Proteins binding NKG2d, CD16 and FLT3

Abstract

To provide proteins that bind NKG2D, CD16, and tumor-associated antigen FLT3.SOLUTION: A protein comprises: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds FLT3; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. In one embodiment, the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain to bind the human NKG2D.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 539,421, filed July 31, 2017, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 30, 2018 is named DFY-027WO_SL.txt and is 103,731 bytes in size.

[0003] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16, and the tumor-associated antigen FLT3. [Background technology]

[0004] Cancer remains a significant health problem, despite considerable research efforts and chemical advances reported in the literature for the treatment of this disease. Blood and bone marrow cancers are frequently diagnosed cancer types, including multiple myeloma, leukemia, and lymphoma. Current treatment options for these cancers are not effective for all patients and / or may have significant adverse side effects. Other types of cancer also remain difficult to treat using existing therapeutic options.

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

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

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

[0008] FMS-like tyrosine kinase-3 (FLT3), a receptor tyrosine kinase expressed in multipotent progenitor cells and common lymphoid progenitor cells, is important for the development of the hematopoietic and immune systems. Signaling through FLT3 plays a key role in cell survival, proliferation, and differentiation. Mutations in the FLT3 receptor can lead to the development of leukemia, such as acute myeloid leukemia, T-cell leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia. Internal tandem duplication of FLT3 (FLT3-ITD) is the most common mutation associated with acute myeloid leukemia (AML). Summary of the Invention

[0009] The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and the tumor-associated antigen FLT3. Such proteins may bind to two or more NK activating receptors and potentially block binding of natural ligands to NKG2D. In certain embodiments, the proteins may agonize human NK cells. In some embodiments, the proteins may agonize human NK cells as well as NK cells of other species, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in further detail below.

[0010] Thus, one aspect of the invention provides a protein incorporating a first antigen-binding site that binds NKG2D, a second antigen-binding site that binds the tumor-associated antigen FLT3, and an antibody Fc domain, a sufficient portion thereof to bind CD16, or a third antigen-binding site that binds CD16.

[0011] The antigen binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged as in an antibody or fused together to form an scFv), or one or more of the antigen binding sites may be a V chain variable domain, such as in a camel antibody. H H antibodies or V antibodies such as those found in cartilaginous fish NAR The antibody may be a single domain antibody such as an antibody.

[0012] In one aspect, the invention provides a multispecific binding protein that binds to the NKG2D and CD16 receptors on natural killer cells and the tumor-associated antigen FLT3. The NKG2D 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.

[0013] A first antigen-binding site that binds to NKG2D may, in some embodiments, incorporate a heavy chain variable domain related to SEQ ID NO: 1, such as by having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1 and / or incorporating amino acid sequences 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. A heavy chain variable domain related to SEQ ID NO: 1 may be paired with a different light chain variable domain to form an NKG2D-binding site. For example, a first antigen binding site incorporating a heavy chain variable domain related to SEQ ID NO: 1 may further incorporate a light chain variable domain selected from any one of the sequences related to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40. For example, the first antigen-binding site incorporates a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:1 and a light chain variable domain having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of 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.

[0014] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 41 and a light chain variable domain related to SEQ ID NO: 42. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 41 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 45) sequences of SEQ ID NO: 41. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:46), CDR2 (SEQ ID NO:47), and CDR3 (SEQ ID NO:48) sequences of SEQ ID NO:42.

[0015] In other embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 49 and a light chain variable domain related to SEQ ID NO: 50. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 49 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 53) sequences of SEQ ID NO: 49. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:55), and CDR3 (SEQ ID NO:56) sequences of SEQ ID NO:50.

[0016] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 57 and a light chain variable domain related to SEQ ID NO: 58, such as by having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 57 and an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 58, respectively.

[0017] In another embodiment, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO:59 and a light chain variable domain related to SEQ ID NO:60, for example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:59 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), and CDR3 (SEQ ID NO:136) 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), and CDR3 (SEQ ID NO:139) sequences of SEQ ID NO:60.

[0018] A first antigen-binding site that binds to NKG2D may, in some embodiments, incorporate a heavy chain variable domain related to SEQ ID NO: 61 and a light chain variable domain related to SEQ ID NO: 62. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 61 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 63), CDR2 (SEQ ID NO: 64), and CDR3 (SEQ ID NO: 65) sequences of SEQ ID NO: 61. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 66), CDR2 (SEQ ID NO: 67), and CDR3 (SEQ ID NO: 68) sequences of SEQ ID NO: 62. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 69 and a light chain variable domain related to SEQ ID NO: 70. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 69 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 71), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73) sequences of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76) sequences of SEQ ID NO: 70.

[0019] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 77 and a light chain variable domain related to SEQ ID NO: 78. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 77 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 79), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81) sequences of SEQ ID NO: 77. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:82), CDR2 (SEQ ID NO:83), and CDR3 (SEQ ID NO:84) sequences of SEQ ID NO:78.

