Proteins that bind to NKG2D, CD16, and fibroblast activation protein

Abstract

To provide multi-specific binding proteins that bind to the NKG2D receptor, CD16 receptor and fibroblast activation protein (FAP) and also provide pharmaceutical compositions and therapeutic methods of the multi-specific binding proteins useful for treatment of cancer, autoimmune disease or fibrosis.SOLUTION: The invention provides multi-specific binding proteins that bind to the NKG2D receptor and CD16 receptor on natural killer cells, and the tumor-associated antigen (FAP). Such proteins can engage two or more kinds of NK-activating receptor, and may block the binding of natural ligands to NKG2D.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 672,299, filed May 16, 2018.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on May 13, 2019, is named DFY_056WO_SL25.txt and is 121,670 bytes in size.

[0003] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16, and fibroblast activation protein (FAP). [Background technology]

[0004] Cancer remains a major health problem, despite considerable research efforts and scientific advances reported in the literature to treat this disease. 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. Current treatment options for these cancers may not be effective for all patients and / or have significant adverse side effects. Other types of cancer also remain difficult to treat using existing treatment options. Cancer-associated fibroblasts in cancer often promote malignancy and inhibit cancer therapy.

[0005] Cancer immunotherapy is desirable because it is highly specific and can use the patient's own immune system to facilitate 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 facilitate the destruction of tumor cells. Antibodies that bind to certain tumor-associated antigens, immune cells, and other cells in the tumor microenvironment (e.g., cancer-associated fibroblasts) have been described in the literature. See, for example, WO2016 / 134371 and WO2015 / 095412.

[0006] Natural killer (NK) cells are components of the innate immune system and comprise approximately 15% of circulating lymphocytes. NK cells were originally characterized by their ability to infiltrate virtually all tissues and effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells (i.e., via death receptor pathways in addition to cytolytic granules containing perforin and granzymes). Activated NK cells also secrete proinflammatory cytokines (e.g., IFN-γ and chemokines) that enhance the recruitment of other leukocytes to target tissues.

[0007] NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited through activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated via activating receptors (e.g., NKG2D, natural cytotoxicity receptors (NCRs), DNAX accessory molecule 1 (DNAM1)). NK cells are also activated by the constant regions of several immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0008] Fibroblast activation protein alpha (FAP) is a homodimeric integral membrane gelatinase belonging to the serine protease family. This protein is thought to be involved in the control of fibroblast proliferation or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. More than 90% of all human carcinomas have FAP expression on activated stromal fibroblasts. Stromal fibroblasts play an important role in carcinoma development, growth, and metastasis. FAP is also expressed in malignant cells of osteosarcoma and soft tissue sarcoma. The present invention offers particular advantages for improving the treatment of the above-mentioned cancers. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention

[0010] The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors and tumor associated antigens (FAPs) on natural killer cells. Such proteins can engage more than one type of NK activating receptor and can block binding of natural ligands to NKG2D. In certain embodiments, the proteins can agonize NK cells in humans. In some embodiments, the proteins can agonize NK cells in humans and other species, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in further detail below.

[0011] Thus, in certain embodiments, the invention provides proteins incorporating a first antigen-binding site that binds to NKG2D; a second antigen-binding site that binds to a FAP; and a sufficient antibody fragment crystallizable (Fc) domain, portion thereof, or a third antigen-binding site that binds to CD16.

[0012] The antigen-binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g. arranged as in an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may comprise a single domain antibody (such as a VFv antibody). H H antibodies, or V antibodies such as those found in cartilaginous fishes NAR The antibody may be a polypeptide such as a polypeptide having a specific structure.

[0013] In certain embodiments, the present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and FAPs on cancer cells. The NKG2D binding site may comprise a heavy chain variable domain at least 90% identical to an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:175, SEQ ID NO:179, SEQ ID NO:183, SEQ ID NO:187, and SEQ ID NO:93.

[0014] The first antigen binding site (which in some embodiments binds to NKG2D) may incorporate a heavy chain variable domain related to SEQ ID NO: 1, such as by 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 / or incorporating identical amino acid sequences to the sequences of CDR1 (SEQ ID NO: 105 or SEQ ID NO: 151), CDR2 (SEQ ID NO: 106), and CDR3 (SEQ ID NO: 107 or SEQ ID NO: 152) of SEQ ID NO: 1. The heavy chain variable domain related to SEQ ID NO: 1 may be coupled with a different light chain variable domain to form the NKG2D binding site. For example, a first antigen binding site incorporating a heavy chain variable domain related to SEQ ID NO:1 may further incorporate a light chain variable domain selected from any one of the sequences related to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40. For example, the first antigen-binding site incorporates a heavy chain variable domain with 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 with 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 the sequences selected from SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40.

[0015] Alternatively, in certain embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 41 and a light chain variable domain related to SEQ ID NO: 42. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 41 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 43 or SEQ ID NO: 153), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45 or SEQ ID NO: 154) of SEQ ID NO: 41. 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:42 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:46), CDR2 (SEQ ID NO:47), and CDR3 (SEQ ID NO:48) of SEQ ID NO:42.

[0016] In certain embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 49 and a light chain variable domain related to SEQ ID NO: 50. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 49 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 51 or SEQ ID NO: 155), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 53 or SEQ ID NO: 156) of SEQ ID NO: 49. 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:50 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:55), and CDR3 (SEQ ID NO:56) of SEQ ID NO:50.

[0017] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO:57 and a light chain variable domain related to SEQ ID NO:58, such as by having amino acid sequences at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:57 and at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:58, respectively. In another embodiment, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO:59 and a light chain variable domain related to SEQ ID NO:60. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 59 and / or incorporates identical amino acid sequences to the CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109) and CDR3 (SEQ ID NO: 110) sequences of SEQ ID NO: 59. 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: 60 and / or incorporates identical amino acid sequences to the CDR1 (SEQ ID NO: 111), CDR2 (SEQ ID NO: 112) and CDR3 (SEQ ID NO: 113) sequences of SEQ ID NO: 60.

[0018] In some embodiments, the first antigen binding site may incorporate a heavy chain variable domain related to SEQ ID NO:101 and a light chain variable domain related to SEQ ID NO:102, such as by having amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:101 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:102, respectively. In some embodiments, the first antigen binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 103 and a light chain variable domain related to SEQ ID NO: 104, such as by having amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 103 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 104, respectively.

[0019] The first antigen-binding site (which in some embodiments binds to NKG2D) may incorporate a heavy chain variable domain related to SEQ ID NO: 61 and a light chain variable domain related to SEQ ID NO: 62. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 61 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 63 or SEQ ID NO: 157), CDR2 (SEQ ID NO: 64), and CDR3 (SEQ ID NO: 65 or SEQ ID NO: 158) of SEQ ID NO: 61. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 62 and / or incorporates amino acid sequences identical to the CDR1 (SEQ ID NO: 66), CDR2 (SEQ ID NO: 67), and CDR3 (SEQ ID NO: 68) sequences of SEQ ID NO: 62. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 69 and a light chain variable domain related to SEQ ID NO: 70. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 69 and / or incorporates identical amino acid sequences to the CDR1 (SEQ ID NO: 71 or SEQ ID NO: 159), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73 or SEQ ID NO: 160) sequences of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 70 and / or incorporates identical amino acid sequences to the CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76) sequences of SEQ ID NO: 70.

[0020] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 77 and a light chain variable domain related to SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 79 or SEQ ID NO: 161), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81 or SEQ ID NO: 162) of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:78 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83), and CDR3 (SEQ ID NO:84) of SEQ ID NO:78.

[0021] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 85 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 163), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89 or SEQ ID NO: 164) of SEQ ID NO: 85. 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:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0022] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 167 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 167 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 168), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 169 or SEQ ID NO: 170) of SEQ ID NO: 167. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0023] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 171 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 171 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 172), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 173 or SEQ ID NO: 174) of SEQ ID NO: 171. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0024] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 175 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 175 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 176), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 177 or SEQ ID NO: 178) of SEQ ID NO: 175. 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:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0025] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 179 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 179 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 180), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 181 or SEQ ID NO: 182) of SEQ ID NO: 179. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0026] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 183 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 183 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 184), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 185 or SEQ ID NO: 186) of SEQ ID NO: 183. 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:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0027] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 187 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 187 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 87 or SEQ ID NO: 188), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 189 or SEQ ID NO: 190) of SEQ ID NO: 187. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) of SEQ ID NO:86.

[0028] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 93 and a light chain variable domain related to SEQ ID NO: 94. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 93 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 95 or SEQ ID NO: 165), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 97 or SEQ ID NO: 166) of SEQ ID NO: 93. 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:94 and / or incorporates amino acid sequences identical to the CDR1 (SEQ ID NO:98), CDR2 (SEQ ID NO:99), and CDR3 (SEQ ID NO:100) sequences of SEQ ID NO:94.

[0029] In certain embodiments, the second antigen-binding site is capable of binding to a FAP and may optionally incorporate a heavy chain variable domain related to SEQ ID NO: 114 and a light chain variable domain related to SEQ ID NO: 118. 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: 114 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 115 or SEQ ID NO: 147), CDR2 (SEQ ID NO: 116 or SEQ ID NO: 148), and CDR3 (SEQ ID NO: 117) of SEQ ID NO: 114. 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:118 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:119 or SEQ ID NO:149), CDR2 (SEQ ID NO:120), and CDR3 (SEQ ID NO:121) of SEQ ID NO:118.

[0030] Alternatively, the second antigen-binding site that binds to the FAP may optionally incorporate a heavy chain variable domain related to SEQ ID NO: 131 and a light chain variable domain related to SEQ ID NO: 135. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 131 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), and CDR3 (SEQ ID NO: 134) of SEQ ID NO: 131. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 135 and / or incorporates amino acid sequences identical to the CDR1 (SEQ ID NO: 136), CDR2 (SEQ ID NO: 137), and CDR3 (SEQ ID NO: 138) sequences of SEQ ID NO: 135.

[0031] Alternatively, the second antigen-binding site that binds to the FAP may optionally incorporate a heavy chain variable domain related to SEQ ID NO: 139 and a light chain variable domain related to SEQ ID NO: 143. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 139 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO: 141), and CDR3 (SEQ ID NO: 142) of SEQ ID NO: 139. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:143 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:144), CDR2 (SEQ ID NO:145), and CDR3 (SEQ ID NO:146) of SEQ ID NO:143.

[0032] Alternatively, the second antigen-binding site that binds to the FAP may optionally incorporate a heavy chain variable domain related to SEQ ID NO: 122 and a light chain variable domain related to SEQ ID NO: 126. 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 incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO: 123), CDR2 (SEQ ID NO: 124), and CDR3 (SEQ ID NO: 125) of SEQ ID NO: 122. 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:126 and / or incorporates amino acid sequences identical to the sequences of CDR1 (SEQ ID NO:127), CDR2 (SEQ ID NO:128), and CDR3 (SEQ ID NO:129) of SEQ ID NO:126.

