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

Abstract

We provide cancer immunotherapy. [Solution] A multispecific binding protein that binds to the NKG2D receptor, a tumor-associated antigen selected from CD16 and CD37, CD20, CD19, CD22, CD30, CD52 and CD133, and pharmaceutical compositions and therapeutic methods useful for treating cancer are described.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 62 / 510,173 filed on 23 May 2017, U.S. Provisional Patent Application No. 62 / 539,396 filed on 31 July 2017, U.S. Provisional Patent Application No. 62 / 539,416 filed on 31 July 2017, U.S. Provisional Patent Application No. 62 / 539,419 filed on 31 July 2017, U.S. Provisional Patent Application No. 62 / 546,292 filed on 16 August 2017, U.S. Provisional Patent Application No. 62 / 546,296 filed on 16 August 2017, and U.S. Provisional Patent Application No. 62 / 552,146 filed on 30 August 2017, the entire contents of each of these are incorporated herein by reference for all purposes.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on 21 May 2018, named DFY-022WO.txt, and is 212kb in size.

[0003] Field of Invention The present invention relates to a multispecific binding protein that binds to NKG2D, CD16, and tumor-associated antigens selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. [Background technology]

[0004] background Cancer remains a significant health problem despite the substantial research efforts and scientific advancements reported in the literature for treating the disease. Blood and bone marrow cancers are among the most frequently diagnosed types of cancer (including multiple myeloma, leukemia, and lymphoma). Current treatment options for these cancers are not effective for all patients and / or may have substantial adverse side effects. Other types of cancer also remain difficult to treat with existing treatment options.

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

[0006] Natural killer (NK) cells are components of the innate immune system and make up about 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were initially characterized by their ability to effectively kill tumor cells without requiring prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells, namely through cytotoxic granules containing perforin and granzymes, as well as through the death receptor pathway. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines, which promote the recruitment of other leukocytes to target tissues.

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

[0008] CD37 (a member of the tetraspanin superfamily of cell surface antigens) is expressed on virtually all mature B lymphocytes but not on pro-B cells or plasma cells. It is a lineage-specific B cell antigen and is absent or minimally expressed on normal T cells, thymocytes, monocytes, granulocytes, platelets, natural killer (NK) cells, and erythrocytes. Furthermore, CD37 is expressed on malignancies derived from peripheral mature B cells (e.g., B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), non-Hodgkin lymphoma, and acute myeloid leukemia).

[0009] CD20 is an activated glycosylated phosphoprotein expressed on the surface of B cells during B cell differentiation from the pro-B cell phase to the mature phase. It plays a role in B cell development and differentiation into plasma cells. CD20 is also found on chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, and B cell malignancies.

[0010] CD19 is a transmembrane glycoprotein expressed on the surface of B lymphocytes from the earliest recognizable B lineage cells during development into B cell blasts. It primarily acts as a B cell coreceptor, along with CD21 and CD81. CD19 is expressed in many cancers (e.g., chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, multiple myeloma, B cell malignancies, and acute myeloid leukemia).

[0011] CD22 (B-cell-restricted phosphoglycoprotein) is expressed on the surface of mature B cells and, to a lower degree, on some immature B cells. It functions as an inhibitory receptor for B-cell receptor (BCR) signaling. Furthermore, CD22 is expressed in cancer cells (e.g., chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, and hairy cell leukemia).

[0012] CD30 is a member of the tumor necrosis factor receptor (TNFR) superfamily, specifically TNFR8. CD30 is expressed on activated lymphocytes and several other normal cells. Its signaling activates the NF-κB transcription factor, resulting in pleiotropic regulation of gene function. CD30 is a characteristic marker of classical Hodgkin lymphoma, anaplastic large cell lymphoma, and embryonic cell carcinoma, and is expressed on a subset of aggressive T-cell and B-cell neoplasms. Its limited expression on normal cells makes it an attractive candidate for targeted therapy.

[0013] CAMPATH-1 (also known as Cluster of Differentiation 52 (CD52)) is a 12-amino acid glycosylphosphatidylinositol (GPI)-anchored peptide. CD52 is expressed on the cell membrane of mature B lymphocytes, T lymphocytes, monocytes, and dendritic cells, but is not expressed on the stem cells from which these lymphocytes are derived. Furthermore, CD52 is found in the male reproductive tract and is present on the surface of mature sperm cells. CD52 is associated with certain cancers including chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, peripheral T-cell lymphoma and T-cell prolymphocytic leukemia, B-cell malignancies, non-Hodgkin's lymphoma, Hodgkin's lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia-lymphoma, mature T / natural killer (NK) cell neoplasms, and thymoma. CD133 is a pentaspan transmembrane glycoprotein that is primarily identified in human hematopoietic stem cells and progenitor cells. To date, the physiological role of this surface receptor remains unknown. However, CD133 has been identified as a cancer stem cell marker in various cancers, including breast cancer, colon cancer, prostate cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, renal cancer, uterine cancer and testicular germ cell cancer, acute myeloid leukemia, acute lymphoblastic leukemia, glioma, glioblastoma, and squamous cell carcinoma of the head and neck. CD133 can interact with p85 to activate the PI3K / AKT / mTOR signaling pathway in cancer stem cells, and this activation consequently stimulates cancer stem cells and promotes their tumorigenic capacity. [Prior Art Document] [Patent Document]

[0014] [Patent Document 1] International Publication No. WO 2016 / 134371 [Patent Document 2] International Publication No. WO 2015 / 095412 [Summary of the Invention] [Means for Solving the Problems]

[0015] Abstract The present invention provides multispecific binding proteins that bind to the NKG2D receptor and the CD16 receptor in natural killer cells, as well as to tumor-associated antigens selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. Such proteins can associate with two or more NK-activating receptors and can block the binding of native ligands to NKG2D. In certain embodiments, the proteins can stimulate NK cells in humans. In some embodiments, the proteins can stimulate NK cells in humans as well as in other species such as rodents and cynomolgus monkeys. Various aspects and embodiments of the present invention are described in further detail below.

[0016] Accordingly, one aspect of the present invention provides a protein incorporating a first antigen-binding site that binds to NKG2D, a second antigen-binding site that binds to a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133, and a third antigen-binding site that binds to CD16, or an antibody Fc domain, a portion thereof, or a third antigen-binding site that binds to CD16.

[0017] Each antigen-binding site may incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (for example, arranged like an antibody or fused together to form an scFv), or one or more antigen-binding sites may be V like a camelid antibody. H H antibodies or V antibodies found in cartilaginous fish NAR It may also be a single-domain antibody, such as an antibody.

[0018] In one embodiment, the present invention provides a multispecific binding protein that binds to the NKG2D receptor and the CD16 receptor on natural killer cells, as well as to a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. The NKG2D binding site includes a heavy chain variable domain that is at least 90% identical to an amino acid selected from SEQ ID NOs. 1, SEQ ID NOs. 41, SEQ ID NOs. 49, SEQ ID NOs. 57, SEQ ID NOs. 59, SEQ ID NOs. 61, SEQ ID NOs. 69, SEQ ID NOs. 77, SEQ ID NOs. 85, and SEQ ID NOs. 93.

[0019] In some embodiments, the first antigen-binding site that binds to NKG2D may have, for example, an amino acid sequence identical to that of SEQ ID NO: 1 by at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), and / or may incorporate a heavy chain variable domain associated with SEQ ID NO: 1 by incorporating an amino acid sequence identical to the CDR1 (SEQ ID NO: 105), CDR2 (SEQ ID NO: 106), and CDR3 (SEQ ID NO: 107) sequences of SEQ ID NO: 1. The heavy chain variable domain associated with SEQ ID NO: 1 can bind to various light chain variable domains to form an NKG2D binding site. For example, the first antigen-binding site incorporating the heavy chain variable domain related to SEQ ID NO: 1 may further incorporate a light chain variable domain selected from any one of the sequences related to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40. For example, the first antigen-binding site incorporates a heavy chain variable domain having an amino acid sequence identical to at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence identical to at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of 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).

[0020] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 41 and a light chain variable domain associated with SEQ ID NO: 42. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 41 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45) sequences of sequence number 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 46), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 48) sequences of SEQ ID NO: 42.

[0021] In other embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 49 and a light chain variable domain associated with SEQ ID NO: 50. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 49 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 53) sequences of SEQ ID NO: 49. Similarly, the light chain variable domain of the second antigen-binding site 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 54), CDR2 (SEQ ID NO: 55), and CDR3 (SEQ ID NO: 56) sequences of SEQ ID NO: 50.

[0022] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 57 and a light chain variable domain associated with SEQ ID NO: 58, for example, by having amino acid sequences that are 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.

[0023] In another embodiment, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 59 and a light chain variable domain associated with SEQ ID NO: 60. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 59 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 324), CDR2 (SEQ ID NO: 325), and CDR3 (SEQ ID NO: 326) 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 327), CDR2 (SEQ ID NO: 328), and CDR3 (SEQ ID NO: 329) sequences of SEQ ID NO: 60.

[0024] In some embodiments, the first antigen-binding site that binds to NKG2D may incorporate a heavy chain variable domain associated with SEQ ID NO: 61 and a light chain variable domain associated with SEQ ID NO: 62. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 61 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 63), CDR2 (SEQ ID NO: 64), and CDR3 (SEQ ID NO: 65) sequences of SEQ ID NO: 61. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 62 and / or incorporate the same amino acid sequence as 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 associated with SEQ ID NO: 69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 69 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 71), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73) sequences of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen-binding site 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76) sequences of SEQ ID NO: 70.

[0025] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 77 and a light chain variable domain associated with SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 79), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81) sequences of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen-binding site 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 82), CDR2 (SEQ ID NO: 83), and CDR3 (SEQ ID NO: 84) sequences of SEQ ID NO: 78.

[0026] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 85 and a light chain variable domain associated with SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 87), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89) sequences of SEQ ID NO: 85. Similarly, the light chain variable domain of the second antigen-binding site 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 90), CDR2 (SEQ ID NO: 91), and CDR3 (SEQ ID NO: 92) sequences of SEQ ID NO: 86.

[0027] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 93 and a light chain variable domain associated with 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 95), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 97) sequences of SEQ ID NO: 93. Similarly, the light chain variable domain of the second antigen-binding site 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 incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 98), CDR2 (SEQ ID NO: 99), and CDR3 (SEQ ID NO: 100) sequences of SEQ ID NO: 94.

[0028] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 101 and a light chain variable domain associated with SEQ ID NO: 102, for example, by having amino acid sequences that are 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. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain associated with SEQ ID NO: 103 and a light chain variable domain associated with SEQ ID NO: 104, for example, by having amino acid sequences that are 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.

[0029] In some embodiments, the second antigen-binding site that binds to CD37 may incorporate a heavy chain variable domain associated with SEQ ID NO: 109 and a light chain variable domain associated with SEQ ID NO: 113. 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: 109 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 110), CDR2 (SEQ ID NO: 111), and CDR3 (SEQ ID NO: 112) sequences of SEQ ID NO: 109. 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: 113 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 114), CDR2 (SEQ ID NO: 115), and CDR3 (SEQ ID NO: 116) sequences of SEQ ID NO: 113.

[0030] The second antigen-binding site that binds to CD37 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 117 and a light chain variable domain associated with SEQ ID NO: 121. 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: 117 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 118), CDR2 (SEQ ID NO: 119), and CDR3 (SEQ ID NO: 120) sequences of SEQ ID NO: 117. 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: 121 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 122), CDR2 (SEQ ID NO: 123), and CDR3 (SEQ ID NO: 124) sequences of SEQ ID NO: 121.

[0031] The second antigen-binding site that binds to CD37 may optionally incorporate the heavy chain variable domain associated with SEQ ID NO: 125 and the light chain variable domain associated with SEQ ID NO: 129. 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: 125 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 126), CDR2 (SEQ ID NO: 127), and CDR3 (SEQ ID NO: 128) sequences of SEQ ID NO: 125. 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: 129 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 130), CDR2 (SEQ ID NO: 131), and CDR3 (SEQ ID NO: 132) sequences of SEQ ID NO: 129.

