dried preparation of 45mg can be contained in a vial. In certain embodiments, the freeze-dried preparation of about 40mg-about 100mg can be contained in a vial. In certain embodiments, the freeze-dried preparations from 12, 27 or 45 vials are merged to obtain the therapeutic dose of the protein in the intravenous drug preparation. In certain embodiments, the preparation can be a liquid preparation and stored at about 250mg / vial to about 1000mg / vial. In certain embodiments, the preparation can be a liquid preparation and stored at about 600mg / vial. In certain embodiments, the preparation can be a liquid preparation and stored at about 250mg / vial.

[0219] The present disclosure may be in the form of an aqueous liquid pharmaceutical formulation comprising a therapeutically effective amount of the protein in a buffered solution forming the formulation.

[0220] 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 use. The pH of the preparation is usually 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 solid-type composition obtained can be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned one or more medicaments. The solid-type composition can also be packaged in a container with a flexible amount.

[0221] In certain embodiments, the present disclosure provides formulations 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.

[0222] In certain embodiments, an aqueous formulation is prepared comprising 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 pH values ​​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.

[0223] In certain embodiments, the formulation 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 formulation is adjusted with sodium hydroxide.

[0224] A polyol that acts as an isotonic agent and can stabilize an antibody may also be included in the formulation. The polyol is added to the formulation in an amount that may vary according to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation 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 may be used as an isotonic agent in the formulation 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 formulation.

[0225] Detergents or surfactants may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulate matter in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include polysorbates as surfactants. In certain embodiments, the formulation 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 formulation may contain polysorbate 80 between about 0.1 mg / mL and about 10 mg / mL or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0226] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation 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 formulation can be diluted with a 0.9% saline solution.

[0227] In certain embodiments, liquid preparations of the present disclosure can be prepared into a solution with a concentration of 10mg / mL, and combined with the sugar of the stabilizer level. In certain embodiments, the liquid preparation can be prepared in an aqueous carrier. In certain embodiments, stabilizers can be added with 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 preparation can also include one or more buffers, surfactants and preservatives.

[0228] In certain embodiments, the pH of the liquid formulation 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.

[0229] 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.

[0230] In certain embodiments, the liquid formulations of the present disclosure may be stored under pH and humidity conditions that prevent deamidation of the protein product.

[0231] 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 formulations. Illustrative 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.

[0232] Preservatives may optionally be added to the formulations of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multi-dose) formulations.

[0233] In certain circumstances, such as when a patient receives all medications by IV route in a hospital after a transplant, an intravenous (IV) formulation may be the preferred route of administration. In certain embodiments, the liquid formulation 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.

[0234] 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.

[0235] Preservatives may optionally be added to the formulations of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multi-dose) formulations.

[0236] 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 formulations. Illustrative 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.

[0237] The present disclosure may be in the form of a freeze-dried formulation comprising 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 formulation may also include one or more buffers, surfactants, bulking agents and / or preservatives.

[0238] 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.

[0239] In certain embodiments, the pH of the formulation 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.

[0240] 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.

[0241] 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.

[0242] 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). Illustrative bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain these bulking agents.

[0243] Preservatives may optionally be added to the formulations of the invention to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multi-dose) formulations.

[0244] 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 formulation after freeze drying. Illustrative 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.

[0245] 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.

[0246] 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).

[0247] 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.

[0248] 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 body 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 in a patient can be measured 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).

[0249] 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.

[0250] 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.

[0251] 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

[0252] The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0253] Example 1 - Binding of the NKG2D binding domain to NKG2D

[0254] NKG2D binding domain binds to purified recombinant NKG2D

[0255] The nucleic acid sequence of the extracellular domain (ectodomains) of human, mouse or cynomolgus monkey NKG2DNKG2D 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, an isotype control, or a positive control (selected from SEQ ID NOs: 47-50, or anti-mouse NKG2D antibody clones MI-6 and CX-5 available at eBioscience) was added.

[0256] 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.

[0257] NKG2D binding domain binds to cells expressing NKG2D

[0258] 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 background multiple (FOB) was calculated using a comparison of the mean fluorescence intensity (MFI) of cells expressing NKG2D to that of parent EL4 cells.

[0259] The NKG2D binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. The positive control antibody (selected from SEQ ID NOs: 45-48, 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.

[0260] Example 2 - NKG2D binding domain blocks natural ligand binding to NKG2D

[0261] Competition with ULBP-6

[0262] 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 450nm and corrected at 540nm. 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 blocked from binding to the NKG2D-Fc protein in the wells. Positive control antibodies (selected from SEQ ID NOs: 47-50) and various NKG2D binding domains blocked ULBP-6 binding to NKG2D, while isotype controls showed little competition with ULBP-6 ( Figure 8 ).