[0033] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having an amino acid sequence identical to that of a light chain variable domain present in the first antigen-binding site.

[0034] In some embodiments, the protein incorporates a sufficient portion of an antibody Fc domain to bind CD16, wherein the antibody Fc domain comprises a hinge domain and a CH2 domain and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0035] In certain embodiments, the protein further incorporates a fourth antigen-binding site that binds to a tumor-associated antigen, which includes any antigen associated with cancer.For example, the fourth antigen binding site may bind to a number of antigens expressed on cancer cells, including human epidermal growth factor receptor 2 (HER2), CD20, CD33, B cell maturation antigen (BCMA), prostate specific membrane antigen (PSMA), delta-like canonical notch ligand (DLL3), ganglioside GD2 (GD2), CD123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signal transducer 2 (TROP2), carcinoembryonic antigen (CEA), claudin-18.2, receptor tyrosine kinase-like orphan receptor tyrosine kinase (ARK), and IFN-γ receptor tyrosine kinase-like ... receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein nonmetastatic melanoma protein B (GPNMB), folate receptor alpha (FRα), pregnancy-associated plasma protein A (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), Receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility complex, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB1), insulin-like growth factor 1 receptor (IGF1R), human epidermal growth factor receptor 3 (HER3) / ERBB3, human epidermal growth factor receptor 4 (HER4) / ERBB4, mucin 1 (MUC1), tyrosine protein kinase MET (cMET), signaling lymphocyte activation molecule F7 (SLAMF7), prostate stem cell antigen (PSCA), MHC class I polypeptide The antibody may bind to the CD4+-associated sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), melanoma-associated antigen 3 (MAGE-A3), B lymphocyte antigen B7.1 (B7.1), B lymphocyte antigen B7.2 (B74.2), cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PD-L1), or CD25 antigen.

[0036] Formulations containing any one of the proteins described herein; cells containing one or more nucleic acids that express the proteins, and methods of using the proteins to promote tumor cell death are also provided.

[0037] Another aspect of the present invention provides a method of treating cancer in a patient. The method comprises administering a therapeutically effective amount of the multispecific binding protein described herein to a patient in need thereof. Cancers that may be treated using FAP-targeting multispecific binding proteins include any cancer that expresses FAP, such as invasive ductal breast carcinoma, pancreatic ductal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, head and neck cancer, mesothelioma, gastric cancer, pancreatic cancer, liver cancer, endometrial cancer, neuroendocrine carcinoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, schwannoma, melanoma, and glioma.

[0038] In certain embodiments, the present invention provides a method of treating an autoimmune disease in a patient. The method comprises administering a therapeutically effective amount of a multispecific binding protein described herein to a patient in need thereof. In certain embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Graves' disease, Sjogren's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, and inflammatory destructive arthritis.

[0039] In certain embodiments, the present invention provides a method of treating fibrosis in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein, hi certain embodiments, the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and myocardial fibrosis. In certain embodiments, for example, the following are provided: (Item 1) below: (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds fibroblast activation protein (FAP); (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; , including protein. (Item 2) The protein of item 1, wherein the first antigen-binding site binds to NKG2D in humans. (Item 3) 3. The protein according to item 1 or 2, wherein the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 4) 4. The protein according to item 3, wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide. (Item 5) 5. The protein according to item 3 or 4, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 6) 6. The protein according to item 5, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are present on the same polypeptide. (Item 7) 7. The protein according to item 5 or 6, wherein the light chain variable domain of the first antigen-binding site has an amino acid sequence identical to an amino acid sequence of the light chain variable domain of the second antigen-binding site. (Item 8) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:175, SEQ ID NO:179, SEQ ID NO:183, SEQ ID NO:187, and SEQ ID NO:93. (Item 9) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 41 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 42. (Item 10) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 49 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 50. (Item 11) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 57 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 58. (Item 12) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 59 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 60. (Item 13) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 61 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 62. (Item 14) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 69 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 70. (Item 15) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 77 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 78. (Item 16) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 85, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183, or SEQ ID NO: 187, and a light chain variable domain that is at least 90% identical to SEQ ID NO: 86. (Item 17) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 93 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 94. (Item 18) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 101 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 102. (Item 19) 8. The protein according to any one of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 103 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 104. (Item 20) 3. The protein according to item 1 or 2, wherein the first antigen-binding site is a single domain antibody. (Item 21) The single domain antibody is H H fragment or V NAR 21. The protein according to item 20, which is a fragment. (Item 22) 22. The protein according to any one of items 1 to 2 or 20 to 21, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 23) 23. The protein according to item 22, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are present on the same polypeptide. (Item 24) 24. The protein according to any one of items 1 to 23, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 114, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 118. (Item 25) 24. The protein according to any one of items 1 to 23, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 131, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 135. (Item 26) 24. The protein according to any one of items 1 to 23, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 139, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 143. (Item 27) 24. The protein according to any one of items 1 to 23, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 122, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 126. (Item 28) 24. The protein according to any one of items 1 to 23, wherein the second antigen-binding site comprises a sequence of CDR1, CDR2, and CDR3 of a heavy chain variable domain and a light chain variable domain selected from the group consisting of SEQ ID NOs: 114 and 118, 131 and 135, 139 and 143, and 122 and 126, respectively. (Item 29) 22. The protein according to any one of items 1 to 4 or 8 to 21, wherein the second antigen-binding site is a single domain antibody. (Item 30) The second antigen-binding site is H H fragment or V NAR 30. The protein according to item 29, which is a fragment. (Item 31) 31. The protein according to any one of items 1 to 30, wherein the protein comprises a portion of an antibody Fc domain sufficient to bind to CD16, the antibody Fc domain comprising a hinge domain and a CH2 domain. (Item 32) 32. The protein according to item 31, wherein the antibody Fc domain comprises the hinge domain and CH2 domain of a human IgG1 antibody. (Item 33) 33. The protein according to item 31 or 32, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 34) 34. The protein according to item 33, wherein the Fc domain comprises an amino acid sequence 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, K439. (Item 35) 35. A formulation comprising the protein according to any one of items 1 to 34 and a pharma- ceutically acceptable carrier. (Item 36) 35. A cell comprising one or more nucleic acids encoding the protein according to any one of items 1 to 34. (Item 37) 35. A method for promoting tumor cell death, comprising exposing tumor cells and natural killer cells to an effective amount of a protein according to any one of items 1 to 34. (Item 38) A method for treating cancer, comprising administering to a patient an effective amount of the protein according to any one of items 1 to 34 or the formulation according to item 35. (Item 39) 39. The method of claim 38, wherein the cancer being treated is selected from the group consisting of invasive ductal carcinoma, pancreatic ductal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, mesothelioma, gastric cancer, pancreatic cancer, head and neck cancer, liver cancer, endometrial cancer, neuroendocrine carcinoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, schwannoma, melanoma, and glioma. (Item 40) A method for treating an autoimmune disease, comprising administering to a patient an effective amount of the protein according to any one of items 1 to 34 or the formulation according to item 35. (Item 41) 41. The method of claim 40, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Graves' disease, Sjogren's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, and inflammatory destructive arthritis. (Item 42) A method for treating fibrosis, comprising administering to a patient an effective amount of the protein according to any one of items 1 to 34 or the formulation according to item 35. (Item 43) 43. The method of claim 42, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and cardiac fibrosis. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 is a diagram of a heterodimeric multispecific binding protein. Each arm can represent either an NKG2D-binding domain or a binding domain for a FAP. The multispecific binding protein further comprises an Fc domain or a portion thereof that binds to CD16. In some embodiments, the NKG2D-binding domain and the FAP-binding domain can share a common light chain. [Diagram 2] FIG. 1: Diagram of heterodimeric multispecific binding proteins. Either the NKG2D binding domain or the binding domain to the FAP can be in scFv format (right arm). [Diagram 3] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay. [Figure 4] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D in an ELISA assay. [Diagram 5]1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to mouse recombinant NKG2D in an ELISA assay. [Figure 6] 1 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D, measured by flow cytometry as fold mean fluorescence intensity (MFI) over background (FOB). [Figure 7] 1 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing mouse NKG2D, measured by flow cytometry as fold mean fluorescence intensity (MFI) over background (FOB). [Figure 8] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) for recombinant human NKG2D-Fc in a competitive binding assay with the natural ligand of NKG2D, ULBP-6. [Figure 9] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) for recombinant human NKG2D-Fc in a competitive binding assay with the natural ligand of NKG2D, MICA. [Figure 10] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) for recombinant murine NKG2D-Fc in a competitive binding assay with the natural ligand of NKG2D, Rae-1 delta. [Figure 11] 1 is a bar graph showing activation of cells expressing human NKG2D-CD3ζ fusion protein by NKG2D binding domains (listed as clones) as measured by flow cytometry and quantified as the percentage of TNF-α positive cells. [Figure 12] 1 is a bar graph showing activation of cells expressing mouse NKG2D-CD3ζ fusion protein by NKG2D binding domains (listed as clones) as measured by flow cytometry and quantified as the percentage of TNF-α positive cells. [Figure 13] 1 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones) measured by flow cytometry and quantified as the percentage of IFN-γ+ / CD107a+ cells. [Figure 14] 1 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones) measured by flow cytometry and quantified as the percentage of IFN-γ+ / CD107a+ cells. [Figure 15] 1 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones) measured by flow cytometry and quantified as the percentage of IFN-γ+ / CD107a+ cells. [Figure 16] 1 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones) measured by flow cytometry and quantified as the percentage of IFN-γ+ / CD107a+ cells. [Figure 17] 1 is a bar graph showing the cytotoxic effect of NKG2D binding domains (listed as clones) on THP-1 tumor cells as measured using the Perkin Elmer DELFIA® Cytotoxicity kit assay. [Figure 18] 1 is a bar graph showing the melting temperatures of NKG2D binding domains (listed as clones) as measured by differential scanning fluorimetry. [Figure 19]1 is a bar graph showing synergistic activation of NK cells by binding of CD16 and NKG2D, measured by flow cytometry and quantified as the percentage of cells positive for NK activation markers. A shows the percentage of CD107a+ cells after 4 hours of treatment with plate-bound anti-CD16 monoclonal antibody alone, anti-NKG2D antibody alone, or a combination of anti-CD16 and anti-NKG2D antibodies. B shows the percentage of IFN-γ+ cells after 4 hours of treatment with plate-bound anti-CD16 monoclonal antibody alone, anti-NKG2D antibody alone, or a combination of anti-CD16 and anti-NKG2D antibodies. C shows the percentage of CD107a+ / IFN-γ+ cells after 4 hours of treatment with plate-bound anti-CD16 monoclonal antibody alone, anti-NKG2D antibody alone, or a combination of anti-CD16 and anti-NKG2D antibodies. Graphs show the mean (n=2) ± SD. Data are representative of five independent experiments using five different healthy donors. [Figure 20] FIG. 1 is a representative diagram of a multispecific binding protein in a Triomab format. [Figure 21] FIG. 1 is a representative diagram of a multispecific binding protein in the KiH common light chain (LC) format. [Figure 22] FIG. 1 is a representative diagram of a multispecific binding protein in the form of a dual variable domain immunoglobulin (DVD-Ig™). [Figure 23] FIG. 1 is a representative diagram of a multispecific binding protein in an orthogonal Fab interface (Ortho-Fab) format. [Figure 24] Representative diagram of a multispecific binding protein in a two-in-one Ig format. [Diagram 25] Representative diagram of a multispecific binding protein in an electrostatic steering (ES) configuration. [Figure 26] FIG. 1 is a representative diagram of a multispecific binding protein in a controlled Fab arm exchange (cFAE) format. [Figure 27]Representative diagram of a multispecific binding protein in a strand exchange engineered domain (SEED) body format. [Figure 28] FIG. 1 is a representative diagram of a multispecific binding protein in the LuZ-Y format. [Figure 29] Representative diagram of multispecific binding proteins in Cov-X body form. [Diagram 30] Representative diagrams of multispecific binding proteins in κλ-bodies: A is an exemplary representative diagram of one form of κλ-body; B is an exemplary representative diagram of another κλ-body. [Diagram 31] Representative diagram of a multispecific binding protein in one-arm single chain (OAsc)-Fab format. [Diagram 32] FIG. 1 is a representative diagram of a multispecific binding protein in a DuetMab format. [Diagram 33] FIG. 1 is a representative diagram of a multispecific binding protein in a CrossmAb format. [Diagram 34] FIG. 1 is a representative diagram of a multispecific binding protein in the Fit-Ig format. [Diagram 35] Histograms showing FAP expression on human cell lines LL86 (A), COLO 829 (B) and U-87 MG (C). [Diagram 36] 1 is a line graph showing the binding affinity of anti-FAP monoclonal antibody (FAP-mAb) and anti-FAP multispecific binding protein (FAP-multispecific BP) for FAP expressed on human cell lines LL86 (A), COLO 829 (B) and U-87 MG (C). [Figure 37A] 1 is a line graph showing the cytotoxic activity of primary human NK cells from two separate donors (donors RR01612, A-C; and donor 55109, D) stimulated with multispecific binding protein (FAP-multispecific BP), monoclonal antibody (FAP-mAb), or isotype control antibody against FAP-expressing LL86 (A), COLO829 (B), U-87 MG (C), and COLO829 (D) cells. [Figure 37B] 1 is a line graph showing the cytotoxic activity of primary human NK cells from two separate donors (donors RR01612, A-C; and donor 55109, D) stimulated with multispecific binding protein (FAP-multispecific BP), monoclonal antibody (FAP-mAb), or isotype control antibody against FAP-expressing LL86 (A), COLO829 (B), U-87 MG (C), and COLO829 (D) cells. [Figure 37C] 1 is a line graph showing the cytotoxic activity of primary human NK cells from two separate donors (donors RR01612, A-C; and donor 55109, D) stimulated with multispecific binding protein (FAP-multispecific BP), monoclonal antibody (FAP-mAb), or isotype control antibody against FAP-expressing LL86 (A), COLO829 (B), U-87 MG (C), and COLO829 (D) cells. [Figure 37D] 1 is a line graph showing the cytotoxic activity of primary human NK cells from two separate donors (donors RR01612, A-C; and donor 55109, D) stimulated with multispecific binding protein (FAP-multispecific BP), monoclonal antibody (FAP-mAb), or isotype control antibody against FAP-expressing LL86 (A), COLO829 (B), U-87 MG (C), and COLO829 (D) cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and FAPs on cancer cells. In certain embodiments, the multispecific binding proteins further comprise an additional antigen binding site that binds to a tumor-associated antigen. The present invention also provides pharmaceutical compositions comprising such multispecific binding proteins, as well as therapeutic methods using such multispecific binding proteins and pharmaceutical compositions, for purposes such as the treatment of cancer. Various aspects of the present invention are described in each section below, although an aspect of the invention described in one particular section is not limited to any particular section.