[0032] In some embodiments, the second antigen-binding site that binds to CD20 may incorporate a heavy chain variable domain associated with SEQ ID NO: 134 and a light chain variable domain associated with SEQ ID NO: 138. 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: 134 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 135), CDR2 (SEQ ID NO: 136), and CDR3 (SEQ ID NO: 137) sequences of SEQ ID NO: 134. 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: 138 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 139), CDR2 (SEQ ID NO: 140), and CDR3 (SEQ ID NO: 141) sequences of SEQ ID NO: 138.

[0033] Alternatively, the second antigen-binding site that binds to CD20 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 142 and the light chain variable domain associated with SEQ ID NO: 146. 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: 142 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 143), CDR2 (SEQ ID NO: 144), and CDR3 (SEQ ID NO: 145) sequences of SEQ ID NO: 142. 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: 146 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 147), CDR2 (SEQ ID NO: 148), and CDR3 (SEQ ID NO: 149) sequences of SEQ ID NO: 146.

[0034] The second antigen-binding site that binds to CD20 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 150 and a light chain variable domain associated with SEQ ID NO: 154. 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: 150 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 151), CDR2 (SEQ ID NO: 152), and CDR3 (SEQ ID NO: 153) sequences of SEQ ID NO: 150. 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: 154 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 155), CDR2 (SEQ ID NO: 156), and CDR3 (SEQ ID NO: 157) sequences of SEQ ID NO: 154.

[0035] Alternatively, the second antigen-binding site that binds to CD20 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 158 and the light chain variable domain associated with SEQ ID NO: 162. 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: 158 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 159), CDR2 (SEQ ID NO: 160), and CDR3 (SEQ ID NO: 161) sequences of SEQ ID NO: 158. 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: 163 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 163), CDR2 (SEQ ID NO: 164), and CDR3 (SEQ ID NO: 165) sequences of SEQ ID NO: 162.

[0036] Alternatively, the second antigen-binding site that binds to CD20 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 166 and the light chain variable domain associated with SEQ ID NO: 170. 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: 166 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 167), CDR2 (SEQ ID NO: 168), and CDR3 (SEQ ID NO: 169) sequences of SEQ ID NO: 166. 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: 170 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 171), CDR2 (SEQ ID NO: 172), and CDR3 (SEQ ID NO: 173) sequences of SEQ ID NO: 170.

[0037] In some embodiments, the second antigen-binding site that binds to CD19 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 175 and a light chain variable domain associated with SEQ ID NO: 179. 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: 175 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 176), CDR2 (SEQ ID NO: 177), and CDR3 (SEQ ID NO: 178) sequences 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: 179 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 180), CDR2 (SEQ ID NO: 181), and CDR3 (SEQ ID NO: 182) sequences of SEQ ID NO: 179.

[0038] Alternatively, the second antigen-binding site that binds to CD19 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 183 and the light chain variable domain associated with SEQ ID NO: 187. 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: 183 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 184), CDR2 (SEQ ID NO: 185), and CDR3 (SEQ ID NO: 186) sequences 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: 187 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 188), CDR2 (SEQ ID NO: 189), and CDR3 (SEQ ID NO: 190) sequences of SEQ ID NO: 187.

[0039] Alternatively, the second antigen-binding site that binds to CD19 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 191 and the light chain variable domain associated with SEQ ID NO: 195. 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: 191 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 192), CDR2 (SEQ ID NO: 193), and CDR3 (SEQ ID NO: 194) sequences of SEQ ID NO: 191. 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: 195 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 196), CDR2 (SEQ ID NO: 197), and CDR3 (SEQ ID NO: 198) sequences of SEQ ID NO: 195. Alternatively, the second antigen-binding site that binds to CD19 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 199 and the light chain variable domain associated with SEQ ID NO: 203. 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: 199 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 200), CDR2 (SEQ ID NO: 201), and CDR3 (SEQ ID NO: 202) sequences of SEQ ID NO: 199. 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: 203 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 204), CDR2 (SEQ ID NO: 205), and CDR3 (SEQ ID NO: 206) sequences of SEQ ID NO: 203.

[0040] In some embodiments, the second antigen-binding site that binds to CD22 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 208 and a light chain variable domain associated with SEQ ID NO: 212. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 208 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 209), CDR2 (SEQ ID NO: 210), and CDR3 (SEQ ID NO: 211) sequences of SEQ ID NO: 208. 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: 212 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 213), CDR2 (SEQ ID NO: 214), and CDR3 (SEQ ID NO: 215) sequences of SEQ ID NO: 212.

[0041] Alternatively, the second antigen-binding site that binds to CD22 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 216 and the light chain variable domain associated with SEQ ID NO: 220. 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: 216 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 217), CDR2 (SEQ ID NO: 218), and CDR3 (SEQ ID NO: 219) sequences of SEQ ID NO: 216. 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 that of SEQ ID NO: 220 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 221), CDR2 (SEQ ID NO: 222), and CDR3 (SEQ ID NO: 223) sequences of SEQ ID NO: 220. Alternatively, the second antigen-binding site that binds to CD22 may, as necessary, incorporate the heavy chain variable domain associated with SEQ ID NO: 224 and the light chain variable domain associated with SEQ ID NO: 228. 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: 224 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 225), CDR2 (SEQ ID NO: 226), and CDR3 (SEQ ID NO: 227) sequences of SEQ ID NO: 224. 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: 228 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 229), CDR2 (SEQ ID NO: 230), and CDR3 (SEQ ID NO: 231) sequences of SEQ ID NO: 228.

[0042] Alternatively, the second antigen-binding site that binds to CD30 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 233 and the light chain variable domain associated with SEQ ID NO: 237. 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: 233 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 234), CDR2 (SEQ ID NO: 235), and CDR3 (SEQ ID NO: 236) sequences of SEQ ID NO: 233. 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: 237 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 238), CDR2 (SEQ ID NO: 239), and CDR3 (SEQ ID NO: 240) sequences of SEQ ID NO: 237.

[0043] Alternatively, the second antigen-binding site that binds to CD30 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 241 and the light chain variable domain associated with SEQ ID NO: 245. 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: 241 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 242), CDR2 (SEQ ID NO: 243), and CDR3 (SEQ ID NO: 244) sequences of SEQ ID NO: 241. 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: 245 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 246), CDR2 (SEQ ID NO: 247), and CDR3 (SEQ ID NO: 248) sequences of SEQ ID NO: 245.

[0044] Alternatively, the second antigen-binding site that binds to CD30 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 249 and the light chain variable domain associated with SEQ ID NO: 253. 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: 249 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 250), CDR2 (SEQ ID NO: 251), and CDR3 (SEQ ID NO: 252) sequences of SEQ ID NO: 249. 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: 253 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 254), CDR2 (SEQ ID NO: 255), and CDR3 (SEQ ID NO: 256) sequences of SEQ ID NO: 253.

[0045] Alternatively, the second antigen-binding site that binds to CD30 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 257 and the light chain variable domain associated with SEQ ID NO: 261. 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: 257 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 258), CDR2 (SEQ ID NO: 259), and CDR3 (SEQ ID NO: 260) sequences of SEQ ID NO: 257. 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: 261 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 262), CDR2 (SEQ ID NO: 263), and CDR3 (SEQ ID NO: 264) sequences of SEQ ID NO: 261.

[0046] Alternatively, the second antigen-binding site that binds to CD30 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 265 and the light chain variable domain associated with SEQ ID NO: 269. 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: 265 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 266), CDR2 (SEQ ID NO: 267), and CDR3 (SEQ ID NO: 268) sequences of SEQ ID NO: 265. 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 that of SEQ ID NO: 269 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 270), CDR2 (SEQ ID NO: 271), and CDR3 (SEQ ID NO: 272) sequences of SEQ ID NO: 269.

[0047] In some embodiments, the second antigen-binding site that binds to CD52 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 274 and a light chain variable domain associated with SEQ ID NO: 278. 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: 274 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 275), CDR2 (SEQ ID NO: 276), and CDR3 (SEQ ID NO: 278) sequences of SEQ ID NO: 274. 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: 278 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 279), CDR2 (SEQ ID NO: 280), and CDR3 (SEQ ID NO: 281) sequences of SEQ ID NO: 278.

[0048] Alternatively, the second antigen-binding site that binds to CD52 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 282 and the light chain variable domain associated with SEQ ID NO: 286. 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: 282 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 283), CDR2 (SEQ ID NO: 284), and CDR3 (SEQ ID NO: 285) sequences of SEQ ID NO: 282. 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: 286 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 287), CDR2 (SEQ ID NO: 288), and CDR3 (SEQ ID NO: 289) sequences of SEQ ID NO: 286.

[0049] In some embodiments, the second antigen-binding site that binds to CD133 may optionally incorporate a heavy chain variable domain associated with SEQ ID NO: 291 and a light chain variable domain associated with SEQ ID NO: 295. 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: 291 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 292), CDR2 (SEQ ID NO: 293), and CDR3 (SEQ ID NO: 294) sequences of SEQ ID NO: 291. 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: 295 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 296), CDR2 (SEQ ID NO: 297), and CDR3 (SEQ ID NO: 298) sequences of SEQ ID NO: 295.

[0050] Alternatively, the second antigen-binding site that binds to CD133 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 299 and the light chain variable domain associated with SEQ ID NO: 303. 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: 299 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 300), CDR2 (SEQ ID NO: 301), and CDR3 (SEQ ID NO: 302) sequences of SEQ ID NO: 299. 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: 303 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 304), CDR2 (SEQ ID NO: 305), and CDR3 (SEQ ID NO: 306) sequences of SEQ ID NO: 303.

[0051] Alternatively, the second antigen-binding site that binds to CD133 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 307 and the light chain variable domain associated with SEQ ID NO: 311. 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: 307 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 308), CDR2 (SEQ ID NO: 309), and CDR3 (SEQ ID NO: 310) sequences of SEQ ID NO: 307. 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: 311 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 312), CDR2 (SEQ ID NO: 313), and CDR3 (SEQ ID NO: 314) sequences of SEQ ID NO: 311.

[0052] Alternatively, the second antigen-binding site that binds to CD133 may, as needed, incorporate the heavy chain variable domain associated with SEQ ID NO: 315 and the light chain variable domain associated with SEQ ID NO: 319. 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: 315 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 316), CDR2 (SEQ ID NO: 317), and CDR3 (SEQ ID NO: 318) sequences of SEQ ID NO: 315. 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 that of SEQ ID NO: 319 and / or incorporate the same amino acid sequence as the CDR1 (SEQ ID NO: 320), CDR2 (SEQ ID NO: 321), and CDR3 (SEQ ID NO: 322) sequences of SEQ ID NO: 319.

[0053] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having the same amino acid sequence as the light chain variable domain present in the first antigen-binding site.

[0054] In some embodiments, the protein incorporates a portion of the antibody Fc domain sufficient to bind to CD16, where the antibody Fc domain includes the hinge and CH2 domains and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0055] Also provided are formulations containing any one of the proteins described herein, cells containing one or more nucleic acids expressing this protein, and methods for enhancing tumor cell death using this protein.

[0056] Another aspect of the present invention provides a method for treating a patient's cancer. This method involves administering a therapeutically effective dose of the multispecific binding proteins described herein to a patient in need. Cancers treated with CD37-targeted multispecific binding proteins include any cancer expressing CD37, such as B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), non-Hodgkin lymphoma, and acute myeloid leukemia. Cancers treated with CD20-targeted multispecific binding proteins include any cancer expressing CD20, such as chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, and B-cell malignancies. Cancers treated with CD19-targeted multispecific binding proteins include any cancer expressing CD19, such as chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, multiple myeloma, and acute myeloid leukemia. Cancers treated with multispecific binding proteins targeting CD22 include any cancers expressing chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, and hairy cell leukemia. Cancers treated with multispecific binding proteins targeting CD30 include any cancers expressing CD30, such as Hodgkin lymphoma, anaplastic large cell lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia-lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, and embryonal cell carcinoma. Cancers treated with CD52-targeting multispecific binding proteins include any CD52-expressing cancer, such as chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, peripheral T-cell lymphoma and T-cell prelymphocytic leukemia, B-cell malignancies, non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia-lymphoma, mature T / natural killer (NK) cell neoplasms, and thymoma.Cancers treated with multispecific binding proteins targeting CD133 include any cancer expressing CD133, such as breast cancer, colon cancer, prostate cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, kidney cancer, uterine cancer, testicular germ cell cancer, acute myeloid leukemia, acute lymphoblastic leukemia, glioma, glioblastoma, and squamous cell carcinoma of the head and neck. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 shows a diagram of a heterodimer multispecific antibody. Each arm may represent either an NKG2D binding domain or a CD37, CD20, CD19, CD22, CD30, CD52, or CD133 binding domain. In some embodiments, the NKG2D binding domain and the antigen-binding domain may share a common light chain.