[0020] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 85 and a light chain variable domain related to SEQ ID NO: 86. For example, the heavy chain variable domain of the first antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85 and / or may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 87), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89) sequences of SEQ ID NO: 85. Similarly, the light chain variable domain of the second antigen-binding site 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:90), CDR2 (SEQ ID NO:91), and CDR3 (SEQ ID NO:92) sequences of SEQ ID NO:86.

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

[0022] In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO:101 and a light chain variable domain related to SEQ ID NO:102, such as by having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:101 and an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:102, respectively. In some embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 103 and a light chain variable domain related to SEQ ID NO: 104, such as by having an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 103 and an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 104, respectively.

[0023] In some embodiments, the second antigen-binding site that binds FLT3 may incorporate a heavy chain variable domain related to SEQ ID NO: 109 and a light chain variable domain related to 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 may incorporate amino acid sequences identical to 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:114), CDR2 (SEQ ID NO:115), and CDR3 (SEQ ID NO:116) sequences of SEQ ID NO:113.

[0024] Alternatively, the second antigen-binding site that binds to FLT3 may incorporate a heavy chain variable domain related to SEQ ID NO: 117 and a light chain variable domain related to 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 may incorporate amino acid sequences identical to 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 122), CDR2 (SEQ ID NO: 123), and CDR3 (SEQ ID NO: 124) sequences of SEQ ID NO: 121.

[0025] Alternatively, the second antigen-binding site that binds to FLT3 may incorporate a heavy chain variable domain related to SEQ ID NO: 125 and a light chain variable domain related to 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 may incorporate amino acid sequences identical to 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 may incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 130), CDR2 (SEQ ID NO: 131), and CDR3 (SEQ ID NO: 132) sequences of SEQ ID NO: 129.

[0026] In some embodiments, the second antigen-binding site incorporates a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen-binding site.

[0027] In some embodiments, the protein incorporates a sufficient portion of an antibody Fc domain to bind to CD16, wherein the antibody Fc domain comprises a hinge and CH2 domain, and / or an amino acid sequence at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

[0028] Also provided are formulations containing any one of the proteins described herein, cells containing one or more nucleic acids that express the proteins, and methods of using the proteins to enhance tumor cell death.

[0029] Another aspect of the present invention provides a method of treating cancer in a patient. The method comprises administering a therapeutically effective amount of a multispecific binding protein described herein to a patient in need thereof. Exemplary cancers treated using the multispecific binding protein include leukemias, such as acute myeloid leukemia, T-cell leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. [Brief explanation of the drawings]

[0030] [Figure 1]

[0023] Figure 1 is a diagram of a heterodimeric multispecific antibody. Each arm can represent either an NKG2D-binding domain or an FLT3-binding domain. In some embodiments, the NKG2D and FLT3-binding domains can share a common light chain.

[0031] [Figure 2] Diagram of heterodimeric multispecific antibodies. Either the NKG2D-binding domain or the FLT3-binding domain can be in scFv format (right arm).

[0032] [Figure 3] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

[0033] [Figure 4] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D in an ELISA assay.

[0034] [Figure 5] 1 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to murine recombinant NKG2D in an ELISA assay.

[0035] [Figure 6]1 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D by flow cytometry showing fold over background (FOB) of mean fluorescence intensity (MFI).

[0036] [Figure 7] 1 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing murine NKG2D by flow cytometry showing fold over background (FOB) of mean fluorescence intensity (MFI).

[0037] [Figure 8] 1 is a line graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competing with the natural ligand ULBP-6.

[0038] [Figure 9] 1 is a line graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competing with the natural ligand MICA.

[0039] [Figure 10] 1 is a line graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant murine NKG2D-Fc by competing with the natural ligand Rae-1 delta.

[0040] [Figure 11] 1 is a bar graph showing activation of human NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-α positive cells, which cells express a human NKG2D-CD3 zeta fusion protein.

[0041] [Figure 12]1 is a bar graph showing activation of murine NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-α positive cells, which cells express a murine NKG2D-CD3 zeta fusion protein.

[0042] [Figure 13] 1 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0043] [Figure 14] 1 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0044] [Figure 15] 1 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0045] [Figure 16] 1 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0046] [Figure 17] 1 is a bar graph showing the cytotoxic effect of NKG2D binding domains (listed as clones) on tumor cells.

[0047] [Figure 18] 1 is a bar graph showing melting temperatures of NKG2D binding domains (listed as clones) as measured by differential scanning fluorimetry.

[0048] [Figure 19]19A and 19B are bar graphs of synergistic activation of NK cells using CD16 and NKG2D binding. Figure 19A shows levels of CD107a, Figure 19B shows levels of IFN-γ, and Figure 19C shows levels of CD107a and IFN-γ. Graphs show mean (n=2) ± SD. Data are representative of five independent experiments using five different healthy donors.