[0042] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0043] The terms "a" and "an" as used herein mean "one or more" and include plurals, unless the context is inappropriate.

[0044] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly diverse stretches within the V regions of the heavy and light chains are called "hypervariable regions", which are sandwiched between more conserved adjacent stretches known as "framework regions" or "FR". Thus, the term "FR" refers to the amino acid sequences naturally found 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 positioned 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 antigen to be bound, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single domain antibody". The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody which retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv (using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide).

[0045] The term "tumor-associated antigen" as used herein means any antigen, including but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, or lipid associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment (such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates).

[0046] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein, preferably including, but not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, and the like), and more preferably including humans.

[0047] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial effect or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, such as amelioration, reduction, modulation, amelioration, or elimination of a condition, disease, disorder, and the like, or amelioration of the symptoms thereof.

[0048] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, which combination makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic uses.

[0049] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizing and preservative substances. For examples of carriers, stabilizing substances, and adjuvants, see, for example, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton, PA (1975).

[0050] As used herein, the term "pharmaceutical acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of the compound of the present invention that can provide the compound of the present invention or its active metabolites or residues upon administration to a subject. As known to those skilled in the art, the "salts" of the compounds of the present invention can be derived from inorganic or organic acids and inorganic or organic bases. 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, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids (such as oxalic acid) are not themselves pharmaceutically acceptable, but may be used in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0051] Exemplary bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein, W is C 1-4 alkyl), and the like.

[0052] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include Na + , NH4 + and NW4 + (Wherein, W is C 1-4 Suitable cations include those which are alkyl groups, as well as the anions of the compounds of the present invention combined with the same.

[0053] For therapeutic use, the salts of the compounds of the invention are contemplated as being pharma-ceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharma-ceutically acceptable compound.

[0054] Throughout this specification, 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 additionally contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0055] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if no definition is accompanied by a variable, then the preceding definition of the variable takes precedence.

[0056] I. Protein The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and FAP on cancer cells. The multispecific binding proteins are useful in the pharmaceutical compositions and methods of treatment described herein. Binding of the multispecific binding proteins to the NKG2D and CD16 receptors on natural killer cells promotes the activity of the natural killer cells toward the destruction of tumor cells expressing the FAP antigen. Binding of the multispecific binding proteins to FAP-expressing cells brings the cancer cells into close proximity with the natural killer cells, thereby facilitating the direct and indirect destruction of the cancer cells by the natural killer cells. Further description of some exemplary multispecific binding proteins is provided below.

[0057] In certain other embodiments, the invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and the FAP on fibroblasts. For example, the fibroblasts can be activated stromal fibroblasts in a patient with an autoimmune disease or fibrosis. Binding of the multispecific binding protein to the NKG2D and CD16 receptors on natural killer cells promotes the activity of the natural killer cells toward the destruction of fibroblasts expressing the FAP antigen. Binding of the multispecific binding protein to FAP-expressing cells brings the fibroblasts into close proximity with the natural killer cells, thereby facilitating direct and indirect destruction of the fibroblasts by the natural killer cells.

[0058] The first component of the multispecific binding protein binds to NKG2D receptor expressing cells (NK cells, γδ T cells and CD8 +Upon binding NKG2D, the multispecific binding protein may block natural ligands (such as ULBP6 and MICA) from binding to NKG2D and activating the NKG2D receptor.

[0059] In certain embodiments, the second component of the multispecific binding protein binds to FAP-expressing cells, which may be found, for example, but not limited to, in invasive ductal breast carcinoma, pancreatic ductal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, mesothelioma, gastric cancer, pancreatic cancer, endometrial cancer, neuroendocrine carcinoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, schwannoma, melanoma, and glioma.

[0060] In some embodiments, the multispecific binding proteins described herein further incorporate additional antigen binding sites that bind to tumor-associated antigens (including any antigen associated with cancer, such as, but not limited to, proteins, glycoproteins, gangliosides, carbohydrates, or lipids). Such antigens may be expressed on malignant cells or in the tumor microenvironment (such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates). For example, the additional antigen binding sites may be expressed on cancer cells, such as HER2, CD20, CD33, BCMA, PSMA, DLL3, GD2, CD123, Ano1, mesothelin, CAIX, TROP2, CEA, claudin-18.2, ROR1, 5T4, GPNMB, FRα, PAPP-A, CD37, EpCAM, CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, or the like. , FLT3, GPC3, B7H6, CCR4, CXCR4, ROR2, CD133, HLA-E, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, PD-L1, or CD25 antigens. Thus, in some embodiments, binding of the multispecific binding protein to a tumor-associated antigen expressed on a cancer cell brings the cell into close proximity with natural killer cells, thereby facilitating the destruction of myeloid-derived suppressor cells (MDSCs) and / or tumor-associated macrophages (TAMs) by the natural killer cells, in addition to the direct and indirect destruction of cancer cells by the natural killer cells.

[0061] A third component of the multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0062] The multispecific binding proteins described herein may take a variety of formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain (Figure 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain together with the first immunoglobulin heavy chain forms an antigen binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. In certain embodiments, the second immunoglobulin light chain comprises a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen binding site that binds to the FAP. The first Fc domain and the second Fc domain together are capable of binding to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0063] 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 comprises a first Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single chain variable fragment (scFv) composed of a heavy chain variable domain and a light chain variable domain, which pair to bind to NKG2D or to bind to a FAP. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to NKG2D or to a FAP. The first Fc domain and the second Fc domain together are capable of binding to CD16 (Figure 2).

[0064] 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 variable region or a disulfide-stabilized variable region (scFv), or may form a tetravalent or trivalent molecule.

[0065] In some embodiments, the multispecific binding protein is in a Triomab format, which is a trifunctional bispecific antibody that maintains an IgG-like shape (e.g., the multispecific binding protein depicted in Figure 20). This chimeric bispecific antibody is composed of two half antibodies, each with one light chain and one heavy chain originating from two parent antibodies. The Triomab format can be a heterodimer composed of 1 / 2 rat antibody and 1 / 2 mouse antibody.

[0066] In some embodiments, the multispecific binding protein is a KiH common light chain (LC) form, which employs knobs-into-holes (KiH) technology (e.g., the multispecific binding protein depicted in FIG. 21). The KiH common LC form is a heterodimer that includes a Fab that binds to a first target, a Fab that binds to a second target, and an Fc domain stabilized by a heterodimerization mutation. The two Fabs each include a heavy chain and a light chain, where the heavy chain of each Fab is different from the other, and the light chain that pairs with each respective heavy chain is common to both Fabs.