[0058] [Figure 2] Figure 2 shows a heterodimerized multispecific antibody. Either the NKG2D binding domain or the antigen-binding domain selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133 can take the scFv format (right arm).

[0059] [Figure 3] Figure 3 is a line graph showing the binding affinity of NKG2D-binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

[0060] [Figure 4] Figure 4 is a line graph showing the binding affinity of the NKG2D binding domain (listed as clones) to recombinant cynomolgus monkey NKG2D in an ELISA assay.

[0061] [Figure 5]Figure 5 is a line graph showing the binding affinity of the NKG2D binding domain (listed as clones) to recombinant mouse NKG2D in an ELISA assay.

[0062] [Figure 6] Figure 6 is a bar graph showing the binding of NKG2D-binding domains (listed as clones) to EL4 cells expressing human NKG2D, as determined by flow cytometry, and shows the mean fluorescence intensity (MFI) multiplier (FOB) relative to the background.

[0063] [Figure 7] Figure 7 is a bar graph showing the binding of NKG2D-binding domains (listed as clones) to EL4 cells expressing mouse NKG2D, as determined by flow cytometry, and shows the mean fluorescence intensity (MFI) multiplier (FOB) relative to the background.

[0064] [Figure 8] Figure 8 is a line graph showing the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant human NKG2D-Fc, due to competition with the natural ligand ULBP-6.

[0065] [Figure 9] Figure 9 is a line graph showing the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant human NKG2D-Fc, due to competition with the natural ligand MICA.

[0066] [Figure 10] Figure 10 is a line graph showing the specific binding affinity of the NKG2D binding domain (listed as clones) to recombinant mouse NKG2D-Fc, due to competition with the natural ligand Rae-1 delta.

[0067] [Figure 11]Figure 11 is a bar graph showing the activation of human NKG2D by the NKG2D-binding domain (listed as clones), calculated by quantifying the percentage of TNF-α-positive cells expressing human NKG2D-CD3 zeta fusion protein.

[0068] [Figure 12] Figure 12 is a bar graph showing the activation of mouse NKG2D by the NKG2D-binding domain (listed as clones), calculated by quantifying the percentage of TNF-α-positive cells expressing the mouse NKG2D-CD3 zeta fusion protein.

[0069] [Figure 13] Figure 13 is a bar graph showing the activation of human NK cells by the NKG2D binding domain (listed as clones).

[0070] [Figure 14] Figure 14 is a bar graph showing the activation of human NK cells by the NKG2D binding domain (listed as clones).

[0071] [Figure 15] Figure 15 is a bar graph showing the activation of mouse NK cells by the NKG2D binding domain (listed as clones).

[0072] [Figure 16] Figure 16 is a bar graph showing the activation of mouse NK cells by the NKG2D binding domain (listed as clones).

[0073] [Figure 17] Figure 17 is a bar graph showing the cytotoxic effects of the NKG2D binding domain (listed as clones) on tumor cells.

[0074] [Figure 18]Figure 18 is a bar graph showing the melting temperatures of the NKG2D-binding domain (listed as clones) as measured by differential scanning fluorescence quantification.

[0075] [Figure 19A] Figures 19A–19C are bar graphs showing the synergistic activation of NK cells using CD16 and NKG2D binding. Figure 19A shows the level of CD107a, Figure 19B shows the level of IFNγ, and Figure 19C shows the levels of both CD107a and IFNγ. The graphs show the mean (n=2) ± SD. The data are representative of five independent experiments using five different healthy donors. [Figure 19B] Same as above. [Figure 19C] Same as above.

[0076] [Figure 20] Figure 20 shows TriNKET in Triomab form, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies derived from two parent antibodies, each half-antibody having one light chain and one heavy chain. The Triomab form can be a heterodimer construct containing half rat antibody and half mouse antibody.

[0077] [Figure 21] Figure 21 shows TriNKET in the KiH common light chain (LC) form using the knob-into-holes (KIH) technique. KiH is a heterodimer containing two Fabs that bind to targets 1 and 2, as well as Fc stabilized by a heterodimerizing mutation. TriNKET in KiH format can be a heterodimer construct having two Fabs that bind to targets 1 and 2, containing two distinct heavy chains and a common light chain that pairs with both heavy chains.

[0078] [Figure 22]Figure 22 shows TriNKET in a bivariate variable-domain immunoglobulin (DVD-Ig®) form, which combines the target-binding domains of two monoclonal antibodies via a naturally occurring mobile linker, resulting in a tetravalent IgG-like molecule. DVD-Ig® is a homodimer construct in which a variable domain targeting antigen 2 is fused to the N-terminus of a variable domain of Fab targeting antigen 1. The construct contains normal Fc.

[0079] [Figure 23] Figure 23 shows TriNKET in an ortho-Fab interface (ortho-Fab) morphology, which is a heterodimer construct containing two Fabs fused to target 1 and target 2 at Fc. LC-HC pairing is ensured by the ortho-interface. Heterodimerization is ensured by mutation at Fc.

[0080] [Figure 24] Figure 24 shows TriNKET in 2in1 IG format.

[0081] [Figure 25] Figure 25 shows TriNKET in ES form, a heterodimer construct containing two different Fabs that bind to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in Fc.

[0082] [Figure 26] Figure 26 shows TriNKET in the Fab arm exchange form, i.e., an antibody that has become a bispecific antibody as a result of exchanging the Fab arms by swapping the heavy chain and the bound light chain (half) with the heavy-light chain pair of another molecule. The Fab arm exchange form (cFae) is a heterodimer containing two Fabs that bind to targets 1 and 2, as well as an Fc stabilized by a heterodimerizing mutation.

[0083] [Figure 27]Figure 27 shows TriNKET in SEED Body form, a heterodimer containing two Fabs that bind to targets 1 and 2, as well as Fc stabilized by a heterodimerizing mutation.

[0084] [Figure 28] Figure 28 shows TriNKET in the LuZ-Y morphology, which utilizes a leucine zipper to induce heterodimerization of two different HCs. The LuZ-Y morphology is a heterodimer containing two different scFabs that bind to targets 1 and 2 fused to Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of Fc.

[0085] [Figure 29] Figure 29 shows TriNKET in Cov-X-Body morphology.

[0086] [Figure 30A] Figures 30A and 30B show TriNKET in κλ-Body form, which is a heterodimer construct having two different Fabs fused to an Fc stabilized by a heterodimerizing mutation. Fab1, targeting antigen 1, contains kappa LC, while the second Fab, targeting antigen 2, contains lambda LC. Figure 30A is an exemplary figure of one form of κλ-Body, and Figure 30B is an exemplary figure of another κλ-Body. [Figure 30B] Same as above.

[0087] [Figure 31] Figure 31 shows an Oasc-Fab heterodimer construct containing a Fab that binds to target 1 and an scFab that binds to target 2, fused to Fc. Heterodimerization is confirmed by mutation in Fc.

[0088] [Figure 32]Figure 32 shows DuetMab, a heterodimer construct containing two different Fabs that bind to antigens 1 and 2, as well as Fc stabilized by heterodimerizing mutations. Fabs 1 and 2 contain differential SS crosslinks that ensure precise pairing of the light chain (LC) and heavy chain (HC).

[0089] [Figure 33] Figure 33 shows a CrossmAb, a heterodimer construct having two different Fabs that bind to targets 1 and 2 fused to Fc stabilized by heterodimerization. The CL domain and CH1 domain are switched with the Vh domain and VL domain; for example, CH1 is fused inline with VL, while CL is fused inline with VH.

[0090] [Figure 34] Figure 34 shows Fit-Ig, a homodimer construct in which the Fab that binds to antigen 2 is fused to the N-terminus of the HC of the Fab that binds to antigen 1. This construct contains wild-type Fc.

[0091] [Figure 35] Figure 35 is a histogram showing the binding of CD20-targeted TriNKET to NKG2D expressed on EL4 cells. Unstained EL4 cells were used as a negative control for the fluorescence signal. Unstained: black fill; F04-TriNKET-CD20: solid line; CD26-TriNKET-CD20: dashed line.

[0092] [Figure 36] Figure 36 is a histogram showing the binding of CD20-targeted TriNKET to CD20 expressed on Raji human lymphoma cells. Unstained cells were used as a negative control for the fluorescence signal. Unstained: black fill; F04-TriNKET-CD20: solid line; CD26-TriNKET-CD20: dashed line.

[0093] [Figure 37] Figure 37 is a bar graph showing that human NK cells were activated by TriNKET when co-cultured with CD20+ Raji B-cell lymphoma cells (indicated by an increase in CD107a / IFN-γ double-positive cells).

[0094] [Figure 38] Figure 38 is a line graph showing the TriNKET-mediated cytotoxic activity of human NK cells against CD20-expressing Raji B-cell lymphoma cells.

[0095] [Figure 39] Figure 39 is a line graph showing that TriNKET mediated higher NK cell cytotoxicity against CD20-expressing Raji B-cell lymphoma cells than parental anti-CD20 monoclonal antibodies. [Modes for carrying out the invention]

[0096] Detailed explanation The present invention provides multispecific binding proteins that bind to NKG2D receptors and CD16 receptors on natural killer cells, as well as to tumor-associated antigens selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. In some embodiments, the multispecific proteins further include additional antigen-binding sites that bind to tumor-associated antigens. The present invention also provides pharmaceutical compositions comprising such multispecific binding proteins, as well as therapeutic methods using such multispecific proteins and pharmaceutical compositions for purposes such as the treatment of cancer. Various aspects of the present invention are described below in several sections. However, the aspects of the present invention described in one particular section are not limited to any particular section.

[0097] To facilitate understanding of the present invention, several terms and phrases are defined below.

[0098] As used herein, the terms “a” and “an” mean “one or more,” and include the plural unless the context is appropriate.

[0099] As used herein, the term “antigen-binding site” refers to a portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly branched stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” interposed between more conserved adjacent stretches, known as “framework regions” or “FR.” Thus, the term “FR” refers to the naturally occurring amino acid sequences 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 bound antigen, and the three hypervariable regions of the heavy and light chains are referred to as “complementarity-determining regions” or “CDR.” In certain animals, such as camels and cartilaginous fish, antigen-binding sites are formed by a single antibody chain that provides a "single-domain antibody." Antigen-binding sites are present in intact antibodies, in antigen-binding fragments of antibodies that retain an antigen-binding surface, or in recombinant polypeptides such as scFv, and a peptide linker can be used to link the heavy-chain variable domain to the light-chain variable domain in a single polypeptide.

[0100] As used herein, the term “tumor-associated antigen” means any antigen, including but not limited to cancer-associated proteins, glycoproteins, gangliosides, carbohydrates, and lipids. Such antigens may be expressed in malignant cells or in the tumor microenvironment, such as in tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immunoinfiltrates.

[0101] As used herein, the terms “subject” and “patient” refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably include humans.

[0102] As used herein, the term “effective dose” refers to an amount of the compound (e.g., the compound of the present invention) sufficient to produce a beneficial or desired result. The effective dose may be administered in one or more doses, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term “treat” includes any effect, such as improving, reducing, mitigating, modulating, improving or eliminating a condition, disease, disorder, or any other condition, disease, disorder, or any improvement of its symptoms.

[0103] As used herein, the term “pharmaceutical composition” refers to a combination of an activator and an inactive or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0104] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA

[1975] .

[0105] As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt (e.g., an acid or base) of the compounds of the present invention that, when administered to a subject, can provide the compounds of the present invention or their active metabolites or residues. As is known to those skilled in the art, “salts” of the compounds of the present invention can be derived from inorganic or organic acids and 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 others. Other acids, such as oxalic acid, are not pharmaceutically acceptable in themselves, but can be used to prepare salts that are useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0106] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and formula NW4. + (In the formula, W is C 1~4 This includes compounds that are alkyl.