[0049] [Figure 20] Diagram of TriNKET in triomab form, which is a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain, derived from two parent antibodies. Triomabs can also be heterodimeric constructs containing half rat and half mouse antibodies.

[0050] [Figure 21] Diagram of KiH-common light chain-based TriNKET, which includes knob-into-hole (KIH) technology. KiH is a heterodimer containing two Fabs that bind to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. KiH-based TriNKET can also be a heterodimeric construct with two Fabs that bind to targets 1 and 2, containing two different heavy chains and a common light chain that pairs with both heavy chains.

[0051] [Figure 22] Diagram of a dual variable domain immunoglobulin (DVD-Ig™) form of TriNKET, which combines the target binding domains of two monoclonal antibodies via a naturally occurring flexible linker, resulting in a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of a Fab targeting antigen 1. The DVD-Ig™ form contains a conventional Fc.

[0052] [Figure 23]Diagram of the orthogonal Fab interface (ortho-Fab) version of TriNKET, a heterodimeric construct containing two Fabs that bind target 1 and target 2 fused to an Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0053] [Figure 24] Schematic diagram of the 2-in-1 Ig TriNKET.

[0054] [Figure 25] Diagram of ES-type TriNKET, which is a heterodimeric construct containing two different Fabs that bind target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0055] [Figure 26] 1 is a diagram of a Fab arm-swapped TriNKET antibody, which exchanges Fab arms by swapping a heavy chain and associated light chain (half molecule) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm-swapped (cFae) is a heterodimer containing two Fabs that bind to targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0056] [Figure 27] FIG. 1 is a diagram of the SEED body form of TriNKET, which is a heterodimer containing two Fabs that bind to targets 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0057] [Figure 28]Diagram of the LuZ-Y type of TriNKET, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y type is a heterodimer containing two different scFabs that bind to targets 1 and 2 fused to an Fc. Heterodimerization is ensured by the leucine zipper motif fused to the C-terminus of the Fc.

[0058] [Figure 29] Diagram of Cov-X-body type TriNKET.

[0059] [Figure 30] 30A and 30B are diagrams of kλ-body versions of TriNKET, which are heterodimeric constructs with two different Fabs fused to an Fc stabilized by heterodimerization mutations, one Fab targeting antigen 1 containing a kappa LC and the second Fab targeting antigen 2 containing a lambda LC. Figure 30A is an exemplary diagram of one type of kλ-body, and Figure 30B is an exemplary diagram of another kλ-body.

[0060] [Figure 31] The Oasc-Fab heterodimer construct contains a Fab that binds to target 1 and a scFab that binds to target 2, both fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0061] [Figure 32] DuetMab is a heterodimeric construct containing two different Fabs that bind antigens 1 and 2, and an Fc that is stabilized by heterodimerization mutations. Fab1 and 2 contain differential S-S bridges that ensure correct light and heavy chain pairing.

[0062] [Figure 33]CrossmAb, which is a heterodimeric construct with two different Fabs that bind to targets 1 and 2, and an Fc stabilized by a heterodimerization mutation. The CL and CH1 domains, as well as the VH and VL domains, are swapped, e.g., CH1 is fused in-line with VL, while CL is fused in-line with VH.

[0063] [Figure 34] Fit-Ig, which is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of the HC of a Fab that binds antigen 1. The construct contains wild-type Fc.

[0064] [Figure 35] 1 is a line graph showing binding of FLT3-targeted TriNKET to NKG2D expressed on EL4 cells. The FLT3 monoclonal antibody IMCEB10 was used as a control.

[0065] [Figure 36] 36A and 36B are line graphs showing binding of FLT3-targeted TriNKET to FLT3 expressed on the human AML cell lines Molm-13 (FIG. 36A) and EOL-1 (FIG. 36B). The FLT3 monoclonal antibody IMCEB10 was used as a control.

[0066] [Figure 37] 37A and 37B are line graphs showing the internalization of FLT3-targeted TriNKET on EOL-1 cells (FIG. 37A) and Molm-13 cells (FIG. 37B) after 2 and 20 hours of incubation at 37° C. Lintuzumab was used as a control.

[0067] [Figure 38]38A and 38B are line graphs showing TriNKET-mediated cytotoxicity of human NK cells against FLT3-expressing EOL-1 cells. FLT3 monoclonal antibody IMCEB10 and TriNKET containing the FLT3-binding domain derived from IMCEB10 are shown in Figure 38A. FLT3 monoclonal antibody 4G8 and TriNKET containing the FLT3-binding domain derived from 4G8 are shown in Figure 38B. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and the tumor-associated antigen FLT3. In some embodiments, the multispecific proteins further comprise an additional antigen-binding site that binds to FLT3 or another tumor-associated antigen. 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 treating cancer. Various aspects of the invention are described in the following sections; however, aspects of the invention described in a particular section should not be limited to any particular section.