[0067] KiH technology involves engineering the CH3 domains to generate either a "knob" or a "hole" in each heavy chain to enhance heterodimerization. The introduction of a "knob" in one CH3 domain (CH3A) involves the replacement of a small residue with a bulky one (e.g., T366W in the EU numbering system). CH3A To accommodate the "knob," a complementary "hole" surface is formed by replacing the residues closest to the knob on the other CH3 domain (CH3B) with smaller ones (e.g., T366S / L368A / Y407V). CH3B ) The "hole" mutation was optimized by structure-guided phage library screening (Atwell S., et al. (1997) J. Mol. Biol. 270(1): 26-35.). X-ray crystal structures of KiH Fc variants (Elliott JM, et al. (2014) J. Mol. Biol. 426(9): 1947-57.; Mimoto F., et al. (2014) Mol. Immunol; 58(1): 132-8.) demonstrated that hydrophobic interactions driven by stereochemical complementarity at the interface between the CH3 domain cores thermodynamically favor heterodimerization, whereas knob-knob and hole-hole interfaces do not favor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0068] In some embodiments, the multispecific binding protein is in the dual variable domain immunoglobulin (DVD-Ig™) format, which is a tetravalent IgG-like structure comprising the target binding domains of two monoclonal antibodies and a flexible, naturally occurring linker (e.g., FIG. 22). The DVD-Ig™ format is a homodimer comprising a variable domain targeting antigen 2 fused to the N-terminus of a Fab variable domain targeting antigen 1. The representative multispecific binding protein shown in FIG. 22 comprises an unmodified Fc.

[0069] In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab) (e.g., the multispecific binding protein depicted in Figure 23). In the Ortho-Fab IgG approach (Lewis SM, et (2014), Nat. Biotechnol.;32(2):191-8.), structure-based domain design allows the LC and HC domains to be modified in only one Fab without altering the other Fab. VH-CH1 Complementary mutations are introduced at the interface with

[0070] In some embodiments, the multispecific binding protein is a two-in-one Ig format (eg, the multispecific binding protein depicted in FIG. 24).

[0071] In some embodiments, the multispecific binding protein is in an electrostatic steering (ES) form, which is a heterodimer that contains two different Fab and Fc domains that bind target 1 and target 2 (e.g., the multispecific binding protein depicted in Figure 25). Heterodimerization is ensured by electrostatic steering mutations in the Fc domain.

[0072] In some embodiments, the multispecific binding protein is in a controlled Fab arm exchange (cFAE) format (e.g., the multispecific binding protein depicted in FIG. 26). The cFAE format is a bispecific heterodimer that contains two different Fabs that bind to target 1 and target 2, and the LC-HC pair (half molecule) is swapped with an LC-HC pair from another molecule. Heterodimerization is ensured by mutations in the Fc.

[0073] In some embodiments, the multispecific binding protein is in the form of a strand-exchange engineered domain (SEED) body (e.g., the multispecific binding protein depicted in FIG. 27). The SEED platform was designed to generate asymmetric and bispecific antibody-like molecules to expand the therapeutic applications of natural antibodies. This protein engineering platform is based on swapping structurally related sequences of immunoglobulin classes within the conserved CH3 domain (e.g., alternating segments of the IgA and IgG CH3 domain sequences). The SEED design allows for efficient generation of heterodimers while disfavoring homodimerization of the SEED CH3 domain (Muda M., et al. (2011) Protein Eng. Des. Sel.; 24(5):447-54.). In some embodiments, the multispecific binding protein is in the form of a LuZ-Y (e.g., the multispecific binding protein depicted in FIG. 28). The LuZ-Y form is a heterodimer that contains two different scFabs that bind to target 1 and target 2 fused to an Fc domain. Heterodimerization is ensured through the introduction of a leucine zipper motif fused to the C-terminus of the Fc domain (Wranik, BJ et al. (2012) J. Biol. Chem.; 287: 43331-9.).

[0074] In some embodiments, the multispecific binding protein is in the form of a Cov-X-body (e.g., the multispecific binding protein depicted in FIG. 29). Bispecific CovX-bodies comprise a scaffold antibody with a pharmacophore peptide heterodimer covalently linked to each Fab arm, where one molecule of the peptide heterodimer binds to a first target and the other molecule of the peptide heterodimer binds to a second target, the two molecules being joined by an azetidinone linker. The pharmacophore is responsible for the functional activity, while the antibody scaffold confers a long half-life and Ig-like distribution. The pharmacophore can be chemically optimized or replaced by other pharmacophores to generate optimized or unique bispecific antibodies (Doppalapudi VRet al.(2010)PNAS;107(52):22611-22616.).

[0075] In some embodiments, the multispecific binding protein is in the form of a κλ-body, which is a heterodimer comprising two different Fabs fused to an Fc domain stabilized by a heterodimerization mutation (e.g., the multispecific binding protein depicted in Figure 30). The first Fab that binds target 1 comprises a κLC, and the second Fab that binds target 2 comprises a λLC. Figure 30A is an exemplary representation of one form of a κλ-body; Figure 30B is an exemplary representation of another κλ-body.

[0076] In some embodiments, the multispecific binding protein is in one-arm single chain (OAsc)-Fab format (e.g., the multispecific binding protein depicted in Figure 31). The OAsc-Fab format is a heterodimer comprising a Fab that binds target 1 and a scFab that binds target 2 fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0077] In some embodiments, the multispecific binding protein is in a DuetMab format (e.g., the multispecific binding protein depicted in FIG. 32). The DuetMab format is a heterodimer that contains two different Fabs that bind to target 1 and target 2 and an Fc domain stabilized by a heterodimerization mutation. The two different Fabs contain different S-S bridges that ensure correct LC and HC pairing.

[0078] In some embodiments, the multispecific binding protein is in a CrossmAb format (e.g., the multispecific binding protein depicted in FIG. 33). The CrossmAb format is a heterodimer that includes two different Fabs that bind to target 1 and target 2 and an Fc domain stabilized by a heterodimerization mutation. The CL and CH1 domains and the VH and VL domains are switched, e.g., CH1 is fused in tandem with VL, while CL is fused in tandem with VH.

[0079] In some embodiments, the multispecific binding protein is a Fit-Ig format (such as the multispecific binding protein depicted in Figure 34). The Fit-Ig format is a homodimer that comprises a Fab that binds target 2 fused to the N-terminus of the HC of a Fab that binds target 1. The representative multispecific binding protein in Figure 34 comprises an unmodified Fc domain.

[0080] Table 1 lists peptide sequences of heavy and light chain variable domains that can bind to NKG2D in combination. Unless otherwise indicated, the CDR sequences provided in Table 1 are determined under Kabat. NKG2D binding domains may vary in binding affinity to NKG2D, but nevertheless, they all activate human NKG2D and NK cells. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0081] Alternatively, a heavy chain variable domain represented by SEQ ID NO: 101 can be paired with a light chain variable domain represented by SEQ ID NO: 102 to form an antigen-binding site capable of binding to NKG2D, as exemplified in US Pat. No. 9,273,136. SEQ ID NO:101 QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS SEQ ID NO:102 QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[0082] Alternatively, a heavy chain variable domain represented by SEQ ID NO: 103 can be paired with a light chain variable domain represented by SEQ ID NO: 104 to form an antigen-binding site capable of binding to NKG2D, as exemplified in US Pat. No. 7,879,985. SEQ ID NO:103 QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS SEQ ID NO:104 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0083] In certain embodiments, the present disclosure provides a multispecific binding protein that binds to the NKG2D and CD16 receptors on natural killer cells and the antigen FAP on cancer cells. Table 2 lists some exemplary sequences of heavy and light chain variable domains that can bind to FAP in combination. The CDR sequences of the amino acid sequences of the heavy and light chain variable domains listed in Table 2 below and described in the corresponding patents and publications are incorporated herein by reference. Unless otherwise indicated, the CDR sequences provided in Table 2 are determined under Kabat. [Table 2-1] [Table 2-2]

[0084] Alternatively, novel antigen binding sites capable of binding to a FAP can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:130. SEQ ID NO:130 MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD

[0085] Within the Fc domain, binding of CD16 is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly centered on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, for example, Sondermann P. et al. (2000) Nature; 406(6793):267-273.). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface-displayed cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0086] The assembly of heterodimeric antibody heavy chains can be achieved by expression of two different antibody heavy chain sequences in the same cell, which can lead to the assembly of homodimers of each antibody heavy chain in addition to the assembly of heterodimers. Enhancement of the selective assembly of heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US13 / 494,870, US16 / 028,850, US11 / 533,709, US12 / 875,015, US13 / 289,934, US14 / 773,418, US12 / 811,207, US13 / 866,756, US14 / 647,480, and US14 / 830,336. For example, mutations can be made in the CH3 domain based on human IgG1 and incorporating distinct pairs of amino acid substitutions in the first and second polypeptides that allow the two chains to selectively heterodimerize with each other. All amino acid substitution positions shown below are numbered according to the EU index, as in Kabat.

[0087] In one scenario, the amino acid substitutions in a first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T) or valine (V), such that the larger amino acid substitution (protuberance) fits into the surface of the smaller amino acid substitution (cavity). For example, one polypeptide can incorporate a T366W substitution and another can incorporate three substitutions including T366S, L368A and Y407V.

[0088] The antibody heavy chain variable domain of the present invention may be optionally coupled to an amino acid sequence at least 90% identical to an antibody constant region (such as an IgG constant region comprising a hinge domain, a CH2 domain, and a CH3 domain), with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region (such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region). In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal (such as a rabbit, a dog, a cat, a mouse, or a horse). One or more mutations may be incorporated into the constant region, for example at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439, relative to the human IgG1 constant region. Exemplary 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, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0089] In certain embodiments, mutations that may be incorporated into the CH1 of the human IgG1 constant region may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be incorporated into the Cκ of the human IgG1 constant region may be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0090] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 3. [Table 3]

[0091] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 4]

[0092] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 5. [Table 5]

[0093] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6. [Table 6]

[0094] Alternatively, at least one amino acid substitution is selected from the set of following substitutions in Table 7, where the position(s) indicated in the column of the first polypeptide is replaced by any known negatively charged amino acid, and the position(s) indicated in the column of the second polypeptide is replaced by any known positively charged amino acid. [Table 7]

[0095] Alternatively, at least one amino acid substitution is selected from the set of following substitutions in Table 8, where the position(s) indicated in the column of the first polypeptide is replaced by any known positively charged amino acid, and the position(s) indicated in the column of the second polypeptide is replaced by any known negatively charged amino acid. [Table 8]

[0096] Alternatively, the amino acid substitutions may be selected from the following set in Table 9. [Table 9]

[0097] Alternatively or additionally, the structural stability of a heteromultimeric protein may be increased by introducing S354C on either the first or second polypeptide chain and Y349C on the opposing polypeptide chain, which forms an artificial disulfide bridge within the interface of the two polypeptides.