[0263] The ULBP-6 sequence is represented by SEQ ID NO: 131

[0264] MAAAAIPALLLCLPLLFLLFGWSRARRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARM SCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSGTTQLRATATTLILCCLLIILPCFILPGI(SEQ ID NO:131)

[0265] Competition with MICA

[0266] 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 (selected from SEQ ID NOs: 47-50) and various NKG2D binding domains blocked MICA binding to NKG2D, while the isotype control showed little competition with MICA ( Fig. 9 ).

[0267] Competition with Rae-1δ

[0268] 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. Positive controls (selected from SEQ ID NOs: 47-50, 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 ).

[0269] Example 3 - NKG2D binding domain cloning Activation of NKG2D

[0270] 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.

[0271] 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.

[0272] Example 4 - NKG2D binding domain activates NK cells

[0273] Primary human NK cells

[0274] 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 the cells to assess NK cell activation. An increase in CD107a / IFN-γ double positive cells indicates better NK cell activation by engaging two activating receptors rather than one receptor. The NKG2D binding domain and positive control (selected from SEQ ID NOs: 47-50) showed changes to CD107a compared to the isotype control.+ 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).

[0275] Primary mouse NK cells

[0276] 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 engaging 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).

[0277] Example 5 - NKG2D binding domain is capable of inducing cytotoxicity against target tumor cells

[0278] 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 region (NKG2D binding domain) served as the other 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 labeled THP-1 cells were then combined with NKG2D antibodies and isolated mouse NK cells in a microplate well and incubated at 37°C for 3 hours. 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. 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.

[0279] The positive control ULBP-6, a 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 ).

[0280] Example 6 - NKG2D antibodies show high thermal stability

[0281] The melting temperature of the NKG2D binding domain was determined using differential scanning fluorimetry. The extrapolated apparent melting temperature was higher than that of a typical IgG1 antibody ( Fig.18 ).

[0282] Example 7 - Synergistic activation of human NK cells by cross-linking NKG2D and CD16 Primary human NK cell activation assay Law

[0283] 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 and 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.

[0284] To investigate the relative potency of receptor combinations, crosslinking of NKG2D or CD16 and co-crosslinking of both receptors were performed by plate-bound stimulation. Fig.19 ( Fig.19 As shown in A-19C), combined stimulation of CD16 and NKG2D resulted in greatly increased CD107a levels (degranulation) ( Fig.19 A) and / or IFN-γ production ( Fig.19 B) Dashed lines indicate the additive effect of individual stimulation of each receptor.

[0285] 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.19 A shows the levels of CD107a; Fig.19B shows the levels of IFNγ; Fig.19 C shows the levels of CD107a and IFNγ. Fig.19 The data shown in A-19C are representative of 5 independent experiments using 5 different healthy donors.

[0286] Example 8 - Binding of multispecific binding proteins to NKG2D

[0287] The EL4 mouse lymphoma cell line was engineered to express human NKG2D. Figure 1 The affinity of a trispecific binding protein (TriNKET) each containing a NKG2D binding domain, a tumor-associated antigen binding domain (CAIX binding domain) and an Fc domain bound to CD16 to extracellular NKG2D expressed on EL4 cells is shown in . The binding of the multispecific binding protein to NKG2D is detected using a fluorophore-coupled anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and the mean fluorescence intensity (MFI) of cells expressing NKG2D was compared with that of parent EL4 cells to calculate the background multiple (FOB).

[0288] The TriNKETs tested included A44-TriNKET-CAIX (ADI-27744 and CAIX binding domains), F63-TriNKET-CAIX (ADI-29404 and CD33 binding domains), E79-TriNKET-CAIX (ADI-29379 and CAIX binding domains), F47-TriNKET-CAIX (ADI-29447 and CAIX binding domains), and A49-TriNKET-CAIX (ADI-27749 and CAIX binding domains). The CAIX binding domain consists of the heavy chain variable domain of SEQ ID NO: 80 and the light chain variable domain of SEQ ID NO: 81.

[0289] Fig.35 It was shown that TriNKETs, each including a CAIX binding domain and a different NKG2D binding domain, bind to NKG2D expressed on EL4 cells.