[0069] To facilitate understanding of this invention, a number of terms and phrases are defined below.

[0070] The terms "a" and "an" are used herein to mean "one or more" and include the plural unless the context is inappropriate.

[0071] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three widely divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions," which are interposed between more conserved adjacent stretches known as "framework regions," or "FRs." Thus, the term "FR" refers to amino acid sequences naturally found among and adjacent to the hypervariable regions of immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged 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 a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions," or "CDRs." In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, resulting in a "single domain antibody." The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide, such as an scFv, in which a peptide linker is used to link the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0072] The term "tumor-associated antigen," as used herein, refers to any antigen, including, but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, or lipid, associated with cancer. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates.

[0073] 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., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably humans.

[0074] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect that results in an improvement of a condition, disease, disorder, etc., e.g., a decrease, reduction, modulation, improvement, or elimination, or an improvement in the symptoms thereof.

[0075] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0076] 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 compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0077] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention, which, upon administration to a subject, can provide a compound of the present invention or its active metabolite or residue. As known to those skilled in the art, "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 the like. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, may be used in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0078] Exemplary bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein W is C 1~4 Examples of suitable compounds include, but are not limited to, compounds in which the aryl group is alkyl.

[0079] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, etc. Other examples of salts include those containing a suitable cation, e.g., Na + , NH4 + , and NW4 + (Wherein W is C 1~4 Examples of the anion of the compound of the present invention include an anion of the compound of the present invention that is combined with an alkyl group.

[0080] For therapeutic use, the salts of the compounds of the invention are considered to be pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0081] Throughout the description, when compositions are described as having, containing, or comprising particular components, or when processes and methods are described as having, containing, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of, or consist of, the recited components, and that there are processes and methods of the invention that consist essentially of, or consist of, the recited process steps.

[0082] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of the variable is taken into account.

[0083] I. Protein The present invention provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and the tumor-associated antigen FLT3. The multispecific binding proteins are useful in the pharmaceutical compositions and methods of treatment described herein. Binding of the multispecific binding proteins to the NKG2D and CD16 receptors on natural killer cells enhances the activity of natural killer cells in destroying tumor cells that express FLT3. Binding of the multispecific binding proteins to FLT3-expressing cells brings cancer cells into close proximity with natural killer cells, thereby facilitating direct and indirect cancer cell destruction by natural killer cells. Further description of several exemplary multispecific binding proteins is provided below.

[0084] The first component of the multispecific binding protein binds to NKG2D receptor-expressing cells, including NK cells, γδ T cells, and CD8 + These may include, but are not limited to, αβ T cells. Upon binding to NKG2D, the multispecific binding protein may block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating the NKG2D receptor.

[0085] The second component of the multispecific binding protein binds to FLT3. FLT3-expressing cells can be found in leukemias, such as acute myeloid leukemia and T-cell leukemia.

[0086] The third component of the multispecific binding protein binds to Fc receptors on the surface of cells that express CD16, including leukocytes, such as natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0087] The multispecific binding proteins described herein can take various forms. For example, one form is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain comprises a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen-binding site that binds to FLT3. Both the first Fc domain and the second Fc domain can bind to CD16 (Figure 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0088] Another exemplary format includes a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (Figure 2). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused, via either a linker or an antibody hinge, to a single-chain variable fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pair together to bind to NKG2D or to the FLT3 antigen. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain to bind to NKG2D or to the tumor-associated antigen FLT3. Both the first and second Fc domains are capable of binding to CD16 (Figure 2).

[0089] One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the antigen-binding motif is a single-chain or disulfide-stabilized variable region (scFv) forming a tetravalent or trivalent molecule.

[0090] In some embodiments, the multispecific binding protein is a Triomab type, which is a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light chain and one heavy chain, derived from two parent antibodies.

[0091] In some embodiments, the multispecific binding protein is a KiH common light chain (LC) type, which includes knob-into-hole (KIH) technology. H The idea behind "knob-into-hole (KiH)" Fc technology is to introduce a "knob" into one CH3 domain (CH3A) by substituting a small residue for a bulky one (e.g., T366W in EU numbering). CH3A To accommodate the "knob," a complementary "hole" surface was created on the other CH3 domain (CH3B) by replacing the nearest neighboring residues with a knob with smaller residues (e.g., T366S / L368A / Y407V CH3BThe "hole" mutations were optimized by structure-based phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(1):26-35). X-ray crystal 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) 426(9):1947-57, 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) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the core interface between the CH3 domains, whereas knob-knob and hole-hole interfaces do not favor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0092] In some embodiments, the multispecific binding protein is of the dual variable domain immunoglobulin (DVD-Ig™) type, which combines the target-binding domains of two monoclonal antibodies via a naturally occurring flexible linker, resulting in a tetravalent IgG-like molecule.