[0098] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366 and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0099] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at position T366.

[0100] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0101] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0102] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411.

[0103] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407.

[0104] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, Y349, K360, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405.

[0105] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399, and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, K360, Q347, and K409.

[0106] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399.

[0107] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0108] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409.

[0109] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399.

[0110] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution S354C and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution Y349C.

[0111] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the Y349C substitution and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the S354C substitution.

[0112] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions K360E and K409W, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions Q347R, D399V, and F405T.

[0113] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions Q347R, D399V, and F405T, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions K360E and K409W.

[0114] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitution T366W, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the substitutions T366S, T368A, and Y407V.

[0115] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitutions T366S, T368A, and Y407V, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution T366W.

[0116] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, L351Y, F405A, and Y407V, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, T366L, K392L, and T394W.

[0117] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, T366L, K392L, and T394W, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by the following substitutions: T350V, L351Y, F405A, and Y407V.

[0118] The multispecific binding proteins described above can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, and a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector, and the first expression vector, the second expression vector, and the third expression vector can be stably transfected together into a host cell to produce a multimeric protein.

[0119] To achieve the highest yield of the multispecific protein, different ratios of the first expression vector, the second expression vector, and the third expression vector can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, flow cytometry, microscopy, or Clonepix.

[0120] The clones can be cultured under conditions suitable for scale-up in a bioreactor to maintain expression of the multispecific proteins. The multispecific binding proteins can be isolated 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.

[0121] II. Characteristics of Multispecific Binding Proteins The multispecific binding proteins described herein comprise an NKG2D binding site, a CD16 binding site, and a binding site for a FAP. In certain embodiments, the multispecific binding proteins simultaneously bind to cells expressing NKG2D and / or CD16 (such as NK cells) and tumor cells expressing a FAP. Binding of the multispecific binding proteins to NK cells can promote the activity of NK cells toward the destruction of cancer cells.

[0122] In certain embodiments, the multispecific binding proteins described herein bind to a FAP with an affinity similar to that of a corresponding monoclonal antibody having the same FAP binding site, hi certain embodiments, the multispecific binding proteins described herein may be more effective at reducing tumor growth and killing tumor cells expressing the FAP than the corresponding monoclonal antibody having the same FAP binding site.

[0123] In certain embodiments, the multispecific binding proteins described herein comprising an NKG2D-binding site and a FAP-binding site activate primary human NK cells when co-cultured with tumor cells expressing a FAP. NK cell activation is characterized by increased CD107a expression, degranulation, and IFN-γ cytokine production. Furthermore, the multispecific binding proteins described herein may exhibit superior activation of human NK cells in the presence of tumor cells expressing a FAP, compared to corresponding monoclonal antibodies with the same FAP-binding site.

[0124] In certain embodiments, a multispecific binding protein described herein that contains an NKG2D-binding site and a binding site for a FAP can promote activation of resting IL-2-activated human NK cells in the presence of tumor cells expressing a FAP.

[0125] In certain embodiments, the multispecific binding proteins described herein may have greater cytotoxic activity against tumor cells expressing a FAP compared to the corresponding monoclonal antibody having the same FAP binding site.

[0126] III. Treatment application The present invention provides a method for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The method can be used to treat various cancers that express FAP. Exemplary cancers to be treated can be gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, endometrial cancer, lung cancer, prostate cancer, bladder cancer, cervical cancer, head and neck cancer, ovarian cancer, esophageal cancer, renal cancer, liver cancer, testicular cancer, and oral cancer, multiple myeloma, leukemia, acute myeloid leukemia, melanoma, basal cell carcinoma and squamous cell carcinoma of the skin, glioma, Ewing's sarcoma, Kaposi's sarcoma, and mesothelioma.

[0127] In some other embodiments, exemplary cancers to be treated are acral lentigo melanoma, actinic keratosis, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenoid cystic carcinoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, angiosarcoma, anorectal carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma (e.g., skin), B-cell lymphoma, biliary tract carcinoma, bladder carcinoma, bone cancer, bone marrow carcinoma, brain tumor, breast cancer, bronchial carcinoma, bronchial adenocarcinoma, Burkitt's lymphoma, carcinoid, cervical carcinoma, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / choroid plexus Cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic neutrophilic leukemia, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue cancer, cutaneous T-cell lymphoma, cystadenoma, diffuse large B-cell lymphoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial cancer / endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, enteropathic T-cell lymphoma, ependymal carcinoma, epithelial cell carcinoma, esophageal cancer, Ewing's sarcoma, extranodal marginal zone B-cell lymphoma, extranodal natural killer cell lymphoma / T-cell lymphoma, eye and orbit cancer, female genital tract cancer, focal nodular hyperplasia, follicular lymphoma , gallbladder cancer, gastric antrum cancer, gastric cancer, gastric fundus cancer, gastrinoma, glioblastoma, glioma, glucagonoma, hairy cell leukemia, head and neck cancer, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, blood tumors, liver adenoma, hepatic adenomatosis, hepatocellular carcinoma, hepatobiliary carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, kidney cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo maligna melanoma, leukemia, liver cancer, lung cancer, lymphoma, lymphoplasmacytic lymphoma, male genital tract cancer, malignant melanoma, malignant mesothelioma , mantle cell lymphoma, marginal zone B-cell lymphoma, medulloblastoma, medulloepithelioma, melanoma, meningeal cancer, mesothelial carcinoma, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, myelodysplastic neoplasm, myeloproliferative neoplasm, nasal cancer, nervous system cancer, neuroblastoma, neuroepithelial adenocarcinoma, nodal marginal zone B-cell lymphoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary serous adenocarcinoma, parotid cancer, pelvic cancer, penile cancer, peripheral T-cell lymphoma, pharyngeal cancer, pituitary tumor, plasmacytoma, precursor T-lymphoblastic lymphoma,Primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, prostate cancer, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinonasal cancer, skin cancer, small cell carcinoma, small intestine cancer, small lymphocytic lymphoma, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumors, spinal cancer, splenic marginal zone B-cell lymphoma, squamous cell carcinoma (e.g., skin), rhabdomyocarcinoma, subcutaneous panniculitis-like T-cell lymphoma, submesothelial carcinoma The cancer may be superficial spreading melanoma, T-cell leukemia, T-cell lymphoma, testicular cancer, thyroid cancer, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, uterine cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIP-producing tumor, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0128] In certain embodiments, the present invention provides a method of treating an autoimmune disease in a patient. Exemplary autoimmune diseases that may be treated include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, destructive arthritis with inflammation, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, juvenile diabetes mellitus, multiple sclerosis, osteoarthritis, psoriatic arthritis, psoriasis, dermatitis, systemic lupus erythematosus (SLE), polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic sclerosis and systemic sclerosis, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, and the like. inflammation, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T cell infiltration and chronic inflammatory responses, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, granulomatous diseases including sarcoidosis, Wegener's granulomatosis, granulocytopenia, vasculitis (including ANCA), immune hemolytic anemias including aplastic anemia, Diamond-Blackfan anemia, autoimmune hemolytic anemia (AIHA), Pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Jackson syndrome, Johnsson's syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, autoimmune diseases of the testes and ovaries including idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune orchitis and autoimmune oophoritis, primary hypothyroidism;Autoimmune endocrine disorders, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), primary sclerosing cholangitis, subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, polyimmune polyglandular syndrome (or polyendocrine syndrome), type 1 diabetes (also called insulin-dependent diabetes mellitus (IDDM)), and Sheehan's syndrome; autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, large-vasculitis (including polymyalgia rheumatica and giant cell arteritis (Takayasu's arteritis)), medium-vasculitis (including Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA nephropathy), acute progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinemia, amyotrophic lateral sclerosis (ALS), or coronary artery disease;

[0129] In certain embodiments, the present invention provides a method for treating fibrosis in a patient. The method comprises administering a therapeutically effective amount of the multispecific binding protein described herein to a patient in need thereof. The fibrosis treated using the multispecific binding protein targeting FAP can be associated with interstitial lung disease, liver cirrhosis, kidney disease, heart disease, eye disease, scleroderma, keloid scars and hypertrophic scars, atherosclerosis and restenosis, postoperative scars, chemotherapy drug use, radiation therapy, physical injury, or burns. For example, the fibrosis can be idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, or cardiac fibrosis.

[0130] IV. Combination Therapy Another aspect of the invention provides combination therapy. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0131] Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine ... These include, for example, rifabutin, flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, as well as variants of the aforementioned agents that may exhibit differential binding to their cognate receptors and increased or decreased serum half-lives.

[0132] An additional class of drugs that can be used as part of a combination therapy in the treatment of cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3.The CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration to treat melanoma.

[0133] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (eg, Herceptin) and non-cytotoxic agents (eg, tyrosine kinase inhibitors).

[0134] Further categories of anticancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, ME (ii) an inhibitor selected from a K inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0135] The proteins of the present invention may also be used as an adjunct to surgical removal of the primary lesion.

[0136] The amounts of the multispecific binding protein and additional therapeutic agent, as well as the relative timing of administration, can be selected to achieve a desired combination therapeutic effect. For example, when administering combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or the pharmaceutical composition(s) comprising the therapeutic agents, can be administered in any order (e.g., sequentially, in parallel, together, simultaneously, homogenously, etc.). Further, for example, the multispecific binding protein can be administered while the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa.

[0137] V. Pharmaceutical Compositions The present disclosure also features a pharmaceutical composition containing a therapeutically effective amount of the protein described herein. The composition may be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for appropriate formulation. Formulations suitable for use in the present disclosure are described in Remington's Pharmaceutical Sciences, 17 th Ed. Mack Publishing Company, Easton, PA (1985). For a brief review of methods for drug delivery, see, e.g., Langer T., Science; 249(4976):1527-1533.

[0138] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen or syringe. In certain embodiments, the bag may be connected to a channel containing a tube and / or needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and may be contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulations from 12, 27 or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial.

[0139] The present disclosure may be present in a liquid, aqueous pharmaceutical formulation comprising a therapeutically effective amount of a multispecific protein in a buffered solution.

[0140] The compositions disclosed herein can be sterilized by conventional sterilization techniques or can be filter sterilized. The resulting aqueous solutions can be packaged for direct use or lyophilized, in which case the lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be between 3 and 11, more preferably between 5 and 9 or 6 and 8, and most preferably between 7 and 8 (such as 7 and 7.5). The compositions in solid form can be packaged in a number of single-dose units, each containing a fixed amount of the aforementioned drug(s). The compositions in solid form can also be packaged in containers for flexible amounts.