[0290] Example 9 - Binding of Multispecific Binding Proteins to Human Tumor Antigens

[0291] Trispecific binding protein binds to CAIX

[0292] The binding of TriNKET to the tumor-associated antigen CAIX was determined using the human renal cell carcinoma cell line A498 expressing CAIX. TriNKET and optionally the parent anti-CAIX monoclonal antibody were incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and the background multiple (FOB) was calculated using the mean fluorescence intensity (MFI) from TriNKET and the parent monoclonal anti-CAIX antibody normalized to the secondary antibody control. TriNKET targeting CAIX showed comparable levels of binding to CAIX on A498 cells compared to the parent anti-CAIX antibody ( Fig.36 ). The overall binding signals are comparable between TriNKETs because they contain the same CAIX binding domain.

[0293] Example 10 - Trispecific binding proteins are able to induce cytotoxicity against target cancer cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ), the purity of the isolated NK cells is usually>90%. The isolated NK cells were cultured in a medium containing 100ng / mL IL-2 for activation or rested overnight in the absence of cytokines. IL-2 activated or resting NK cells were used for cytotoxicity assays the next day.

[0294] The NK / NK T-cell line KHYG-1 cells were in 10% HI-FBS-RPMI-1640 containing 10 ng / mL IL-2. One day before use for effector cells, KHYG-1 cells were harvested from the culture and the cells were washed off the medium containing IL-2. After washing, KHYG-1 cells were resuspended in 10% HI-FBS-RPMI-1640 and rested overnight without cytokines.

[0295] DELFIA Cytotoxicity Assay:

[0296] Human A498 cancer cells expressing CAIX were harvested from culture, washed with PBS and incubated for 10 min. 6 / mL resuspended in growth medium for labeling with BATDA reagent (PerkinElmer AD0116). The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed 3x with PBS and lysed at 0.5-1.0x10 5 / mL resuspended in culture medium. To prepare background wells, place an aliquot of labeled cells and centrifuge the cells from the culture medium. Carefully add 100μl of the culture medium to the wells in triplicate to avoid disturbing the deposited cells. Add 100μl of BATDA-labeled cells to each well of a 96-well plate. Reserve wells for spontaneous release from target cells and prepare wells for maximum lysis of target cells by adding 1% Triton-X. Dilute TriNKET for the tumor target of interest in culture medium and add 50μl of diluted TriNKET to each well. Harvest resting NK cells, activated NK cells, or KHYG-1 cells from the culture, wash the cells, and add 10 to 10 cells at the desired ratio of effector cells to target cells (E:T). 5 -2.0x10 6 / mL resuspended in culture medium. Add 50 μl of NK cells to each well to make a total culture volume of 200 μl. Incubate the plate at 37°C and 5% CO2 for 2-3 hours before assay development.

[0297] After culturing for 2-3 hours, the plate is taken out from the incubator and the cells are deposited by centrifugation at 200g for 5 minutes. 20 μl of culture supernatant is transferred to a clean microplate provided by the manufacturer, and 200 μl of europium solution at room temperature is added to each well. The plate is protected from light and incubated at 250rpm for 15 minutes on a plate shaker. Victor 3 or SpectraMax i3X instrument is used to read the plate. The % of specific lysis is calculated as follows: % of specific lysis=((experimental release-spontaneous release) / (maximum release-spontaneous release))*100%.

[0298] TriNKET mediates human NK cell cytotoxicity against CAIX-positive human cancer cells. Co-culture of resting KHYG-1 cells with A498 target cells resulted in ~38% specific tumor lysis (by Fig.37 The addition of three different TriNKETs targeting CAIX (A49-TriNKET-CAIX, E79-TriNKET-CAIX, and A44-TriNKET-CAIX) to the co-culture increased the specific lysis of tumor cells by approximately 20% for all three NKG2D binding domains ( Fig.37 A).

[0299] Resting human NK cells were mixed with A498 cancer cell cells. In the absence of added CAIX mAb or TriNKET, resting human NK cells had no cytotoxic activity against A498 cells. A monoclonal antibody against CAIX (BAY79-4620 variable domain with hIgG1 as mAb (homodimer)) was able to increase specific lysis by about 9%. TriNKET (e.g., A49-TriNKET-CAIX, E79-TriNKET-CAIX, and A44-TriNKET-CAIX) increased the cytotoxic activity of resting human NK cells against the cancer cells compared to the monoclonal antibody ( Fig.37 B).