[0093] In some embodiments, the multispecific binding protein has an orthogonal Fab interface (ortho-Fab). In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191-8), structure-based design is used to engineer the LC and HC domains of only one Fab, without changing the other Fab. VH-CH1 Complementary mutations are introduced at the interface.

[0094] In some embodiments, the multispecific binding protein is a 2-in-1 Ig type. In some embodiments, the multispecific binding protein is an ES type, which is a heterodimeric construct containing two different Fabs that bind to target 1 and target 2 fused to an Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0095] In some embodiments, the multispecific binding protein is a kλ-body type, which is a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains a kappa LC, while the second Fab targeting antigen 2 contains a lambda LC. Figure 30A is an exemplary diagram of one type of kλ-body, and Figure 30B is an exemplary diagram of another kλ-body.

[0096] In some embodiments, the multispecific binding protein is a Fab arm exchanged antibody (an antibody that exchanges a Fab arm by exchanging a heavy chain and associated light chain (half molecule) with a heavy-light chain pair from another molecule, thereby resulting in a bispecific antibody).

[0097] In some embodiments, the multispecific binding protein is a SEED body type. The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules with the potential to expand the therapeutic applications of natural antibodies. This protein engineering platform is based on exchanging structurally related sequences of immunoglobulins within the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while disfavoring homodimerization of the AG and GA SEED CH3 domains. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

[0098] In some embodiments, the multispecific binding protein is of the LuZ-Y type, in which a leucine zipper is used to direct heterodimerization of two different HCs. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0099] In some embodiments, the multispecific binding protein is a Cov-X-body type. In bispecific CovX-bodies, two different peptides are linked to each other using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. The pharmacophore is responsible for functional activity, while the antibody scaffold confers long half-life and Ig-like distribution. The pharmacophore can be chemically optimized or replaced with another pharmacophore to generate optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107(52), 22611-22616).

[0100] In some embodiments, the multispecific binding protein is an Oasc-Fab heterodimer comprising a Fab that binds target 1 and a scFab that binds target 2, fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0101] In some embodiments, the multispecific binding protein is of the DuetMab type, which is a heterodimeric construct containing two different Fabs that bind to antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fab1 and 2 contain differential S-S bridges that ensure correct LC and HC pairing.

[0102] In some embodiments, the multispecific binding protein is a CrossmAb type, which is a heterodimeric construct with two different Fabs that bind to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are swapped, e.g., CH1 is fused in-line with VL, while CL is fused in-line with VH.

[0103] In some embodiments, the multispecific binding protein is of the Fit-Ig type, which is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of the HC of a Fab that binds antigen 1. The construct contains a wild-type Fc.

[0104] Table 1 lists peptide sequences of heavy and light chain variable domains that, in combination, can bind to NKG2D. NKG2D-binding domains may differ in their binding affinity to NKG2D, but they all nevertheless activate human NKG2D and NK cells. [Table 1] TIFF2025131681000003.tif222150 TIFF2025131681000004.tif222150 TIFF2025131681000005.tif219150 TIFF2025131681000006.tif219150 TIFF2025131681000007.tif219150 TIFF2025131681000008.tif70150

[0105] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 101 may be paired with the light chain variable domain represented by SEQ ID NO: 102 to form an antigen-binding site capable of binding to NKG2D, as exemplified in US Pat. No. 9,273,136. [ka]

[0106] Alternatively, the heavy chain variable domain represented by SEQ ID NO: 103 may be paired with the light chain variable domain represented by SEQ ID NO: 104 to form an antigen-binding site capable of binding to NKG2D, as exemplified in US Pat. No. 7,879,985. [ka]

[0107] In one aspect, the present disclosure provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and the antigen FLT3. Table 2 lists some exemplary sequences of heavy and light chain variable domains that, in combination, can bind to FLT3. [Table 2] TIFF2025131681000012.tif219150 TIFF2025131681000013.tif45150

[0108] Alternatively, novel antigen binding sites capable of binding to FLT3 can be identified by screening for binding to the amino acid sequence defined in SEQ ID NO:133. [ka]

[0109] Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, interaction with CD16 is primarily focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 in the CH2 domain, and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or decrease binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0110] The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which may result in the assembly of heterodimers in addition to the assembly of homodimers of each antibody heavy chain. Promoting the preferential assembly of heterodimers can be achieved by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in US 13 / 494870, US 16 / 028850, US 11 / 533709, US 12 / 875015, US 13 / 289934, US 14 / 773418, US 12 / 811207, US 13 / 866756, US 14 / 647480, and US 14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1 by incorporating different pairs of amino acid substitutions within the two chains that allow the first and second polypeptides to selectively heterodimerize with each other. All amino acid substitution positions illustrated below are numbered according to the EU index as in Kabat.

[0111] In one configuration, the amino acid substitutions in the first polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in the second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (protrusion) fits onto the surface of the smaller amino acid substitution (cavity). For example, one polypeptide may incorporate a T366W substitution and the other may incorporate three substitutions including T366S, L368A, and Y407V.