[0141] In certain embodiments, the disclosure provides extended shelf life formulations comprising a multispecific protein of the disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0142] In certain embodiments, aqueous formulations are prepared containing the proteins of the present disclosure in a pH buffer solution. The buffers of the present invention may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Ranges intermediate to the pHs listed above are also intended to be part of the present disclosure. For example, ranges of values using any combination of the values listed above as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate) buffers, succinate (e.g., sodium succinate) buffers, gluconate buffers, histidine buffers, citrate buffers, and other organic acid buffers.

[0143] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range can be a pH range of about 4.5 to about 6.0, or about 4.8 to about 5.5, or about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system 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 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, 0.25-0.5 mg / mL sodium citrate, 1.25-1.75 mg / mL disodium phosphate dihydrate, 0.7-1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / mL sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0144] Polyols that act as isotonicity agents and can stabilize the antibodies can also be included in the formulations described herein. The polyols are added to the formulation in amounts that can vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also be altered relative to the molecular weight of the polyol. For example, monosaccharides (e.g., mannitol) can be added in smaller amounts compared to disaccharides (e.g., trehalose). In certain embodiments, the polyol that can be used in the formulation as an isotonicity agent is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / mL. In certain embodiments, the mannitol concentration can be about 7.5 to 15 mg / mL. In certain embodiments, the mannitol concentration can be about 10 to 14 mg / mL. In certain embodiments, the mannitol concentration can be about 12 mg / mL. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0145] Detergents or surfactants may also be added to the formulations of the invention. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20 and polysorbate 80) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see, e.g., Fiedler HP, Lexikon der Hifsstoffe fur Pharmazie, Kosmetik und andrenzende Gebiete, 4 thEd., Editio Cantor, Aulendorf, Germany (1996). In certain embodiments, the formulation may contain between about 0.1 mg / mL and about 10 mg / mL or between about 0.5 mg / mL and about 5 mg / mL of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.

[0146] In certain embodiments, the multispecific protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be present at a concentration of 10 mg / mL in any USP / Ph Eur Type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of an elastomer compliant with USP and Ph Eur. In certain embodiments, the vial may be filled with 61.2 mL of the multispecific protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.

[0147] In certain embodiments, the liquid formulation of the present disclosure can be prepared as a solution with a concentration of 10mg / mL in combination with sugar at a stabilizing level.In certain embodiments, the liquid formulation can be prepared in an aqueous carrier.In certain embodiments, the stabilizing substance can be added in an amount that does not exceed the amount that may cause undesirable or unsuitable viscosity for intravenous administration.In certain embodiments, the sugar can be a disaccharide (e.g., sucrose).In certain embodiments, the liquid formulation can also include one or more of a buffer, a surfactant, and a preservative.

[0148] In certain embodiments, the pH of the liquid formulation can be set by the addition of a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0149] In addition to aggregation, deamidation is a common product variation of peptides and proteins that can occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage, and sample analysis. Under physiological conditions, deamidation is the loss of ammonia (NH3) from asparagine residues of proteins, resulting in a loss of mass of 17 daltons and the formation of a succinimide intermediate. Subsequent hydrolysis of succinimide results in an increase in mass of 18 daltons and the formation of aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain can also affect the deamidation rate (e.g., Gly and Ser following an Asn residue result in higher susceptibility to deamidation).

[0150] In certain embodiments, the liquid formulations of the present disclosure may be stored under conditions of pH and humidity to prevent deamidation of the protein product.

[0151] The aqueous carrier of interest herein is pharma- ceutically acceptable (i.e., safe and non-toxic for human administration) and useful for preparing liquid formulations.Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0152] Preservatives may optionally be added to the formulations herein to reduce microbial action. The addition of a preservative may, for example, facilitate the manufacture of a multi-use (multi-dose) formulation.

[0153] Intravenous (IV) formulations may be the preferred route of administration in certain cases, such as when hospitalized patients receive all medications via the IV route after transplant. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the drug product diluted for injection is isotonic and suitable for administration by intravenous infusion.

[0154] In certain embodiments, salts or buffer components may be added in amounts of about 10 mM to about 200 mM. The salts and / or buffer substances are pharma- ceutically acceptable and are derived from a variety of known acids (inorganic and organic) along with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate buffer, a carbonate buffer, or a citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counter ions.

[0155] Preservatives can optionally be added to the formulations herein to reduce microbial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (i.e., multi-dose) formulation.

[0156] The aqueous carriers of interest herein are those that are pharma- ceutically acceptable (i.e., safe and non-toxic for administration to humans) and useful for preparing liquid formulations.Exemplary carriers include SWFI, BWFI, pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0157] The present disclosure may be in a lyophilized formulation comprising a protein and a cryoprotectant. The cryoprotectant may be a sugar (e.g., a disaccharide). In certain embodiments, the cryoprotectant may be sucrose or maltose. The lyophilized formulation may also comprise one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0158] The amount of sucrose or maltose useful for stabilizing a lyophilized drug 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 between 1:2 and 1:5.

[0159] In certain embodiments, the pH of the lyophilized formulation prior to lyophilization can be set by the addition of a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharma-ceutically acceptable base can be sodium hydroxide.

[0160] Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range for the lyophilized drug product may be between 7 and 8.

[0161] In certain embodiments of the lyophilized formulation, salts or buffer components may be added in amounts between 10 mM and 200 mM. The salts and / or buffer substances are pharma- ceutically acceptable and are derived from a variety of known acids (inorganic and organic) along with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate buffer, a carbonate buffer, a citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0162] In certain embodiments, a "bulking agent" may be added to the lyophilized formulation. A "bulking agent" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulation of the present invention may contain such a bulking agent.

[0163] Preservatives can optionally be added to the lyophilized formulations herein to reduce microbial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (i.e., multi-dose) formulation.

[0164] In certain embodiments, lyophilized drug product may be composed of aqueous diluent.The aqueous diluent of interest herein is pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing reconstituted liquid formulation after lyophilization.Exemplary diluents include SWFI, BWFI, pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0165] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either SWFI or 0.9% Sodium Chloride for Injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

[0166] In certain embodiments, the lyophilized protein product of the present disclosure is made up into about 4.5 mL of water for injection and diluted with 0.9% saline (sodium chloride solution).

[0167] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

[0168] The specific dose may be a uniform dose for each patient, for example 50-5000 mg of protein. Alternatively, the patient's dose may be adjusted to the patient's approximate body weight or surface area. Other factors that determine the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, as well as the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those of skill in the art, especially in light of the dosage information and assays disclosed herein. Dosage may also be determined through the use of known assays for determining dosage used in conjunction with appropriate dose-response data. Dosage for individual patients may be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex in the patient may be measured to ascertain whether dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, and their dosages, are most likely to be effective for a given individual (see, e.g., Schmitz et al. (2001) Clinica Chimica Acta; 308:43-53.; Steimer et al. (2001) Clinica Chimica Acta; 308:33-41.).

[0169] Generally, dosages based on body weight range from about 0.01 μg to about 100 mg / kg body weight (about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0.1 μg / 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 100 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 / k g body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight 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, or about 50 mg to about 100 mg / kg body weight, etc.

[0170] Doses may be given one or more times per day, one or more times per week, one or more times per month, or one or more times per year, or in some cases once every 2-20 years. One of skill in the art can easily estimate repetition rates for dosing based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by perfusion via a catheter, or by direct intralesional injection. It may be administered one or more times per day, one or more times per week, one or more times per month, or one or more times per year.

[0171] The above description describes multiple aspects and embodiments of the present invention. This patent application specifically contemplates all combinations and permutations of these aspects and embodiments. EXAMPLES

[0172] The invention generally described herein will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0173] Example 1 – NKG2D binding domain binds to NKG2D The NKG2D-binding domain binds to purified recombinant NKG2D Nucleic acid sequences of human, mouse, or cynomolgus NKG2D ectodomain were fused to a nucleic acid sequence encoding a human IgG1 Fc domain and introduced into mammalian cells where they were to be expressed. After purification, the NKG2D-Fc fusion proteins were adsorbed to wells of a microplate. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D binding domains were titrated and added to the wells pre-adsorbed with the NKG2D-Fc fusion proteins. Binding of the primary antibody was detected using a secondary antibody conjugated to horseradish peroxidase, which specifically recognizes the human kappa light chain and avoids Fc cross-reactivity. Binding signals were visualized by adding 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, to the wells, and the absorbance was measured at 450 nM and corrected at 540 nM. NKG2D binding domain clones, isotype controls, or positive controls (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 (eBioscience, San Diego, Calif.)) were added to each well.

[0174] The isotype control showed minimal binding to recombinant NKG2D-Fc protein, whereas the positive control bound most strongly to the recombinant antigen. The NKG2D binding domains produced by all clones were demonstrated to bind across human, mouse and cynomolgus recombinant NKG2D-Fc proteins, with affinities varying between clones. In general, each anti-NKG2D clone bound with similar affinity to human (Figure 3) and cynomolgus (Figure 4) recombinant NKG2D-Fc, but with lower affinity to mouse (Figure 5) recombinant NKG2D-Fc.

[0175] The NKG2D-binding domain binds to cells expressing NKG2D EL4 mouse lymphoma cell lines were 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 EL4 cells. Antibody binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and fold over background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[0176] NKG2D binding domains were produced by all clones that bound to EL4 cells expressing human and mouse NKG2D. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 (eBioscience, San Diego, Calif.)) provided the best FOB binding signals. NKG2D binding affinity for each clone was similar between cells expressing human NKG2D (FIG. 6) and mouse NKG2D (FIG. 7).

[0177] Example 2 – NKG2D-binding domains block binding of natural ligands to NKG2D Competition with ULBP-6 Recombinant human NKG2D-Fc protein was adsorbed to the wells of a 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 addition of the NKG2D-binding domain clones. After 2 hours of incubation, the wells were washed, and ULBP-6-His-biotin that remained bound to the NKG2D-Fc-coated wells was detected by streptavidin conjugated to horseradish peroxidase and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin in the wells that was blocked from binding to the NKG2D-Fc protein. A positive control antibody (comprising a heavy chain variable domain and a light chain variable domain selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked binding of ULBP-6 to NKG2D, while an isotype control showed little competition with ULBP-6 (Figure 8).

[0178] The ULBP-6 sequence is represented by SEQ ID NO:150. MAAAAIPALLLCLPLLFLLFGWSRARRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSGTTQLRATATTLILCCLLIILPCFILPGI (SEQ ID NO: 150)

[0179] Conflict with MICA Recombinant human MICA-Fc protein was adsorbed to the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. NKG2D-Fc-biotin was added to the wells, followed by the NKG2D binding domain. After incubation and washing, the NKG2D-Fc-biotin that remained bound to the MICA-Fc-coated wells was detected by streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected to 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 that was blocked from binding to the MICA-Fc-coated wells. A positive control antibody (comprising a heavy chain variable domain and a light chain variable domain selected from SEQ ID NOs: 101-104) and various NKG2D binding domains blocked binding of MICA to NKG2D, while an isotype control showed little competition with MICA (Figure 9).