[0300] Incorporated by Reference

[0301] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0302] Equivalence

[0303] 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) a first antigen binding site comprising a fab region comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of an anti-NKG2D antibody that activates NKG2D; (b) a second antigen binding site comprising a single chain variable fragment (scFv), wherein the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) of an anti-CEA antibody; (c) a first polypeptide that is an antibody Fc domain or a portion thereof; and (d) a second polypeptide of the antibody Fc domain or a portion thereof; wherein the first polypeptide of the antibody Fc domain or a portion thereof and the second polypeptide of the antibody Fc domain or a portion thereof jointly bind to CD16; wherein the fab region of the anti-NKG2D antibody is fused to the N-terminus of a first polypeptide of the antibody Fc domain or a portion thereof, and the scFv of the anti-CEA antibody is fused to the N-terminus of a second polypeptide of the antibody Fc domain or a portion thereof; and The binding of NKG2D and CD16 on NK cells results in synergistic activation of NK cells, and the synergistic activation is determined by increased expression of one or both of CD107a and IFNγ.

2. The protein of claim 1, wherein the first antigen binding site binds to NKG2D in humans and non-human primates.

3. The protein of claim 1, wherein the VH and VL of the anti-NKG2D antibody are present on the same polypeptide. The protein of claim 1 , wherein the VH and VL of the anti-CEA antibody are present on the same polypeptide. 5 . The protein of claim 1 , wherein the VL of the anti-NKG2D antibody has an amino acid sequence identical to that of the VL of the anti-CEA antibody.

6. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 140, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

141.

7. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

2.

8. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:41, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

42.

9. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:45, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

46.

10. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 132, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

133.

11. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 148, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

149.

12. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 156, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

157.

13. The protein of claim 1, wherein the VH of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 164, and the VL of the anti-NKG2D antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO:

165.

14. The protein of claim 1, wherein the second antigen binding site comprises: (a) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 197; the heavy chain CDR2 amino acid sequence of SEQ ID NO: 198; the heavy chain CDR3 amino acid sequence of SEQ ID NO: 199; the light chain CDR1 amino acid sequence of SEQ ID NO: 200; The light chain CDR2 amino acid sequence of SEQ ID NO: 201; and the light chain CDR3 amino acid sequence of SEQ ID NO:202; (b) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 205; the heavy chain CDR2 amino acid sequence of SEQ ID NO: 206; the heavy chain CDR3 amino acid sequence of SEQ ID NO: 207; the light chain CDR1 amino acid sequence of SEQ ID NO: 208; The light chain CDR2 amino acid sequence of SEQ ID NO: 209; and The light chain CDR3 amino acid sequence of SEQ ID NO: 210; or (c) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 213; the heavy chain CDR2 amino acid sequence of SEQ ID NO: 214; the heavy chain CDR3 amino acid sequence of SEQ ID NO:215; The light chain CDR1 amino acid sequence of SEQ ID NO: 216; The light chain CDR2 amino acid sequence of SEQ ID NO: 217; and The light chain CDR3 amino acid sequence of SEQ ID NO:

218.

15. The protein of claim 1, wherein the VH of the anti-CEA antibody is at least 90% identical to SEQ ID NO: 195, and the VL of the anti-CEA antibody is at least 90% identical to SEQ ID NO:

196.

16. The protein of claim 1, wherein the VH of the anti-CEA antibody is at least 90% identical to SEQ ID NO: 203, and the VL of the anti-CEA antibody is at least 90% identical to SEQ ID NO:

204.

17. The protein of claim 1, wherein the VH of the anti-CEA antibody is at least 90% identical to SEQ ID NO: 211, and the VL of the anti-CEA antibody is at least 90% identical to SEQ ID NO:

212.

18. The protein of claim 1, wherein the antibody Fc domain or portion thereof comprises a hinge and a CH2 domain.

19. The protein of claim 18, wherein the antibody Fc domain or portion thereof comprises the hinge and CH2 domains of a human IgG1 antibody.

20. The protein of claim 18, wherein the Fc domain or portion thereof comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody.

21. The protein of claim 20, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, all numbered according to the EU index.

22. A formulation comprising the protein of any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

23. A cell comprising one or more nucleic acids encoding the protein of any one of claims 1-21.

24. Use of the protein according to any one of claims 1 to 21 in the preparation of a medicament for directly and / or indirectly enhancing tumor cell death, wherein the tumor cells express CEA and are selected from the group consisting of gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer and breast cancer cells.

25. The use according to claim 24, wherein the tumor cells are colorectal cancer cells.

26. Use of the protein of any one of claims 1 to 21 in the preparation of a medicament for treating a cancer expressing CEA, wherein the cancer is selected from the group consisting of gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer and breast cancer.

27. The use according to claim 26, wherein the cancer is colorectal cancer.

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