[0112] The antibody heavy chain variable domains of the present invention may optionally be linked to an amino acid sequence at least 90% identical to that of an antibody constant region, such as an IgG constant region comprising the hinge, CH2, and CH3 domains, with or without the CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to that of a human antibody constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to that of an antibody constant region of another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439 when compared to the human IgG1 constant region. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, and T366S. , L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0113] In certain embodiments, mutations that may be incorporated into CH1 of the human IgG1 constant region may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be incorporated into Cκ of the human IgG1 constant region may be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0114] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 3. [Table 3]

[0115] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 4]

[0116] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 5. [Table 5]

[0117] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6. [Table 6]

[0118] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 7, where the position(s) shown in the first polypeptide row are replaced with any known negatively charged amino acid and the position(s) shown in the second polypeptide row are replaced with any known positively charged amino acid. [Table 7]

[0119] Alternatively, at least one amino acid substitution may be selected from the following set of Table 8, where the position(s) shown in the first polypeptide row are replaced with any known positively charged amino acid and the position(s) shown in the second polypeptide row are replaced with any known negatively charged amino acid. [Table 8]

[0120] Alternatively, the amino acid substitutions may be selected from the following set in Table 9. [Table 9]

[0121] Alternatively, or in addition, the structural stability of the heteromultimeric protein may be increased by introducing S354C on either the first or second polypeptide chain and Y349C on the opposing polypeptide chain, which forms an artificial disulfide bridge within the interface of the two polypeptides.

[0122] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407.

[0123] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0124] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0125] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0126] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411.

[0127] 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, 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 at one or more positions selected from the group consisting of L351, D399, S400, and Y407.

[0128] 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, 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 at one or more positions selected from the group consisting of Q347, E357, D399, and F405.

[0129] 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, 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 at one or more positions selected from the group consisting of Y349, K360, Q347, and K409.

[0130] 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, 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 at one or more positions selected from the group consisting of D356, E357, and D399.

[0131] 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, 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 at one or more positions selected from the group consisting of K370, K392, K409, and K439.

[0132] 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, 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 at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409.

[0133] In some embodiments, the amino acid sequence of one polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392, and K409, and the amino acid sequence of the other polypeptide chain in the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366, and D399.

[0134] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by a Y349C substitution.

[0135] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by a S354C substitution.

[0136] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by O347R, D399V, and F405T substitutions.

[0137] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by O347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by K360E and K409W substitutions.

[0138] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by T366S, T368A, and Y407V substitutions.

[0139] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by T366S, T368A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by a T366W substitution.

[0140] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by T350V, T366L, K392L, and T394W substitutions.

[0141] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by T350V, T366L, K392L, and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of an IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions.

[0142] The multispecific proteins described above can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, and a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector, and the first, second, and third expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0143] To achieve the highest yield of the multispecific protein, 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.

[0144] The clones can be cultured under conditions suitable for scale-up in a bioreactor to maintain expression of the multispecific protein. The multispecific protein can be isolated and purified using methods known in the art, including, for example, centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0145] II. Properties of Multispecific Proteins The multispecific proteins described herein comprise an NKG2D-binding site, a CD16-binding site, and an FLT3-binding site. In some embodiments, the multispecific proteins simultaneously bind to cells expressing NKG2D and / or CD16, such as NK cells, and tumor cells expressing FLT3. Binding of the multispecific binding protein to NK cells can enhance the activity of NK cells for the destruction of tumor cells.

[0146] In some embodiments, the multispecific protein binds to FLT3 with similar affinity as the corresponding FLT3 monoclonal antibody (i.e., a monoclonal antibody that contains the same FLT3 binding site as that incorporated into the multispecific protein). In some embodiments, the multispecific protein is more effective at killing tumor cells that express FLT3 than the corresponding FLT3 monoclonal antibody.

[0147] In certain embodiments, the multispecific proteins described herein contain a binding site for NKG2D and a binding site for FLT3 and activate primary human NK cells when co-cultured with cells expressing FLT3. NK cell activation is characterized by CD107a degranulation and increased IFN-γ cytokine production. Furthermore, compared to the corresponding FLT3 monoclonal antibody, the multispecific proteins may exhibit superior activation of human NK cells in the presence of cells expressing FLT3.

[0148] In certain embodiments, the multispecific proteins described herein contain a binding site for NKG2D and a binding site for FLT3 and enhance the activity of resting and IL-2-activated human NK cells when co-cultured with cells expressing FLT3.

[0149] In certain embodiments, compared to corresponding monoclonal antibodies that bind to FLT3, the multispecific proteins have the advantage of targeting tumor cells that express intermediate and low levels of FLT3.

[0150] III. Therapeutic applications The present invention provides methods of treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods may be used to treat a variety of cancers that express FLT3. In some embodiments, the cancer is leukemia, such as acute myeloid leukemia, T-cell leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, or hairy cell leukemia.