[0180] Competition with Rae-1δ Recombinant mouse Rae-1 delta-Fc (R&D Systems, Minneapolis, MN) was adsorbed to the wells of a 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, the NKG2D-Fc-biotin that remained bound to the Rae-1 delta-Fc-coated wells was detected by streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected to 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 that was blocked from binding to the Rae-1 delta-Fc-coated wells. Positive controls (including heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 (eBioscience, San Diego, CA)) and various NKG2D binding domain clones blocked binding of Rae-1δ to mouse NKG2D, while an isotype control antibody showed little competition with Rae-1δ (Figure 10).

[0181] Example 3 –NKG2D binding domain clones activate NKG2D The nucleic acid sequences of human and mouse NKG2D were fused to the nucleic acid sequence encoding the CD3ζ signaling domain to obtain chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into retroviral vectors using Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with the virus containing NKG2D-CAR together with 8μg / mL polybrene. 24 hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0182] To determine whether the NKG2D-binding domains activate NKG2D, they were adsorbed to microplate wells and NKG2D-CAR EL4 cells were cultured on the antibody fragment-coated wells in the presence of brefeldin-A and monensin for 4 hours. Intracellular TNF-α production (an indicator for NKG2D activation) was assayed by flow cytometry. The percentage of TNF-α positive cells was normalized to cells treated with a positive control. All NKG2D-binding domains activated both human NKG2D (Figure 11) and mouse NKG2D (Figure 12).

[0183] Example 4 –NKG2D binding domains activate NK cells Primary human NK cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated and the purity of isolated NK cells was typically >95%. The isolated NK cells were then cultured for 24-48 hours in medium containing 100 ng / mL IL-2, after which they were transferred to wells of a microplate adsorbed with NKG2D binding domain and cultured in medium containing a fluorophore-conjugated anti-CD107a antibody, brefeldin-A, and monensin. Following culture, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-γ. CD3 - CD56 +NK cell activation was assessed by analyzing CD107a staining and IFN-γ staining in cells. An increase in CD107a / IFN-γ double positive cells represents better NK cell activation through engagement of two activating receptors rather than one. The NKG2D binding domain and positive control (e.g., heavy chain variable domain represented by SEQ ID NO: 101 or SEQ ID NO: 103, and light chain variable domain represented by SEQ ID NO: 102 or SEQ ID NO: 104) showed that a higher percentage of NK cells express CD107a than the isotype control. + and IFN-γ + (FIGS. 13 and 14 represent data from two independent experiments, each using PBMCs from a different donor for the preparation of NK cells).

[0184] Primary mouse NK cells Spleens were obtained from C57Bl / 6 mice and disrupted through a 70 μm cell strainer to obtain a single cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (Thermo Fisher Scientific #A1049201, Carlsbad, CA; 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 before being harvested and prepared for NK cell isolation. NK cells (CD3 - NK1.1 + ) were isolated from splenocytes using a negative depletion technique with magnetic beads, which typically resulted in NK cell populations with a purity of >90%. Purified NK cells were cultured in medium containing 100 ng / mL mIL-15 for 48 hours, after which they were transferred to wells of microplates adsorbed with NKG2D binding domains and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin-A, and monensin. Following culture in the NKG2D binding domain-coated wells, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-γ. CD3 - NK1.1+ NK cell activation was assessed by analyzing CD107a staining and IFN-γ staining in cells. An increase in CD107a / IFN-γ double positive cells represents better NK cell activation through engagement of two activating receptors rather than one. NKG2D binding domains and positive controls (selected from anti-mouse NKG2D clones MI-6 and CX-5, eBioscience, San Diego, CA) showed that a higher percentage of NK cells express CD107a than the isotype control. + and IFN-γ + (FIGS. 15 and 16 represent data from two independent experiments, each using different mice for the preparation of NK cells).

[0185] Example 5 –NKG2D-binding domains promote cytotoxicity against target tumor cells Human and mouse primary NK cell activation assays demonstrate an increase in cytotoxicity markers on NK cells after incubation with the NKG2D binding domain. To examine whether this translates to increased 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) acted as another targeting arm to activate NK cells. THP-1 cells (of human origin and expressing high levels of Fc receptors) were used as tumor targets, using the Perkin Elmer DELFIA® Cytotoxicity Kit (Waltham, MA). THP-1 cells were labeled with BATDA reagent and incubated for 10 min at 4°C for 1 h. 5The labeled THP-1 cells were then combined with NKG2D antibody and isolated mouse NK cells in a well of a microtiter plate for 3 hours at 37°C. After incubation, 20 μl of culture supernatant was removed and mixed with 200 μl of europium solution and incubated for 15 minutes in the dark 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.

[0186] A positive control (ULBP-6, the natural ligand for NKG2D) showed increased 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 shows the specific lysis of THP-1 cells by mouse NK cells without added antibody (Figure 17).

[0187] Example 6 –NKG2D antibodies have high thermal stability The melting temperature of the NKG2D-binding domain was assayed using differential scanning fluorimetry. The predicted extrapolated melting temperature of the NKG2D-binding domain was higher than that of a typical IgG1 antibody (FIG. 18).

[0188] Example 7 –Synergistic activation of human NK cells by cross-linking NKG2D and CD16 Primary human NK cell activation assay Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral human blood buffy coats using density gradient centrifugation. NK cells were purified from PBMCs using negative selection magnetic beads (StemCell Technologies, Vancouver, Canada; Cat. No. 17955). NK cells were >90% CD3+ / - cells as determined by flow cytometry. - CD56 +The cells were then expanded in medium containing 100 ng / mL hIL-2 (PeproTech, Inc., Rocky Hill, NJ, Catalog No. 200-02) for 48 hours before being used in the activation assay. Antibodies were coated onto 96-well flat-bottom plates overnight at 4° C. at concentrations of 2 μg / mL (anti-CD16, BioLegend, San Diego, CA; Catalog No. 302013) and 5 μg / mL (anti-NKG2D, R&D Systems, Minneapolis, MN; Catalog No. MAB139) in 100 μl of sterile phosphate-buffered saline (PBS), followed by thorough washing of the wells to remove excess antibody. For assessment of degranulation, IL-2-activated NK cells were cultured at 5 × 10 in culture medium supplemented with 100 ng / mL hIL2 and 1 μg / mL APC-conjugated anti-CD107a mAb (BioLegend, San Diego, CA; catalog number 328619). 5 The cells were then resuspended at 1 × 10 5 Cells / well were added onto the antibody-coated plates. Protein transport inhibitors brefeldin A (BFA, BioLegend, San Diego, CA; Catalog No. 420601) and monensin (BioLegend, San Diego, CA; Catalog No. 420701) were added at final dilutions of 1:1000 and 1:270, respectively. Plated cells were incubated for 4 hours at 37°C in 5% CO2. For intracellular staining of IFN-γ, NK cells were labeled with anti-CD3 (BioLegend, San Diego, CA; Catalog No. 300452) and anti-CD56 mAb (BioLegend, San Diego, CA; Catalog No. 318328), followed by fixation, permeabilization, and labeling with anti-IFN-γ mAb (BioLegend, San Diego, CA, Catalog No. 506507). NK cells were analyzed for viable CD56 + CD3 - After gating on cells, they were analyzed by flow cytometry for expression of CD107a and IFN-γ.

[0189] To investigate the relative potency of receptor combinations, crosslinking of NKG2D or CD16 and co-crosslinking of both receptors by plate-bound stimulation was performed.

[0190] As shown in Figure 19, the expression of CD107a and intracellular IFN-γ of IL-2-activated NK cells was analyzed after 4 hours of plate-bound stimulation with a combination of anti-CD16, anti-NKG2D, or both monoclonal antibodies. Combination stimulation of CD16 and NKG2D resulted in a greater percentage of CD107a upregulation than the additive effect of individual stimulation of CD16 or NKG2D alone (indicated by the dotted line). + Cells (Figure 19A) and IFN-γ + Similarly, combined stimulation with CD16 and NKG2D resulted in a higher percentage of CD107a cells compared to the additive effect of each receptor alone individually. + IFN-γ + This resulted in double positive cells (Figure 19C). Bar graphs show the mean (n=2) ± SD and are representative of 5 independent experiments using 5 different healthy donors.

[0191] Example 8 –Expression of FAP on human cell lines FAP expression was confirmed on three human cell lines: LL86 fibroblasts derived from normal tissue from a patient with osteogenic sarcoma; COLO829 melanoma cancer cells; and U-87 MG epithelial carcinoma cells from glioblastoma. FAP expression was measured using flow cytometry analysis by staining cells with a fluorophore-conjugated anti-human FAP antibody (R&D Systems, Minneapolis, Minn.).

[0192] As shown in FIG. 35, FAP expression was detected on LL86 (FIG. 35A), COLO 829 (FIG. 35B), and U-87 MG (FIG. 35C) cells compared to antibody isotype controls.

[0193] Example 9 –Binding of anti-FAP multispecific binding proteins and anti-FAP monoclonal antibodies to FAP-expressing cell lines FAP-expressing human cell lines (LL86, COLO 829 and U-87MG) were used to assess tumor antigen binding of multispecific binding proteins with FAP binding sites, including a heavy chain variable domain sequence identical to SEQ ID NO: 114 paired with a light chain variable domain sequence identical to SEQ ID NO: 118 (FAP multispecific BP Sibrotuzumab); a heavy chain variable domain sequence identical to SEQ ID NO: 131 paired with a light chain variable domain sequence identical to SEQ ID NO: 135 (FAP multispecific BP 4G8); or a heavy chain variable domain sequence identical to SEQ ID NO: 139 paired with a light chain variable domain sequence identical to SEQ ID NO: 143 (FAP multispecific BP 29B11). Multispecific binding proteins with the same FAP binding sites or corresponding monoclonal antibodies (mAbs) were diluted and incubated with the cells. Binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and expressed as mean fluorescence intensity (MFI) normalized to a human recombinant IgG1 stained control to obtain fold over background (FOB) values.

[0194] As shown in Figures 36A-36C, FAP multispecific BP Sibrotuzumab, FAP multispecific BP 4G8, FAP multispecific BP 29B11, and corresponding mABs with the same FAP binding site bind to FAP-expressing human LL86 cells (Figure 36A), COLO829 cells (Figure 36B), and U-87 MG cells (Figure 36C). The overall binding signal was higher for the multispecific binding proteins compared to the corresponding mAbs.