[0107] Exemplary salts include, but are not limited to, acetates, adipines, alginates, aspartates, benzoates, benzenesulfons, bisulfates, butyrates, citrates, camphorates, camphor sulfons, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfons, fumarates, flucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobroms, hydroiodides, 2-hydroxyethanesulfons, lactates, maleates, methanesulfons, 2-naphthalenesulfons, nicotinates, oxalates, palmates, pectins, persulfates, phenylpropionates, picrinates, pivalates, propions, succinates, tartrates, thiocyans, tosylates, and undecanoates. Other examples of salts include Na + NH4+ and NW4 + (In the formula, W is C 1~4 The present invention includes anions of the compound formulated with appropriate cations (such as alkyl groups).

[0108] For therapeutic use, salts of the compounds of the present invention are intended to be pharmaceutically acceptable. However, salts of pharmaceutically unacceptable acids and bases can also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0109] Throughout the descriptions in which a composition is described as having, including, or comprising a particular component, or a process and method having, including, or comprising a particular step, it is intended that there exist compositions of the present invention that are essentially composed of or comprise the listed components, and processes and methods of the present invention that are essentially composed of or comprise the listed process steps.

[0110] As a general rule, unless otherwise specified, percentages of compositions are given by weight. Furthermore, if a variable is not defined, the previous definition of the variable takes precedence.

[0111] I. Proteins The present invention provides a multispecific binding protein that binds to NKG2D receptor and CD16 receptor on natural killer cells, and a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52 and CD133. The multispecific binding protein is useful in the pharmaceutical compositions and treatment methods described herein. When the multispecific binding protein binds to NKG2D receptor and CD16 receptor on natural killer cells, the activity of natural killer cells aimed at destroying tumor cells expressing CD37, CD20, CD19, CD22, CD30, CD52 or CD133 antigen is enhanced. When the multispecific binding protein binds to CD37, CD20, CD19, CD22, CD30, CD52 or CD133-expressing cells, the cancer cells are brought into proximity to natural killer cells, which facilitates direct and indirect destruction of cancer cells by natural killer cells. Further description of some exemplary multispecific binding proteins is provided below.

[0112] The first component of the multispecific binding protein is NK cells, γδ T cells and CD8 + binds to NKG2D receptor-expressing cells, which may include, but are not limited to, αβ T cells. When bound to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating the NKG2D receptor.

[0113] The second component of the multispecific binding protein binds to CD37, CD20, CD19, CD22, CD30, CD52, or CD133. CD37-expressing cells may be found, for example, in B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), non-Hodgkin lymphoma, and acute myeloid leukemia. CD20-expressing cells may be found, for example, in chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, and B-cell malignancies. CD19-expressing cells may be found, for example, in chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, multiple myeloma, and acute myeloid leukemia. CD22-expressing cells may be found, for example, in chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, and hairy cell leukemia. CD30-expressing cells may be found, for example, in Hodgkin lymphoma, anaplastic large cell lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia-lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, and embryonal cell carcinoma. CD52-expressing cells may be found in, for example, chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, peripheral T-cell lymphoma and T-cell prelymphocytic leukemia, B-cell malignancies, non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia-lymphoma, mature T / natural killer (NK) cell neoplasms, and thymoma. CD133-expressing cells may be found in, for example, breast cancer, colon cancer, prostate cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, kidney cancer, uterine cancer, testicular germ cell carcinoma, acute myeloid leukemia, acute lymphoblastic leukemia, glioma, glioblastoma, and squamous cell carcinoma of the head and neck.

[0114] The third component of the multispecific binding protein binds to cells that express CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0115] The multispecific binding proteins described herein can take various formats. For example, one format is a heterodimer multispecific antibody (Figure 1) comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain 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. Together with the first immunoglobulin heavy chain, the first immunoglobulin light 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. The second immunoglobulin light chain comprises a second light chain variable domain and a second light chain constant domain. Together with the second immunoglobulin heavy chain, the second immunoglobulin light chain forms an antigen-binding site that binds to CD37, CD20, CD19, CD22, CD30, CD52, or CD133. The first Fc domain and the second Fc domain can bind to CD16 together (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0116] Another exemplary format relates to a heterodimer multispecific antibody (Figure 2) comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. 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-strand variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain that pair and bind to NKG2D or to an antigen selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. 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. This 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 tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52, and CD133. The first and second Fc domains can bind together to CD16 (Figure 2).

[0117] One or more additional binding motifs, as needed, via the linker sequence, are stationary It can be fused to the C-terminus of the CH3 domain. In certain embodiments, the antigen-binding site may be a single-chain or disulfide-stabilized variable region (scFv), or it may form a tetravalent or trivalent molecule.

[0118] In some embodiments, the multispecific binding protein is in the form of a triomab, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two semiantibodies derived from two parent antibodies, each semiantibody having one light chain and one heavy chain.

[0119] In some embodiments, the multispecific binding protein is in the form of a KiH common light chain (LC) using the knob-into-hole (KIH) technique. KIH promotes heterodimerization, C HThis involves manipulating three domains to create either a "knob" or a "hole" in each heavy chain. The concept behind the "knob-into-hole (KiH)" Fc technology is to create a "knob" in one CH3 domain (CH3A) by substituting a small residue with a bulky residue (e.g., T366W in EU numbering). CH3A The goal was to introduce a "knob". To accommodate this "knob", the other CH3 domain (CH3B) was modified by replacing the adjacent residue closest to the knob with a smaller residue (for example, T366S / L368A / Y407V CH3BA complementary "hole" surface was created by ). The "hole" mutation was optimized by phage library screening based on structural information (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J Mol Biol (1997) vol. 270 (no. 1): pp. 26-35). X-ray crystal structure of KiH Fc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) Vol. 426 (No. 9): pp. 1947-1957; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs. Mol Immunol (2014) Vol. 58 (No. 1): pp. 132-138 showed that at core interfaces between CH3 domains, hydrophobic interactions driven by steric complementarity are thermodynamically favorable for heterodimerization, while knob-knob and hole-hole interfaces are not favorable for homodimerization due to steric hindrance and interference with favorable interactions, respectively.

[0120] In some embodiments, the multispecific binding protein is in the form of a bivariable domain immunoglobulin (DVD-Ig®), which combines the target-binding domains of two monoclonal antibodies via a naturally occurring mobile linker, resulting in a tetravalent IgG-like molecule.

[0121] In some embodiments, the multispecific binding protein is in an orthogonal Fab interface (ortho-Fab) configuration. The ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) Vol. 32 (No. 2): pp. 191-198) uses structure-based region design to enable LC and HC in one Fab. VH-CH1 Complementary mutations are introduced only at the interface, and the other Fab remains unchanged.

[0122] In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in an ES form, a heterodimer construct containing two different Fabs that bind to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in Fc.

[0123] In some embodiments, the multispecific binding protein is in the κλ-Body form, which is a heterodimer construct having two different Fabs fused to an Fc stabilized by a heterodimerizing mutation. Fab1, which targets antigen 1, contains kappa LC, while a second Fab, which targets antigen 2, contains lambda LC. Figure 30A is an exemplary diagram of one form of the κλ-Body, and Figure 30B is an exemplary diagram of another κλ-Body.

[0124] In some embodiments, the multispecific binding protein is in the form of a Fab arm exchange (an antibody that has become a bispecific antibody as a result of exchanging the Fab arms by swapping the heavy chain and the bound light chain (half) with a heavy chain-light chain pair of another molecule).

[0125] In some embodiments, the multispecific binding protein is in the form of a SEED Body. The SEED (strand-exchange engineered domain) platform was designed to generate asymmetric and bispecific antibody-like molecules that could expand the therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins in a conserved CH3 domain. The SEED design enables the effective generation of AG / GA heterodimers while avoiding homodimerization of the AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, Vol. 24 (No. 5): pp. 447-454)).

[0126] In some embodiments, the multispecific binding protein is in a LuZ-Y form, using a leucine zipper to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), vol. 287: pp. 43331-4339).

[0127] In some embodiments, the multispecific binding protein is in the Cov-X-Body form. In the bispecific CovX-Body, a branched azetidinone linker is used to fused two different peptides into one, which are then site-directly fused to a scaffold antibody under mild conditions. The pharmacophore is responsible for functional activity, while the antibody scaffold results in a long half-life and Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with another pharmacophore (Doppalapudi VR et al., PNAS (2010), vol. 107 (no. 52); pp. 22611-22616).

[0128] In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer, comprising a Fab fused to Fc that binds to target 1 and an scFab that binds to target 2. Heterodimerization is ensured by mutation in Fc.

[0129] In some embodiments, the multispecific binding protein is in DuetMab form, a heterodimer construct containing two different Fabs that bind to antigens 1 and 2, as well as an Fc stabilized by a heterodimerizing mutation. Fabs 1 and 2 contain specific SS crosslinks that ensure accurate pairing of LC and HC.

[0130] In some embodiments, the multispecific binding protein is in a CrossmAb form, which is a heterodimer construct having two different Fabs that bind to targets 1 and 2, fused to an Fc stabilized by heterodimerization. The CL domain and CH1 domain are switched with the VH domain and VL domain, for example, CH1 is fused inline with VL and CL is fused inline with VH.

[0131] In some embodiments, the multispecific binding protein is in the Fit-Ig form, which is a homodimer construct in which the Fab that binds to antigen 2 is fused to the N-terminus of the HC of the Fab that binds to antigen 1. This construct contains wild-type Fc.

[0132] Table 1 lists the peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to NKG2D. The NKG2D-binding domains may vary in their binding affinity to NKG2D, but 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]

[0133] Alternatively, as described in US9,273,136, the heavy chain variable domain represented by SEQ ID NO: 101 may be paired with the light chain variable domain represented by SEQ ID NO: 102 to form an antigen-binding site that can bind to NKG2D. [ka]

[0134] Alternatively, as described in US7,879,985, the heavy chain variable domain represented by SEQ ID NO: 103 may be paired with the light chain variable domain represented by SEQ ID NO: 104 to form an antigen-binding site that can bind to NKG2D. [ka]

[0135] In one embodiment, the present disclosure provides NKG2D receptor and CD16 receptor on natural killer cells, as well as multispecific binding proteins that bind to the antigen CD37. Table 2 lists some exemplary sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD37. [Table 2-1] [Table 2-2]

[0136] Alternatively, novel antigen-binding sites capable of binding to CD37 can be identified by screening for binding to amino acid sequences defined by SEQ ID NO: 133. [ka]

[0137] In one embodiment, the present disclosure provides NKG2D receptor and CD16 receptor on natural killer cells, as well as multispecific binding proteins that bind to the antigen CD20. Table 3 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD20. [Table 3-1] [Table 3-2] [Table 3-3]

[0138] Alternatively, novel antigen-binding sites capable of binding to CD20 can be identified by screening for binding to amino acid sequences defined by SEQ ID NO: 174. [ka]

[0139] In one embodiment, the present disclosure provides NKG2D receptor and CD16 receptor on natural killer cells, as well as multispecific binding proteins that bind to the antigen CD19. Table 4 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD19. [Table 4-1] [Table 4-2] [Table 4-3]

[0140] Alternatively, novel antigen-binding sites capable of binding to CD19 can be identified by screening for binding to amino acid sequences defined by SEQ ID NO: 207. [ka]

[0141] In one embodiment, the present disclosure provides NKG2D receptor and CD16 receptor on natural killer cells, as well as multispecific binding proteins that bind to the antigen CD22. Table 5 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD22. [Table 5-1] [Table 5-2]

[0142] The antigen-binding site that binds to CD22 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 232. [ka]

[0143] In one embodiment, the present disclosure provides multispecific binding proteins that bind to the NKG2D receptor and CD16 receptor on natural killer cells, as well as the antigen CD30. Table 6 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD30. [Table 6-1] [Table 6-2] [Table 6-3]

[0144] Alternatively, novel antigen-binding sites capable of binding to CD30 can be identified by screening for binding to amino acid sequences defined by SEQ ID NO: 273. [ka]

[0145] In one embodiment, the present disclosure provides multispecific binding proteins that bind to the NKG2D receptor and CD16 receptor on natural killer cells, as well as the antigen CD52. Table 7 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD52. [Table 7-1] [Table 7-2]

[0146] Alternatively, novel antigen-binding sites capable of binding to CD52 can be identified by screening for binding to amino acid sequences defined by SEQ ID NO: 290. [ka]

[0147] In one embodiment, the present disclosure provides multispecific binding proteins that bind to the NKG2D receptor and CD16 receptor on natural killer cells, as well as the antigen CD133. Table 8 lists some exemplary peptide sequences of heavy-chain and light-chain variable domains that can be combined to bind to CD133. [Table 8-1] [Table 8-2]

[0148] Alternatively, novel antigen-binding sites capable of binding to CD133 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 323. [ka]

[0149] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, interactions with CD16 are primarily focused on the amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, vol. 406 (no. 6793): pp. 267-273). Based on known domains, mutations can be selected to enhance or reduce binding affinity to CD16, for example, by using phage display libraries or yeast surface display cDNA libraries, or they can be designed based on the known three-dimensional structure of the interaction.