[0151] In some other embodiments, the cancer is breast cancer, ovarian cancer, esophageal cancer, bladder cancer, or stomach cancer, salivary gland duct carcinoma, salivary gland duct carcinoma(s), adenocarcinoma of the lung, or an aggressive form of uterine cancer such as uterine serous endometrial cancer. In some other embodiments, the cancer is brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer.In still other embodiments, the cancer is selected from the group consisting of squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid gland carcinoma, biliary tract carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract carcinoma, bone cancer, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic myeloid leukemia, and thyroid carcinoma. Lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, cancer of the eye and orbit, cancer of the female genital tract, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatocellular adenoma, hepatocellular adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, Srinoma, intraepithelial neoplasia, intraepithelial squamous cell tumor, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyocarcinoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0152] In some other embodiments, the cancer to be treated is a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0153] IV. Combination Therapy Another aspect of the present invention provides for combination therapy. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0154] Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, Drugs that may be used include but are not limited to: drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma (IFN-γ), colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and modified forms of the above-listed agents that may exhibit differential binding to the agent's cognate receptor and increased or decreased serum half-life.

[0155] An additional class of agents that may be used as part of a combination therapy in cancer treatment are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. Food and Drug Administration for the treatment of melanoma.

[0156] Additionally, other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0157] Furthermore, other categories of anticancer drugs include, for example, the following: (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, m (ii) an inhibitor selected from a TOR inhibitor, a MEK inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iv) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0158] The protein of the present invention may also be used as an adjuvant for surgical removal of the primary lesion.

[0159] The amounts of the multispecific binding protein and additional therapeutic agent and the relative timing of administration may be selected to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the combined therapeutic agents, or pharmaceutical composition(s) comprising the therapeutic agents, may be administered in any order, e.g., sequentially, in parallel, together, simultaneously, etc. Further, for example, the multispecific binding protein may be administered while the additional therapeutic agent(s) is exerting its prophylactic or therapeutic effect, or vice versa.

[0160] V. Pharmaceutical Compositions The present disclosure also features a pharmaceutical composition containing a therapeutically effective amount of the protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for appropriate formulation. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0161] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a conduit containing tubing and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be frozen and dried (lyophilized) and contained in about 12 to 60 vials. In certain embodiments, the formulation may be lyophilized, and 45 mg of lyophilized formulation may be contained in a single vial. In certain embodiments, about 40 mg to about 100 mg of lyophilized formulation may be contained in a single vial. In certain embodiments, lyophilized formulation from 12, 27, or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial.

[0162] The protein may be present in a liquid, aqueous pharmaceutical formulation, comprising a therapeutically effective amount of the protein in a buffer solution that forms the formulation.

[0163] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, such as 7 to 7.5. The resulting solid form of the composition may be packaged in a plurality of single-dose units, each containing a fixed amount of the aforementioned agent(s). The solid form of the composition may also be packaged in containers for varying amounts.

[0164] In certain embodiments, the present disclosure provides an extended shelf life formulation comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0165] In certain embodiments, aqueous formulations are prepared containing a protein of the present disclosure in a pH buffer solution. The buffers of the present invention may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or from about 5.0 to about 5.2. Intermediate pH ranges within the above-listed ranges are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination of the above-listed values ​​as upper and / or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0166] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range can be within a pH range of 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 citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg / mL citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / mL), and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In certain embodiments, the buffer system includes 1-1.5 mg / mL citric acid, 0.25-0.5 mg / mL sodium citrate, 1.25-1.75 mg / mL disodium phosphate dihydrate, 0.7-1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / mL sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0167] Polyols may act as tonicity agents and stabilize the antibody and may also be included in the formulation. The polyol is added to the formulation in an amount that may vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary relative to 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 embodiments, the polyol used in the formulation as an isotonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the mannitol concentration may be about 7.5 to 15 mg / mL. In certain embodiments, the mannitol concentration may be about 10 to 14 mg / mL. In certain embodiments, the mannitol concentration may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0168] Detergents or surfactants may also be added to the formulation. 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 that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0169] In embodiments, the protein products of the present disclosure are formulated as liquid formulations. The liquid formulations may be presented at a concentration of 10 mg / mL in USP / Ph Eur Type I 50R vials closed with rubber stoppers and sealed with aluminum crimp seal closures. The stoppers may be made of elastomers that comply with USP and Ph Eur. In certain embodiments, the vials may be filled with 61.2 mL of protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulations may be diluted with 0.9% saline.

[0170] In certain embodiments, the liquid formulation of the present disclosure may be prepared as a 10 mg / mL solution in combination with a stable level of sugar. In certain embodiments, the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount that does not exceed the amount that may cause undesirable or inappropriate viscosity 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.

[0171] 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.