[0195] Example 10 –Promotion of NK cell-mediated lysis of FAP-expressing target cells by multispecific binding proteins PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. Isolated PBMCs were washed and prepared for isolation of NK cells. NK cells were isolated using negative selection with magnetic beads. NK cells were >90% CD3+ as determined by flow cytometry. - CD56 +Isolated NK cells were incubated overnight in cytokine-free medium and then used in the cytotoxicity assay.

[0196] DELFIA Cytotoxicity Assay: FAP-expressing human cancer cell lines were harvested from culture. Cells were washed with PBS and incubated for 10 6 The cells were resuspended in growth medium at 10 cells / mL and labeled with BATDA reagent (Perkin Elmer, Waltham, MA, Cat. No. AD0116) according to the manufacturer's instructions. After labeling, the cells were washed 3× with HEPES-buffered saline and diluted with 5×10 4 Resuspended in culture medium at cells / mL, 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Designated wells were reserved for spontaneous release from target cells, and all other wells were prepared for maximum lysis of target cells by the addition of 1% Triton-X.

[0197] Anti-FAP multispecific binding proteins and corresponding mAbs with the same FAP binding site were diluted in culture medium. 50 μl of diluted anti-FAP mAbs or anti-FAP multispecific binding proteins were added to designated wells. Purified primary NK cells were harvested from culture, washed, and diluted to 1×10 5 ~2.0×10 6 Primary NK cells were resuspended in culture medium at a concentration of 1000 cells / mL. 50 μl of primary NK cell suspension was added to designated wells of a 96-well plate for a total culture volume of 200 μl to achieve an effector to target cell ratio of 10:1. Plates were incubated at 37°C with 5% CO2 for 2-4 hours before proceeding with the assay.

[0198] Following co-culture, cells were pelleted by centrifugation at 500×G for 5 min. 20 μl of culture supernatant was transferred to a clean microplate and 200 μl of room temperature europium solution was added to each well. The microplate was protected from light and incubated for 15 min at 250 rpm on a plate shaker. The microplate was read on a SpectraMax i3X instrument (Molecular Devices, San Jose, CA). % specific lysis was calculated as follows: % specific dissolution = [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x 100%

[0199] FIG. 37A shows that FAP multispecific BP Sibrotuzumab, FAP multispecific BP 4G8, and FAP-multispecific BP 29B11 mimicked the cytotoxic activity of primary human NK cells isolated from donor RR01612 against FAP-expressing LL86 cells.

[0200] Similarly, FIG. 37D shows that FAP multispecific BP sibrotuzumab, FAP multispecific BP 4G8, and FAP-multispecific BP 29B11 mimicked the cytotoxic activity of primary human NK cells isolated from donor 55109 against FAP-expressing LL86 cells.

[0201] FIG. 37B shows that FAP multispecific BP sibrotuzumab, FAP multispecific BP 4G8, and FAP-multispecific BP 29B11 mimicked the cytotoxic activity of primary human NK cells isolated from donor RR01612 against FAP-expressing COLO829 cells.

[0202] FIG. 37C shows that FAP multispecific BP sibrotuzumab, FAP multispecific BP 4G8, and FAP-multispecific BP 29B11 mimicked the cytotoxic activity of primary human NK cells isolated from donor RR01612 against FAP-expressing U-87 MG cells.

[0203] All anti-FAP multispecific binding proteins stimulated primary NK cell cytotoxicity against human cancer cells more effectively than the corresponding mAbs bearing the same FAP-binding site.

[0204] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes.

[0205] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is thus indicated by the appended claims, rather than by the above description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. (a) A Fab that specifically binds to NKG2D and comprises an antibody heavy chain variable domain (VH) and a light chain variable domain (VL), (i) the VH of the Fab comprises a heavy chain CDR1 amino acid sequence of SEQ ID NO: 168, a heavy chain CDR2 amino acid sequence of SEQ ID NO: 88, and a heavy chain CDR3 amino acid sequence of SEQ ID NO: 170, and the VL of the Fab comprises a light chain CDR1 amino acid sequence of SEQ ID NO: 90, a light chain CDR2 amino acid sequence of SEQ ID NO: 91, and a light chain CDR3 amino acid sequence of SEQ ID NO: 92, or (ii) a Fab, wherein the VH of the Fab comprises a heavy chain CDR1 amino acid sequence of SEQ ID NO: 163, a heavy chain CDR2 amino acid sequence of SEQ ID NO: 88, and a heavy chain CDR3 amino acid sequence of SEQ ID NO: 164, and the VL of the Fab comprises a light chain CDR1 amino acid sequence of SEQ ID NO: 90, a light chain CDR2 amino acid sequence of SEQ ID NO: 91, and a light chain CDR3 amino acid sequence of SEQ ID NO: 92; (b) a single chain variable fragment (scFv) that specifically binds to fibroblast activation protein (FAP) and comprises a VH and a VL; (c) a first antibody Fc domain and a second antibody Fc domain that form a heterodimer that specifically binds to CD16; A multispecific binding protein comprising: A multispecific binding protein, wherein the first and second antibody Fc domains comprise different amino acid mutations that promote heterodimerization.

2. The multispecific binding protein of claim 1, wherein the Fab specifically binds to NKG2D in humans and cynomolgus monkeys.

3. A multispecific binding protein as described in claim 1 or 2, wherein the VH of the Fab is at least 90% identical to the amino acid sequence of SEQ ID NO:

167.

4. The multispecific binding protein of claim 1, wherein the VH of the Fab is at least 90% identical to SEQ ID NO:167 and the VL of the Fab is at least 90% identical to SEQ ID NO:

86. (a) the VH of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO:114, the VL of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO:118, the VH of the scFv comprises a heavy chain CDR1 amino acid sequence of SEQ ID NO:147, a heavy chain CDR2 amino acid sequence of SEQ ID NO:148, and a heavy chain CDR3 amino acid sequence of SEQ ID NO:117, and the VL of the scFv comprises a light chain CDR1 amino acid sequence of SEQ ID NO:149, a light chain CDR2 amino acid sequence of SEQ ID NO:120, and a light chain CDR3 amino acid sequence of SEQ ID NO:121; (b) the VH of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 131, and the VL of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 135, wherein the VH of the scFv comprises a heavy chain CDR1 amino acid sequence of SEQ ID NO: 132, a heavy chain CDR2 amino acid sequence of SEQ ID NO: 133, and a heavy chain CDR3 amino acid sequence of SEQ ID NO: 134, and the VL of the scFv comprises a light chain CDR1 amino acid sequence of SEQ ID NO: 136, a light chain CDR2 amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 amino acid sequence of SEQ ID NO: 138; (c) the VH of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 139, and the VL of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 143, wherein the VH of the scFv comprises a heavy chain CDR1 amino acid sequence of SEQ ID NO: 140, a heavy chain CDR2 amino acid sequence of SEQ ID NO: 141, and a heavy chain CDR3 amino acid sequence of SEQ ID NO: 142, and the VL of the scFv comprises a light chain CDR1 amino acid sequence of SEQ ID NO: 144, a light chain CDR2 amino acid sequence of SEQ ID NO: 145, and a light chain CDR3 amino acid sequence of SEQ ID NO: 146; or (d) the VH of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 122, and the VL of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 126, the VH of the scFv comprising a heavy chain CDR1 amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 amino acid sequence of SEQ ID NO: 124, and a heavy chain CDR3 amino acid sequence of SEQ ID NO: 125, and the VL of the scFv comprising a light chain CDR1 amino acid sequence of SEQ ID NO: 127, a light chain CDR2 amino acid sequence of SEQ ID NO: 128, and a light chain CDR3 amino acid sequence of SEQ ID NO: 129; The multispecific binding protein of any one of claims 1 to 4.

6. The scFv, (a) VH and VL CDR1, CDR2, and CDR3 sequences selected from the group consisting of SEQ ID NOs: 114 and 118, 131 and 135, 139 and 143, and 122 and 126, respectively; (b) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 147; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 148; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 117; The light chain CDR1 amino acid sequence of SEQ ID NO: 149; The light chain CDR2 amino acid sequence of SEQ ID NO: 120; and The light chain CDR3 amino acid sequence of SEQ ID NO: 121; (c) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 123; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 124; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 125; The light chain CDR1 amino acid sequence of SEQ ID NO: 127; The light chain CDR2 amino acid sequence of SEQ ID NO: 128; and The light chain CDR3 amino acid sequence of SEQ ID NO: 129; (d) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 132; Heavy chain CDR2 amino acid sequence of SEQ ID NO: 133; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 134; Light chain CDR1 amino acid sequence of SEQ ID NO: 136; The light chain CDR2 amino acid sequence of SEQ ID NO: 137; and The light chain CDR3 amino acid sequence of SEQ ID NO: 138; or (e) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 140; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 141; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 142; Light chain CDR1 amino acid sequence of SEQ ID NO: 144; The light chain CDR2 amino acid sequence of SEQ ID NO: 145; and Light chain CDR3 amino acid sequence of SEQ ID NO: 146 6. The multispecific binding protein of claim 1, comprising:

7. The multispecific binding protein of claim 1, wherein each antibody Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody.

8. A multispecific binding protein according to any one of claims 1 to 6, wherein each antibody 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.

9. A formulation comprising the multispecific binding protein of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

10. A cell comprising one or more nucleic acids encoding a multispecific binding protein according to any one of claims 1 to 8.

11. A composition comprising a multispecific binding protein according to any one of claims 1 to 8 or a formulation according to claim 9 for use in therapy.

12. A composition comprising a multispecific binding protein according to any one of claims 1 to 8 or a formulation according to claim 9 for use in a method for promoting the death of tumor cells, the method comprising exposing the tumor cells and natural killer cells to an effective amount of the multispecific binding protein.

13. A composition comprising a multispecific binding protein according to any one of claims 1 to 8 or a formulation according to claim 9 for use in a method for treating cancer, the method comprising administering to a patient an effective amount of the multispecific binding protein or formulation.

14. The composition or formulation for use according to claim 13, wherein the cancer is selected from the group consisting of invasive ductal carcinoma, pancreatic ductal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, mesothelioma, gastric cancer, pancreatic cancer, head and neck cancer, liver cancer, endometrial cancer, neuroendocrine carcinoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, neurilemmoma, melanoma, and glioma.

15. A composition comprising a multispecific binding protein according to any one of claims 1 to 8 or a formulation according to claim 9 for use in a method for treating an autoimmune disease, the method comprising administering to a patient an effective amount of the multispecific binding protein or formulation.

16. The composition or formulation for use according to claim 15, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Graves' disease, Sjogren's syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, and inflammatory destructive arthritis.

17. A composition comprising a multispecific binding protein according to any one of claims 1 to 8 or a formulation according to claim 9 for use in a method for treating fibrosis, the method comprising administering to a patient an effective amount of the multispecific binding protein or formulation.

18. The composition or formulation for use according to claim 17, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and cardiac fibrosis.

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