[0150] The construction of heterodimerized antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences within the same cell, thereby resulting in the construction of both homodimers and heterodimers of each antibody heavy chain. Facilitating the selective construction of heterodimers can be achieved by incorporating different mutations within the CH3 domain of the constant region of each antibody heavy chain, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, mutations can be produced within the CH3 domain, incorporating different pairs of amino acid substitutions within a first and second polypeptide, based on human IgG1, enabling these two chains to selectively heterodimerize from each other. The amino acid substitution positions shown below are all numbered according to the EU index, as in Kabat.

[0151] In one scenario, an amino acid substitution in the first polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in the second polypeptide replaces the original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (protrusion) fits on the surface of the smaller amino acid substitution (cavity). For example, one polypeptide may incorporate the T366W substitution, while the other polypeptide may incorporate three substitutions, including T366S, L368A, and Y407V.

[0152] The antibody heavy chain variable domain of the present invention may be linked, as needed, to an amino acid sequence that is at least 90% identical to the antibody constant region, such as an IgG constant region containing a hinge with or without a CH1 domain, and CH2 and CH3 domains. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region derived from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region of human IgG1 compared to the constant region at, for example, Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Examples of 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, This includes 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.

[0153] In certain embodiments, mutations that can be incorporated into the CH1 constant region of human IgG1 may be amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into the Cκ constant region of human IgG1 may be amino acids E123, F116, S176, V163, S174, and / or T164.

[0154] Alternatively, amino acid substitutions may be selected from the set of substitutions shown in Table 9 below. [Table 9]

[0155] Alternatively, amino acid substitutions may be selected from the set of substitutions shown in Table 10 below. [Table 10]

[0156] Alternatively, amino acid substitutions may be selected from the set of substitutions shown in Table 11 below. [Table 11]

[0157] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 12. [Table 12]

[0158] Alternatively, at least one amino acid substitution may be selected from the set of substitutions in Table 13 below, where the position indicated in the “First polypeptide” column is replaced by any known negatively charged amino acid, and the position indicated in the “Second polypeptide” column is replaced by any known positively charged amino acid. [Table 13]

[0159] Alternatively, at least one amino acid substitution may be selected from the set in Table 14 below, where the position indicated in the “First polypeptide” column is replaced by any known negatively charged amino acid, and the position indicated in the “Second polypeptide” column is replaced by any known negatively charged amino acid. [Table 14]

[0160] Alternatively, amino acid substitutions may be selected from the sets shown in Table 15 below. [Table 15]

[0161] Alternatively or additionally, the structural stability of heteromultimeric proteins can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, thereby forming an artificial disulfide crosslink at the interface of the two polypeptides.

[0162] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, where the amino acid sequence of the other polypeptide chain in 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.

[0163] In some embodiments, the amino acid sequence of one polypeptide chain in 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, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0164] In some embodiments, the amino acid sequence of one polypeptide chain in 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 E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, where the amino acid sequence of the other polypeptide chain in 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 Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0165] In some embodiments, the amino acid sequence of one polypeptide chain in 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 Y349, E357, S364, L368, K370, T394, D401, F405, and T411, where the amino acid sequence of the other polypeptide chain in 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 E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0166] In some embodiments, the amino acid sequence of one polypeptide chain in 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 L351, D399, S400, and Y407, where the amino acid sequence of the other polypeptide chain in 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, N390, K392, K409, and T411.

[0167] In some embodiments, the amino acid sequence of one polypeptide chain in 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, N390, K392, K409, and T411, where the amino acid sequence of the other polypeptide chain in 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 L351, D399, S400, and Y407.

[0168] In some embodiments, the amino acid sequence of one polypeptide chain in 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 Q347, Y349, K360, and K409, where the amino acid sequence of the other polypeptide chain in 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 Q347, E357, D399, and F405.

[0169] In some embodiments, the amino acid sequence of one polypeptide chain in 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 Q347, E357, D399, and F405, where the amino acid sequence of the other polypeptide chain in 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 Y349, K360, Q347, and K409.

[0170] In some embodiments, the amino acid sequence of one polypeptide chain in 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 K370, K392, K409, and K439, where the amino acid sequence of the other polypeptide chain in 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 D356, E357, and D399.

[0171] In some embodiments, the amino acid sequence of one polypeptide chain in 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 D356, E357, and D399, where the amino acid sequence of the other polypeptide chain in 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 K370, K392, K409, and K439.

[0172] In some embodiments, the amino acid sequence of one polypeptide chain in 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 L351, E356, T366, and D399, where the amino acid sequence of the other polypeptide chain in 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 Y349, L351, L368, K392, and K409.

[0173] In some embodiments, the amino acid sequence of one polypeptide chain in 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 Y349, L351, L368, K392, and K409, where the amino acid sequence of the other polypeptide chain in 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 L351, E356, T366, and D399.

[0174] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region is S35 The amino acid sequence of the IgG1 constant region differs only from that of the 4C substitution, and here the amino acid sequence of the other polypeptide chain in the antibody constant region differs only from that of the IgG1 constant region by the Y349C substitution.

[0175] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the Y349C substitution, and the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the S354C substitution.

[0176] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the K360E and K409W substitutions, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the O347R, D399V and F405T substitutions.

[0177] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the O347R, D399V and F405T substitutions, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the K360E and K409W substitutions.

[0178] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T366W substitution, while the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T366S, T368A, and Y407V substitutions.

[0179] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T366S, T368A, and Y407V substitutions, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T366W substitution.

[0180] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T350V, L351Y, F405A, and Y407V substitutions, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T350V, T366L, K392L, and T394W substitutions.

[0181] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T350V, T366L, K392L, and T394W substitutions, where the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of the IgG1 constant region only by the T350V, L351Y, F405A, and Y407V substitutions.

[0182] The above-mentioned multispecific proteins can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector, and the first, second, and third expression vectors can be stably transfected together into host cells to produce a multimeric protein.

[0183] To achieve the highest yield of multispecific proteins, different ratios of the first, second, and third expression vectors can be investigated to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

[0184] Clones can be cultured under conditions suitable for bioreactor scale-up, and can maintain the expression of the multispecific protein. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0185] II. Characteristics of the multispecific protein The multispecific protein described herein comprises an NKG2D binding site, a CD16 binding site, and a binding site for a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52 and CD133. In some embodiments, the multispecific protein binds to cells expressing NKG2D and / or CD16 (e.g., NK cells), and tumor cells that simultaneously express any one of the above antigens. Binding of the multispecific protein to NK cells can enhance NK cell activity towards destruction of cancer cells.

[0186] In some embodiments, the multispecific protein binds to a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52 and CD133 with an affinity similar to that of a monoclonal antibody having the same respective antigen binding site. In some embodiments, the multispecific protein is more effective than the corresponding respective monoclonal antibody in killing tumor cells expressing the antigen(s).

[0187] In certain specific embodiments, the multispecific proteins described herein that comprise an NKG2D binding site and a binding site for a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52 and CD133 activate primary human NK cells when co-cultured with cells expressing CD37, CD20, CD19, CD22, CD30, CD52 and CD133, respectively. NK cell activation is indicated by increased CD107a degranulation and IFNγ cytokine production. Furthermore, compared with the corresponding individual monoclonal antibodies, the multispecific protein can exhibit superior activation of human NK cells in the presence of cells expressing the antigen CD37, CD20, CD19, CD22, CD30, CD52 or CD133.

[0188] In certain specific embodiments, the multispecific proteins described herein that comprise an NKG2D binding site and a binding site for a tumor-associated antigen selected from CD37, CD20, CD19, CD22, CD30, CD52 and CD133 are co-cultured with cells expressing CD37, CD20, CD19, CD22, CD30, CD52 and CD133 to enhance the activity of human resting and IL-2-activated NK cells.

[0189] In certain specific embodiments, compared with the corresponding monoclonal antibody that binds to CD37, CD20, CD19, CD22, CD30, CD52 or CD133, the multispecific protein provides advantages in targeting tumor cells expressing moderate and low levels of CD37, CD20, CD19, CD22, CD30, CD52 and CD133.

[0190] III. Therapeutic Uses The present invention provides a method for treating cancer using the multispecific binding proteins and / or pharmaceutical compositions described herein, the method may be used to treat various cancers expressing CD37, CD20, CD19, CD22, CD30, CD52, or CD133. Exemplary cancers treated with multispecific binding proteins targeting CD37 may be B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), non-Hodgkin lymphoma, or acute myeloid leukemia. Exemplary cancers treated with multispecific binding proteins targeting CD20 may be chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, or B-cell malignancies. Exemplary cancers treated with multispecific binding proteins targeting CD19 may be chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, multiple myeloma, or acute myeloid leukemia. Exemplary cancers treated with multispecific binding proteins targeting CD22 may include chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancies, or hairy cell leukemia. Exemplary cancers treated with multispecific binding proteins targeting CD30 may include Hodgkin lymphoma, anaplastic large cell lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia-lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, or embryonic cell carcinoma. Exemplary cancers treated with multispecific binding proteins targeting CD52 may include chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, peripheral T-cell lymphoma and T-cell prelymphoblastic leukemia, B-cell malignancies, non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia-lymphoma, mature T / natural killer (NK) cell neoplasms, or thymoma. Exemplary cancers treated with multispecific binding proteins targeting CD133 may include breast cancer, colon cancer, prostate cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, kidney cancer, uterine cancer, testicular germ cell carcinoma, acute myeloid leukemia, acute lymphoblastic leukemia, glioma, glioblastoma, or squamous cell carcinoma of the head and neck.

[0191] In some other embodiments, cancers treated include brain cancer, rectal cancer, and uterine cancer. In yet other embodiments, cancers include squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, and adenoid cystic carcinoma. Carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choroid plexus papilloma / cell tumor, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, gastrointestinal cancer, duodenal cancer, endocrine cancer, yolk sac tumor, endometrial hyperplasia, endometrial stromal sarcoma Endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, cancers of the eye and orbit, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric cardia cancer, gastric fundus cancer, gastrin-producing tumors, glioblastoma, glucagon-producing tumors, cardiac cancer, hemangiblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, carcinoma in situ, interepithelial squamous cell neoplasmsNeoplasty, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, melanoma originating from lentigo malignant, lymphoma, male reproductive organ cancer, malignant mesothelioma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin lymphoma, oat cell carcinoma, oligodendroglia, oral cancer, osteosarcoma, serous papillary adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor These include tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cell carcinoma, paranasal sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyocyte carcinoma, soft tissue cancer, somatostatin-secreting tumors, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial cancer, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary tract cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIP-producing tumor, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0192] In certain other embodiments, the cancer to be treated is a non-Hodgkin lymphoma, such as B-cell lymphoma or T-cell lymphoma. In certain embodiments, non-Hodgkin lymphomas are B-cell lymphomas such as diffuse large B-cell lymphoma, mediastinal primary B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, non-Hodgkin lymphoma is a T-cell lymphoma such as progenitor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0193] IV. Combination Therapy Another aspect of the present invention provides combination therapy. The multispecific binding proteins described herein may be used in combination with further therapeutic agents to treat cancer.

[0194] Examples of therapeutic agents that may be used as part of combination therapy when treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carbocon, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole, larcitrexed, daunorubicin, fadrozol, fotemustine, thymalfacin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, eriptinium acetate, ketanserine, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, and droguetidine. This includes Nil, Butosin, Carmofur, Lazoxane, Schizophyllan, Carboplatin, Mitractol, Tegafur, Ifosfamide, Prednimustine, Picibanil, Levamizole, Teniposide, Improsulfan, Enocitabine, Risurido, Oxymetholone, Tamoxifen, Progesterone, Mepitiostane, Epitiostanol, Formestan, Interferon-alpha, Interferon-2-alpha, Interferon-beta, Interferon-gamma (IFN-γ), Colony-stimulating factor-1, Colony-stimulating factor-2, Denileukin-diffitox, Interleukin-2, Luteinizing hormone-releasing factor, and variants of the above-mentioned drugs that may exhibit specific binding to their alloceptors and increased or decreased serum half-lives.

[0195] A further class of drugs that can be used as part of combination therapy when treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of the following: (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Ipilimumab, a CTLA4 inhibitor, is approved by the U.S. Food and Drug Administration for the treatment of melanoma.

[0196] Other drugs that may be used as part of combination therapy when treating cancer include monoclonal antibody drugs that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0197] 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 + 2-chlorodeoxyadenosine, HDAC inhibitors, Hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, and MEK inhibitors. The following are included: (ii) inhibitors selected from MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0198] The protein of the present invention can also be used as an adjunct to the surgical removal of primary lesions.

[0199] The amounts and relative timing of administration of multispecific binding proteins and additional therapeutic agents may be selected to achieve the desired combination therapy effect. For example, when administering combination therapy to a patient requiring such administration, the combined therapeutic agents, or one or more pharmaceutical compositions containing therapeutic agents, may be administered in any order, such as sequentially, together, together, or simultaneously. Furthermore, for example, the multispecific binding protein may be administered for the duration that the additional therapeutic agent exerts its prophylactic or therapeutic effect, or vice versa.

[0200] V. Pharmaceutical Compositions This disclosure also features pharmaceutical compositions containing therapeutically effective amounts of the proteins described herein. The compositions can be formulated for use in various drug delivery systems. To create a suitable formulation, one or more physiologically acceptable excipients or carriers may be included in the composition. Suitable formulations used in this disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief overview of methods for drug delivery, see, for example, Langer (Science, vol. 249: pp. 1527–1533, 1990).

[0201] The intravenous drug delivery formulations of this disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including 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 and 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 may be combined to obtain a therapeutic dose of protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored as about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored as about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at approximately 250 mg / vial.

[0202] Proteins can be present in liquid aqueous pharmaceutical formulations containing a therapeutically effective amount of protein in the buffer solution that forms the formulation.

[0203] These compositions may be sterilized by conventional sterilization techniques or by filter sterilization. The resulting aqueous solutions may be packaged for immediate use or lyophilized, and the lyophilized preparations may be combined with a sterile aqueous carrier before administration. The pH of the preparations is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, for example, 7 to 7.5. The resulting solid compositions may be packaged in multiple single-dose units, each containing a certain amount of one or more of the above-mentioned agents. The solid compositions may also be packaged in containers for flexible quantities.

[0204] In certain embodiments, the Disclosure provides formulations having an extended shelf life, comprising the protein of the Disclosure in combination with mannitol, citrate monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium oxide.

[0205] In certain embodiments, an aqueous formulation containing the protein of the Disclosure is prepared in a pH buffer solution. The buffer of the Disclosure may have a pH in the range of about 4 to about 8, for example, about 4.5 to about 6.0 or about 4.8 to about 5.5, or about 5.0 to about 5.2. A range of pH intermediates between those listed above is also intended to be part of the Disclosure. For example, a range of values ​​using any combination of the values ​​listed above as upper and / or lower limits is intended to be included. Examples of buffers that control pH within this range include acetates (e.g., sodium acetate), succinates (such as sodium succinate), glucons, histidine, citrates, and other organic acid buffers.

[0206] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range may be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about 5.0 to about 5.2. In certain embodiments, the buffer system includes citrate monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises approximately 1.3 mg / ml of citric acid (e.g., 1.305 mg / ml), approximately 0.3 mg / ml of sodium citrate (e.g., 0.305 mg / ml), approximately 1.5 mg / ml of disodium phosphate dihydrate (e.g., 1.53 mg / ml), approximately 0.9 mg / ml of sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / ml), and approximately 6.2 mg / ml of sodium chloride (e.g., 6.165 mg / ml). In certain embodiments, the buffer system comprises 1–1.5 mg / ml of citric acid, 0.25–0.5 mg / ml of sodium citrate, 1.25–1.75 mg / ml of disodium phosphate dihydrate, 0.7–1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0–6.4 mg / ml of sodium chloride. In certain embodiments, the pH of the formulation is adjusted using sodium hydroxide.

[0207] Polyols that act as tonicifiers and can stabilize antibodies can also be included in the preparation. Polyols are added to the preparation in an amount that may vary depending on the desired isotonicity of the preparation. In certain specific embodiments, the aqueous preparation may be isotonic. The amount of polyol added may also vary depending on the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain specific embodiments, the polyol that can be used in the preparation as an isotonic agent is mannitol. In certain specific embodiments, the mannitol concentration may be from about 5 to about 20 mg / ml. In certain specific embodiments, the mannitol concentration may be from about 7.5 to 15 mg / ml. In certain specific embodiments, the mannitol concentration may be from about 10 to 14 mg / ml. In certain specific embodiments, the mannitol concentration may be about 12 mg / ml. In certain specific embodiments, the polyol sorbitol can be included in the preparation.

[0208] Detergents or surfactants may also be added to the preparation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such as to reduce aggregation of the formulated antibody, and / or minimize the formation of particulates in the preparation, and / or reduce adsorption. In certain specific embodiments, the preparation may comprise a surfactant that is a polysorbate. In certain specific embodiments, the preparation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to refer to polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996). In certain specific embodiments, the preparation may contain between about 0.1 mg / mL and about 10 mg / mL of polysorbate 80, or between about 0.5 mg / mL and about 5 mg / mL. In certain specific embodiments, about 0.1% of polysorbate 80 may be added to the preparation.

[0209] In some embodiments, the protein product of this disclosure is formulated as a liquid formulation. The liquid formulation may be supplied at a concentration of 10 mg / mL in a USP / Ph Eur Type I 50R vial, which is 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, 61.2 mL of the protein product solution may be filled into the vial to allow a 60 mL dispensing volume. In certain embodiments, the liquid formulation may be diluted with 0.9% saline.

[0210] In certain embodiments, the liquid formulation of the present disclosure may be prepared as a 10 mg / mL concentration solution combined with a sugar at a stabilizing level. In certain embodiments, the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount less than or equal to the amount that would result in a viscosity undesirable or inappropriate for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0211] In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0212] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from a protein to form a succinimide intermediate that can be hydrolyzed. The succinimide intermediate results in a mass decrease of 17 daltons of the parent peptide. Subsequent hydrolysis results in a mass increase of 18 daltons. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Therefore, deamidation is typically detectable as a mass increase of 1 dalton. Deamidation of asparagine yields either aspartic acid or isoaspartic acid. Parameters influencing the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the rate of deamidation. Gly and Ser following Asn in the protein sequence result in higher sensitivity to deamidation.

[0213] In certain embodiments, the liquid formulations of the present disclosure may be stored under pH and humidity conditions to prevent deamination of protein products.

[0214] The aqueous carriers of interest as used herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0215] Preservatives may be added to the formulations herein as needed to reduce bacterial activity. The addition of preservatives can facilitate, for example, the manufacture of multi-use (multiple-dose) formulations.

[0216] Intravenous (IV) formulations may be a preferred route of administration in certain cases, such as when a patient is hospitalized after transplantation and receiving all medications via the IV route. In certain embodiments, the liquid formulation is diluted with a 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0217] In certain embodiments, the salt or buffering component may be added in an amount of 10 mM to 200 mM. The salt and / or buffer may be pharmaceutically acceptable and derived from various known acids (inorganic and organic) using a "base-forming" metal or amine. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may act as counterions.

[0218] Preservatives may be added to the formulations herein as needed to reduce bacterial activity. The addition of preservatives can facilitate, for example, the manufacture of multi-use (multiple-dose) formulations.

[0219] The aqueous carriers of interest as used herein are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0220] The proteins of this disclosure may also exist as lycoprotectant formulations comprising the protein and a lycoprotectant. The lycoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lycoprotectant may be sucrose or maltose. The lycoprotectant formulation may contain one or more of the following: buffers, surfactants, fillers, and / or preservatives.

[0221] The amount of sucrose or maltose useful for stabilizing lyophilized drug products may be at least a protein-to-sucrose or maltose weight ratio of 1:2. In certain embodiments, the protein-to-sucrose or maltose weight ratio may be 1:2 to 1:5.

[0222] In certain embodiments, the pH of the formulation before lyophilization may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[0223] Before freeze-drying, the pH of the solution containing the protein of this disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range for the freeze-dried drug product may be between 7 and 8.

[0224] In certain embodiments, the salt or buffering component may be added in an amount of 10 mM to 200 mM. The salt and / or buffer may be pharmaceutically acceptable and derived from various known acids (inorganic and organic) using a "base-forming" metal or amine. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may act as counterions.

[0225] In certain embodiments, a “bulking agent” may be added. The “bulking agent” is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (for example, facilitating the production of an essentially uniform lyophilized cake that maintains an open porosity structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.

[0226] Preservatives may be added to the formulations herein as needed to reduce bacterial activity. The addition of preservatives can facilitate, for example, the manufacture of multi-use (multiple-dose) formulations.

[0227] In certain embodiments, the lyophilized drug product may consist of an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) and, after lyophilization, useful for the preparation of liquid formulations. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0228] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either sterile water for injection, USP (SWFI), or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder is dissolved in the solution.

[0229] In a particular embodiment, the lyophilized protein product of the present disclosure is composed of approximately 4.5 mL of water for injection and diluted with a 0.9% saline solution (sodium chloride solution).

[0230] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that does not cause toxicity to the patient and is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration.

[0231] A 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 in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical status. Further refinements to the calculations required to determine the appropriate dosage for treatment are customarily made by those skilled in the art, in particular, taking into account the dosage information and assays disclosed herein. The dosage can also be determined by the use of known assays for determining dosage, used in conjunction with appropriate dose-response data. The dosage for individual patients may be adjusted as the progression of the disease is monitored. Blood levels of the targetable construct or complex in a patient may be measured to determine whether an effective concentration has been reached or whether the dosage needs to be adjusted to maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes, and their dosages, are likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta vol. 308: pp. 43-53, 2001; Steimer et al., Clinica Chimica Acta vol. 308: pp. 33-41, 2001).

[0232] Generally, the dosage based on body weight is approximately 0.01 μg to 100 mg / kg body weight, for example, approximately 0.01 μg to 100 mg / kg body weight, approximately 0.01 μg to 50 mg / kg body weight, approximately 0.01 μg to 10 mg / kg body weight, approximately 0.01 μg to 1 mg / kg body weight, approximately 0.01 μg to 100 μg / kg body weight, approximately 0.01 μg to 50 μg / kg body weight, approximately 0.01 μg to 10 μg / kg body weight, approximately 0.01 μg to 1 μg / kg body weight, and approximately 0.01 μg to 0.1 μg. g / 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~about 10μg / kg body weight, about 0.1μg~about 1μg / kg body weight, about 1μg~about 100mg / kg body weight, about 1μg~about 50mg / kg body weight, about 1μg~about 10mg / kg body weight, about 1μg~about 1mg / kg body weight, about 1μg~about 1 00 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 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 The dosage ranges are 1 mg / kg body weight, approximately 50 μg to 100 μg / kg body weight, approximately 100 μg to 100 mg / kg body weight, approximately 100 μg to 50 mg / kg body weight, approximately 100 μg to 10 mg / kg body weight, approximately 100 μg to 1 mg / kg body weight, approximately 1 mg to 100 mg / kg body weight, approximately 1 mg to 50 mg / kg body weight, approximately 1 mg to 10 mg / kg body weight, approximately 10 mg to 100 mg / kg body weight, approximately 10 mg to 50 mg / kg body weight, and approximately 50 mg to 100 mg / kg body weight.

[0233] Dosages may be administered once or more times daily, once or more times weekly, once or more times monthly, once or more times annually, or even once every 2 to 20 years. Those skilled in the art can easily estimate the frequency of administration based on the measured residence time and concentration of the targetable construct or complex in body fluids or tissues. The administration of the present invention may be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, or intracavitary, and may be by catheter-mediated perfusion, or by direct intrafocal injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.

[0234] The above description outlines several aspects and embodiments of the present invention. This application is particularly intended to cover all combinations and substitutions of aspects and embodiments. [Examples]

[0235] The present invention, as generally described herein, will be more readily understood by referring to the following examples, which are included solely for illustrative purposes of certain aspects and embodiments of the invention and are not intended to limit the invention.

[0236] (Example 1) The NKG2D binding domain binds to NKG2D. The NKG2D binding domain binds to purified recombinant NKG2D. Nucleic acid sequences of the extracellular domains of human, mouse, or cynomolgus monkey NKG2D were fused with nucleic acid sequences encoding the human IgG1 Fc domain and introduced into mammalian cells expressing these sequences. After purification, the NKG2D-Fc fusion protein was adsorbed into the wells of a microplate. To prevent nonspecific binding, the wells were blocked with bovine serum albumin, and then the NKG2D binding domain was titrated and added to the wells to which the NKG2D-Fc fusion protein had been pre-adsorbed. Primary antibody binding was conjugated with horseradish peroxidase and detected using a secondary antibody that specifically recognizes the human kappa light chain to avoid Fc cross-reactivity. 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, was added to the wells to visualize the binding signal, and its absorbance was measured at 450 nM and corrected to 540 nM. NKG2D binding domain clones, isotype controls, or positive controls (sequence numbers 101-104, or heavy and light chain variable domains selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) were added to each well.

[0237] Isotype controls showed slight binding to recombinant NKG2D-Fc protein, while positive controls showed the strongest binding to the recombinant antigen. Although affinity varied among clones, the NKG2D-binding domain produced by all clones showed binding to all recombinant NKG2D-Fc proteins from human, mouse, and cynomolgus monkeys. Generally, each anti-NKG2D clone bound to recombinant NKG2D-Fc from human (Figure 3) and cynomolgus monkeys (Figure 4) with similar affinity, but showed relatively low affinity to recombinant NKG2D-Fc from mouse (Figure 5). The NKG2D binding domain binds to cells that express NKG2D.

[0238] EL4 mouse lymphoma cell lines were engineered to express human or mouse NKG2D-CD3 zeta signaling domain chimeric antigen receptors. Extracellular NKG2D expressed in EL4 cells was stained using NKG2D-conjugated clones, isotype controls, or positive controls at a concentration of 100 nM. Antibody binding was detected using fluorophore conjugate anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry, and the factor of background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[0239] The NKG2D binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. Positive control antibodies (sequences 101-104, or heavy and light chain variable domains selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) yielded the best FOB binding signal. The NKG2D binding affinity of each clone was similar between cells expressing human NKG2D (Figure 6) and cells expressing mouse NKG2D (Figure 7).

[0240] (Example 2) The NKG2D binding domain prevents the binding of natural ligands to NKG2D. Competition with ULBP-6 Recombinant human NKG2D-Fc protein was adsorbed into microplate wells, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. Saturated ULBP-6-His-biotin was added to the wells, followed by the addition of 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 using horseradish peroxidase and streptavidin conjugated with a TMB substrate. Absorbance was measured at 450 nM and corrected to 540 nM. After subtracting background, 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 whose binding to the NKG2D-Fc protein was blocked. Positive control antibody (containing heavy and light chain variable domains selected from SEQ ID NOs. 101-104) and various NKG2D binding domains The inhibitor blocked ULBP-6 binding to NKG2D, while the isotype control showed little competition with ULBP-6 (Figure 8).

[0241] The ULBP-6 sequence is represented by sequence number 108. [ka]

[0242] Competition with MICA Recombinant human MICA-Fc protein was adsorbed into microplate wells, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the addition of the NKG2D-binding domain. After incubation and washing, NKG2D-Fc-biotin that remained bound to the MICA-Fc-coated wells was detected using streptavidin-HRP and TMB substrates. Absorbance was measured at 450 nM and corrected to 540 nM. After subtracting background, the specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the MICA-Fc-coated wells was blocked. Positive control antibodies (including heavy and light chain variable domains selected from SEQ ID NOs. 101-104) and various NKG2D-binding domains blocked MICA binding to NKG2D, while isotype controls showed little competition with MICA (Figure 9).

[0243] Competition with Rae-1 Delta Recombinant mouse Rae-1 delta-Fc (purchased from R&D Systems) was adsorbed into microplate wells, 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 addition of the NKG2D-binding domain. After incubation and washing, NKG2D-Fc-biotin that remained bound to the Rae-1 delta-Fc coated wells was detected using streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected to 540 nM. After subtracting background, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin whose binding to the Rae-1 delta-Fc coated wells was blocked. Positive control antibodies (sequences 101-104, or heavy and light chain variable domains selected from anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) and various NKG2D-binding domain clones blocked Rae-1 delta binding to mouse NKG2D, while isotype control antibodies showed little competition with Rae-1 delta (Figure 10).

[0244] (Example 3) The NKG2D binding domain clone activates NKG2D. Chimeric antigen receptor (CAR) constructs were obtained by fusing the nucleic acid sequences of human and mouse NKG2D to the nucleic acid sequence encoding the CD3 zeta signaling domain. Next, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with the virus containing NKG2D-CAR along with 8 μg / mL of polyblen. 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.

[0245] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed onto microplate wells, and NKG2D-CAR EL4 cells were cultured for 4 hours in wells coated with antibody fragments in the presence of brefeldin-A and monensin. Intracellular TNF-α production, an indicator of 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).

[0246] (Example 4) The NKG2D binding domain activates NK cells. Primary human NK cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood pia using density gradient centrifugation. NK cells (CD3) were then selected from the PBMCs using negative selection with magnetic beads. - CD56 + ) were isolated. The purity of the isolated NK cells was typically >95%. Next, the isolated NK cells were cultured for 24–48 hours in a medium containing 100 ng / mL of IL-2, and then transferred to wells of a microplate adsorbed with NKG2D binding domains and cultured in a medium containing fluorophore conjugate anti-CD107a antibody, brefelzin-A, and monensin. After culturing, the NK cells were assayed by flow cytometry using fluorophore conjugate antibodies against CD3, CD56, and IFN-γ. CD3 - CD56 +NK cell activation was assessed by analyzing the staining of cells for CD107a and IFN-γ. An increase in CD107a / IFN-γ double-positive cells indicates better NK cell activation due to the association of two activating receptors rather than one. The NKG2D binding domain and positive controls (e.g., the heavy chain variable domain represented by SEQ ID NO: 101 or SEQ ID NO: 103, and the light chain variable domain represented by SEQ ID NO: 102 or SEQ ID NO: 104) resulted in a higher percentage of NK cells activating CD107a compared to isotype controls. + and IFN-γ + This demonstrated the following (Figures 13 and 14 represent data from two independent experiments using PBMCs from different donors, respectively, for NK cell preparation).

[0247] Primary mouse NK cells Spleens were obtained from C57Bl / 6 mice and crushed through a 70 μm cell strainer to obtain a single-cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove erythrocytes. The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours, then harvested and prepared for NK cell isolation. Next, NK cells (CD3) were extracted from the spleen cells using negative depletion techniques with magnetic beads, typically with >90% purity. - NK1.1 + ) was isolated. Purified NK cells were cultured for 48 hours in a medium containing 100 ng / mL of mL-15, then transferred to wells of a microplate adsorbed with NKG2D-binding domains, and cultured in a medium containing fluorophore-conjugate anti-CD107a antibody, brefeldin-A, and monensin. After culturing in wells coated with NKG2D-binding domains, NK cells were assayed by flow cytometry using fluorophore-conjugate antibodies against CD3, NK1.1, and IFN-γ. CD3 - NK1.1 +NK cell activation was assessed by analyzing the staining of cells for CD107a and IFN-γ. An increase in CD107a / IFN-γ double-positive cells indicates better NK cell activation due to the association of two activating receptors rather than just one. The NKG2D binding domain and positive controls (selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) showed a higher percentage of NK cells activating CD107a compared to the isotype control. + and IFN-γ + This demonstrated the following (Figures 15 and 16 represent data from two independent experiments using different mice, respectively, for NK cell preparation).

[0248] (Example 5) The NKG2D binding domain enables cytotoxicity of target tumor cells. Human and mouse primary NK cell activation assays show increased cytotoxic markers in NK cells after incubation with NKG2D-binding domains. To address whether this leads to increased tumor cell lysis, cell-based assays were utilized in which each NKG2D-binding domain becomes a monospecific antibody. The Fc region was used as one targeting arm, while the Fab region (NKG2D-binding domain) acted as another targeting arm for activating NK cells. Human-derived THP-1 cells expressing high levels of the Fc receptor were used as the tumor target, and the Perkin Elmer DELFIA cytotoxicity kit was used. THP-1 cells were labeled with BATDA reagent and 10 5 The cells were resuspended in culture medium at 1 / mL. Next, labeled THP-1 cells were combined with NKG2D antibody, and mouse NK cells were isolated in microtiter plate wells at 37°C for 3 hours. After incubation, 20 μl of the culture supernatant was taken out, mixed with 200 μl of europium solution, and incubated in the dark with shaking for 15 minutes. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (excitation 337 nm, emission 620 nm), and specific lysis rates were calculated according to the kit instructions.

[0249] ULBP-6, a positive control and natural ligand for NKG2D, increased the specific lysis rate of THP-1 target cells by mouse NK cells. The NKG2D antibody also increased the specific lysis rate of THP-1 target cells, while isotype control antibodies showed a decrease in specific lysis rate. The dotted line shows the specific lysis rate of THP-1 cells by mouse NK cells without antibody treatment (Figure 17).

[0250] (Example 6) NKG2D antibodies exhibit high thermal stability. The melting temperature of the NKG2D binding domain was assayed using differential scanning fluorescence (SMRI). The extrapolated apparent melting temperature was higher compared to that of a typical IgG1 antibody (Figure 18).

[0251] (Example 7) Synergistic activation of human NK cells by crosslinking of NKG2D and CD16 Primary Human NK Cell Activation Assay Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral human blood pia using density gradient centrifugation. NK cells were purified from PBMCs using negative magnetic beads (StemCell #17955). NK cells were determined by flow cytometry to have >90% CD33 - CD56 + Next, the cells were grown for 48 hours in a medium containing 100 ng / mL of hIL-2 (Peprotech #200-02) before use in the activation assay. Antibodies were coated onto 96-well flat-bottom plates overnight at 4°C in 100 μl of sterile PBS at concentrations of 2 μg / mL (anti-CD16, Biolegend #302013) and 5 μg / mL (anti-NKG2D, R&D #MAB139), followed by thorough washing of the wells to remove excess antibody. For evaluation of degranulation, IL-2 activated NK cells were cultured in 5 × 10⁶ cells in a medium supplemented with 100 ng / mL of human IL-2 (hIL2) and 1 μg / mL of APC-conjugated anti-CD107a mAb (Biolegen #328619). 5 The cells were resuspended at 1 × 10⁶ cells / mL. Next, 1 × 10⁶ cells were resuspended. 5Individual cells / wells were added onto an antibody-coated plate. The protein transport inhibitor was Brefelzin A (BFA, Biolegend #420601) and monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively. The seeded cells were incubated at 37°C for 4 hours in 5% CO2. For intracellular staining of IFN-γ, NK cells were labeled with anti-CD3 (Biolegend #300452) and anti-CD56 mAb (Biolegend #318328), subsequently fixed, permeabilized, and labeled with anti-IFN-γ mAb (Biolegend #506507). NK cells were then labeled with live CD56 + CD3 - After gating the cells, the expression of CD107a and IFN-γ was analyzed by flow cytometry.

[0252] To investigate the relative efficacy of receptor combinations, plate-binding stimulation was used to crosslink NKG2D or CD16, as well as co-crosslink both receptors. As shown in Figure 19 (Figures 19A-19C), combined stimulation of CD16 and NKG2D resulted in a significant increase in CD107a (degranulation) levels (Figure 19A) and / or IFN-γ production levels (Figure 19B). The dotted lines represent the additive effect of individual stimulation of each receptor.

[0253] CD107a levels and intracellular IFN-γ production in IL-2 activated NK cells were analyzed after 4 hours of plate-binding stimulation with anti-CD16, anti-NKG2D, or a combination of both monoclonal antibodies. Graphs show mean (n=2) ± SD. Figure 19A shows CD107a levels, Figure 19B shows IFNγ levels, and Figure 19C shows CD107a and IFN-γ production levels. The data shown in Figures 19A–19C are representative of five independent experiments using five different healthy donors.

[0254] Example 8 - Evaluation of TriNKET binding to human NKG2D expressed in cells EL4 mouse lymphoma cell lines were engineered to express human NKG2D. A triple-specific binding protein (TriNKET), containing an NKG2D-binding domain, a tumor-associated antigen-binding domain (e.g., a CD20-binding domain), and a CD16-binding Fc domain as shown in Figure 1, was tested for its affinity to extracellular NKG2D expressed on EL4 cells. TriNKET was diluted to 20 μg / mL and then serially diluted. Binding of TriNKET to NKG2D was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were then analyzed by flow cytometry, and histograms were plotted. The TriNKET strains tested were CD26-TriNKET-CD20 (NKG2D-binding domain derived from clone ADI-28226 and CD20-binding domain derived from rituximab) and F04-TriNKET-CD20 (NKG2D-binding domain derived from clone ADI-29404 and CD20-binding domain derived from rituximab). The binding profiles of CD26-TriNKET-CD20 (dashed line) and F04-TriNKET-CD20 (solid line) are shown in Figure 35, along with unstained samples. The results show different levels of binding to NKG2D by clones ADI-28226 and ADI-29404.

[0255] Example 9 - Evaluation of TriNKET binding to human cancer antigens expressed in cells The binding of TriNKET to the tumor-associated antigen CD20 was assayed using Raji human lymphoma cells expressing CD20. TriNKET was incubated with the cells, and its binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry, and histograms were plotted. As shown in Figure 36, F04-TriNKET-CD20 and CD26-TriNKET-CD20 bind equally and sufficiently to CD20.

[0256] Example 10 - TriNKET activates NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood bufficoat using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated from PBMCs using negative selection with magnetic beads, and the purity of the isolated NK cells was typically >90%. The isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation, or rested overnight without cytokines. IL-2 activated NK cells were used within 24-48 hours after activation. Restless NK cells were always used on the same day after purification.

[0257] Human cancer cells expressing tumor antigens were collected and cultured in 2 × 10⁶ cells in culture medium. 6 The cells were resuspended at / mL. Monoclonal antibodies targeting tumor antigens or TriNKET targeting tumor antigens were diluted in culture medium. Restless and / or activated NK cells were harvested, washed, and placed in culture medium at a rate of 2 × 10⁶. 6 The cells were resuspended in 1 / mL. Cancer cells were then mixed with monoclonal antibody / TriNKET, and NK cells were activated in the presence of IL-2. Brefeldin A and monensin were also added to the mixed culture to block protein transport from the cells for intracellular cytokine staining. Fluorophore conjugate anti-CD107a was added to the mixed culture, and the culture was incubated for 4 hours before sample preparation for FACS analysis using fluorophore conjugate antibodies against CD3, CD56, and IFN-γ. CD107a and IFN-γ staining was performed on CD3 - CD56 + Cellular analysis was performed to evaluate NK cell activation. An increase in CD107a / IFN-γ double-positive cells indicates better NK cell activation due to the association of two activating receptors rather than just one.

[0258] When primary human NK cells were co-cultured with CD20-positive human cancer cells, TriNKET-mediated activation of primary human NK cells occurred (Figure 37). TriNKET-targeted CD20 (e.g., C26-TriNKET-CD20 and F04-TriNKET-CD20) mediated the activation of human NK cells co-cultured with CD20-positive Raji cells, as indicated by increased CD107a degranulation and IFN-γ cytokine production (Figure 37). Compared to the monoclonal antibody rituximab, both TriNKETs (e.g., C26-TriNKET-CD20 and F04-TriNKET-CD20) showed superior activation of human NK cells.

[0259] Example 11 - TriNKET enhances the cytotoxicity of human NK cells against cancer cells. To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, human NK cell line KHYG-1 cells transduced to express human CD16a-158v were used as effector cells. All cytotoxic assays were prepared as follows: Human cancer cell lines expressing the target of interest (e.g., CD20-positive Raji cells) were harvested from culture, washed with PBS, and labeled with BATDA reagent (Perkin Elmer AD0116) for 10 minutes. 6 The cells were resuspended in growth medium at a concentration of / mL. Target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with PBS and then rehydrated in culture medium at a density of 0.5–1.0 × 10⁶. 5The cells were resuspended at / mL. Aliquots of the labeled cells were set aside to prepare background wells, and the cells were spun out of the medium. 100 μl of the medium was carefully added to the wells in triplicates to avoid disturbing the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Wells were preserved for spontaneous release from target cells, and wells were prepared for maximum lysis of target cells by adding 1% Triton-X. Monoclonal antibody or TriNKET against the target tumor was diluted in culture medium, and 50 μl of the diluted monoclonal antibody or TriNKET was added to each well. KHYG-1-CD16-158V cells were washed and added to the culture medium in 10 units according to the desired effector cell to target cell ratio. 5 ~2.0×10 6 The cells were resuspended in 1 / mL. 50 μl of NK cells were added to each well of the plate, bringing the total culture volume to 200 μl. The plate was then... Before developing the assay, the samples were incubated with 5% CO2 at 37°C for 2–3 hours.

[0260] After 2-3 hours of incubation, the plate was removed from the incubator, and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μl of the culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μl of room temperature europium solution was added to each well. The plate was shielded from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plate was read using either a Victor 3 or SpectraMax i3X instrument. % Specific lysis was calculated as follows: % Specific lysis = ((Experimental release - Spontaneous release) / (Maximum release - Spontaneous release)) × 100%.

[0261] CD20-targeted TriNKET mediates the cytotoxicity of human NK cells against CD20-positive Raji B-cell lymphoma cells. As shown in Figure 39, both TriNKETs (C26-TriNKET-CD20 and F04-TriNKET-CD20) were able to enhance the cytotoxic activity of resting human KHYG-1-CD16a-158V effector cells against their cancer cells in a dose-response manner. KHYG-1-CD16a-158V cells were weakly active against Raji cells without the addition of TriNKET. The dotted line indicates specific lysis of Raji target cells without the addition of TriNKET.

[0262] F04-TriNKET-CD20 (which mediates NK cell cytotoxicity against CD20-expressing cancer cells) was compared to the parental monoclonal antibody rituximab. F04-TriNKET-CD20 or the anti-CD20 monoclonal antibody rituximab were mixed with KHYG-1-CD16a-158V cells (KHYG-1 cells transduced to express human CD16a-158V) and Raji cells, and NK cell-mediated cytotoxicity was measured as described above. Figure 39 shows that F04-TriNKET-CD20 enhanced the potency and maximal killing of NK cells against Raji cells compared to the anti-CD20 monoclonal antibody. The dotted line indicates specific lysis of Raji target cells by KHYG-1-CD16a-158V cells without the addition of TriNKET or the anti-CD20 monoclonal antibody.

[0263] Embedding by reference Each of the entire disclosures of the patent documents and scientific papers referenced herein is incorporated by reference for all purposes.

[0264] Equivalents The present invention may be realized in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein should be considered illustrative in all respects and not limiting the invention as described herein. Accordingly, the scope of the invention is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the equivalent meaning and scope of the claims are intended to be encompassed therein.

Claims

1. (a) The first antigen-binding site that binds to NKG2D, (b) A second antigen-binding site that binds to CD37, CD20, CD19, CD22, CD30, CD52, or CD133, (c) an antibody Fc domain or a portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16 Proteins containing these proteins.

2. The protein according to claim 1, wherein the first antigen-binding site binds to human NKG2D.

3. The protein according to claim 1 or 2, wherein the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

4. The protein according to claim 3, wherein the heavy chain variable domain and the light chain variable domain are located on the same polypeptide.

5. The protein according to claim 3 or 4, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

6. The protein according to claim 5, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are located on the same polypeptide.

7. The protein according to claim 5 or 6, wherein the light chain variable domain of the first antigen-binding site has the same amino acid sequence as the light chain variable domain of the second antigen-binding site.

8. The protein according to any one of the preceding claims, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 41, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 85, and SEQ ID NO:

93.

9. The protein according to any one of claims 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.

10. The protein according to any one of claims 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.

11. The protein according to any one of claims 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.

12. The protein according to any one of claims 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.

13. The protein according to any one of claims 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.

14. The protein according to any one of claims 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to that of SEQ ID NO: 69 and a light chain variable domain that is at least 90% identical to that of SEQ ID NO:

70.

15. The protein according to any one of claims 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.

16. The protein according to any one of claims 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 and a light chain variable domain that is at least 90% identical to SEQ ID NO:

86.

17. The protein according to any one of claims 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.

18. The protein according to any one of claims 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.

19. The protein according to any one of claims 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.

20. The protein according to claim 1 or 2, wherein the first antigen-binding site is a single-domain antibody.

21. The single-domain antibody, Ｈ H fragment or V ＮＡＲ A protein according to claim 20, which is a fragment.

22. The protein according to any one of claims 1, 2, or 20 to 21, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

23. The protein according to claim 22, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are located on the same polypeptide.

24. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD37, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 109, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

113.

25. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD37, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 117, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

121.

26. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD37, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 125, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

129.

27. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD20, 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: 134, 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:

138.

28. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD20, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 142, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

146.

29. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD20, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 150, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

154.

30. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD20, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 158, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

162.

31. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD20, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 166, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

170.

32. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD19, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 175, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

179.

33. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD19, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 183, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

187.

34. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD19, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 191, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

195.

35. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD19, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 199, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

203.

36. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD22, 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: 208, 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:

212.

37. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD22, 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: 216, 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:

220.

38. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD22, 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: 224, 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:

228.

39. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD30, 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: 233, 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:

237.

40. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD30, 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: 241, 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:

245.

41. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD30, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 249, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

253.

42. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD30, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 257, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

261.

43. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD30, 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: 265, 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:

269.

44. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD52, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 274, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

278.

45. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD52, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 282, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

286.

46. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD133, 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: 291, 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:

295.

47. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD133, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 299, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

303.

48. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD133, the heavy chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 307, and the light chain variable domain of the second antigen-binding site contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

311.

49. The protein according to any one of claims 1 to 23, wherein the second antigen-binding site is bound to CD133, 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: 315, 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:

319.

50. The protein according to any one of claims 1 to 4 or 8 to 21, wherein the second antigen-binding site is a single-domain antibody.

51. The second antigen-binding site is V Ｈ H fragment or V ＮＡＲ A protein according to claim 50, which is a fragment.

52. The protein according to any one of the preceding claims, wherein the protein comprises a portion of an antibody Fc domain sufficient to bind to CD16, and the antibody Fc domain comprises a hinge and a CH2 domain.

53. The protein according to claim 52, wherein the antibody Fc domain comprises the hinge and CH2 domain of a human IgG1 antibody.

54. The protein according to claim 52 or 53, wherein the Fc domain contains an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody.

55. The protein according to claim 54, wherein the Fc domain has an amino acid sequence that is at least 90% identical to that of 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.

56. A formulation comprising the protein and a pharmaceutically acceptable carrier as described in any one of the preceding claims.

57. A cell comprising one or more nucleic acids expressing the protein described in any one of claims 1 to 55.

58. A method for enhancing tumor cell death, comprising the step of exposing tumor cells and natural killer cells to an effective amount of the protein described in any one of claims 1 to 55.

59. A method for treating cancer, comprising administering to a patient an effective amount of the protein according to any one of claims 1 to 55 or the formulation according to claim 56.

60. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD37, and the cancer to be treated is selected from the group consisting of B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), non-Hodgkin lymphoma, and acute myeloid leukemia.

61. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD20, and the cancer to be treated is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, and B-cell malignancy.

62. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD19, and the cancer to be treated is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancy, multiple myeloma, and acute myeloid leukemia.

63. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD22, and the cancer to be treated is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin lymphoma, follicular lymphoma, acute lymphoblastic leukemia, B-cell malignancy, and hairy cell leukemia.

64. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD30, and the cancer to be treated is selected from the group consisting of Hodgkin lymphoma, anaplastic large cell lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia-lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, and embryonal cell carcinoma.

65. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD52, and the cancer to be treated is selected from the group consisting of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, peripheral T-cell lymphoma and T-cell prelymphocytic leukemia, B-cell malignancies, non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia-lymphoma, mature T / natural killer (NK) cell neoplasms, and thymoma.

66. The method according to claim 59, wherein the second antigen-binding site of the protein binds to CD133, and the cancer to be treated is selected from the group consisting of breast cancer, colon cancer, prostate cancer, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, kidney cancer, uterine cancer, testicular germ cell cancer, acute myeloid leukemia, acute lymphoblastic leukemia, glioma, glioblastoma, and squamous cell carcinoma of the head and neck.

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