[0172] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, storage of drug substances / drug products, and sample analysis. Deamidation is the loss of NH3 from proteins to form a succinimide intermediate that can be hydrolyzed. The succinimide intermediate results in a 17-dalton mass reduction of the parent peptide. Subsequent hydrolysis results in an 18-dalton mass increase. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Therefore, deamidation is usually detectable as a 1-dalton mass increase. Deamidation of asparagine leads to either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. The amino acid residue adjacent to Asn in the peptide chain affects the rate of deamidation. Gly and Ser following Asn in the protein sequence are more susceptible to deamidation.

[0173] In certain embodiments, the liquid formulations of the present disclosure may be stored under conditions of pH and humidity to prevent deamination of the protein product.

[0174] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0175] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-use (multi-dose) formulation.

[0176] An intravenous (IV) formulation may be the preferred route of administration in certain cases, such as when a patient is hospitalized after transplant and receives all medications by the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the pharmaceutical preparation diluted for injection is isotonic and suitable for administration by intravenous infusion.

[0177] In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0178] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-use (multi-dose) formulation.

[0179] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0180] The protein of the present disclosure may be present in a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant may be a sugar, e.g., a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0181] The amount of sucrose or maltose useful for stabilizing a lyophilized pharmaceutical product can be a weight ratio of protein to sucrose or maltose of at least 1:2, in certain embodiments, the weight ratio of protein to sucrose or maltose can be between 1:2 and 1:5.

[0182] 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.

[0183] Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to 6-8. In certain embodiments, the pH range of the lyophilized pharmaceutical product may be 7-8.

[0184] In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) with "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0185] In certain embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.

[0186] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-use (multi-dose) formulation.

[0187] In certain embodiments, the lyophilized pharmaceutical product may be composed of an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and is useful for preparing a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0188] In certain embodiments, the lyophilized pharmaceutical products of the present disclosure are reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves in solution.

[0189] In certain embodiments, the lyophilized protein product of the present disclosure is constituted in about 4.5 mL of water for injection and diluted with 0.9% saline (sodium chloride solution).

[0190] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to provide an amount of the active ingredient that is non-toxic to the patient and effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration.

[0191] A specific dose may be a uniform dose for each patient, e.g., 50-5000 mg of protein. Alternatively, patient doses may be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and health. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages may also be determined by the use of known assays designed to determine dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted while monitoring disease progression. Blood levels of the targetable construct or complex in the patient may be measured to determine whether the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and / or complexes, and their dosages, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0192] Generally, the dosage based on body weight is from about 0.01 μg to about 100 mg / kg body weight, e.g., from about 0.01 μg to about 100 mg / kg body weight, from about 0.01 μg to about 50 mg / kg body weight, from about 0.01 μg to about 10 mg / kg body weight, from about 0.01 μg to about 1 mg / kg body weight, from about 0.01 μg to about 100 μg / kg body weight, from about 0.01 μg to about 50 μg / kg body weight, from about 0.01 μg to about 10 μg / kg body weight, from about 0.01 μg to about 1 μg / kg body weight, from about 0.01 μg to about 0.1 μg 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 10 0 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 m g / 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 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight, etc.

[0193] Dosages may be administered one or more times daily, weekly, monthly, or yearly, or once every 2 to 20 years. One skilled in the art can readily estimate repetition rates for dosing based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by catheter perfusion, or by direct intralesional injection. It may be administered one or more times daily, one or more times weekly, one or more times monthly, and one or more times yearly.

[0194] The above description describes several aspects and embodiments of the present invention, and this patent application specifically contemplates all combinations and permutations of aspects and embodiments. [Example]

[0195] The invention generally described herein will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0196] Example 1 - NKG2D binding domain binds to NKG2D The NKG2D-binding domain binds to purified recombinant NKG2D. The nucleic acid sequence of the human, mouse or cynomolgus NKG2D ectodomain was fused to a nucleic acid sequence encoding a human IgG1 Fc domain and introduced into mammalian cells where it was expressed. After purification, the NKG2D-Fc fusion protein was adsorbed to microplate wells. After blocking the wells with bovine serum albumin to prevent nonspecific binding, titrated NKG2D-binding domains were added to the pre-adsorbed wells. Primary antibody binding was detected using a secondary antibody conjugated with horseradish peroxidase that specifically recognizes the human kappa light chain to avoid Fc cross-reactivity. 3,3',5,5'-tetramethylbenzidine (TMB), a horseradish peroxidase substrate, was added to the wells to visualize the binding signal, and the absorbance was measured at 450 nM and corrected at 540 nM. An NKG2D-binding domain clone, an isotype control, or a positive control (containing heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) was added to each well.

[0197] The isotype control showed minimal binding to recombinant NKG2D-Fc protein, while the positive control bound most strongly to the recombinant antigen. The NKG2D-binding domains produced by all clones demonstrated binding to human, mouse, and cynomolgus monkey recombinant NKG2D-Fc protein, although the affinity varied among clones. In general, each anti-NKG2D clone bound with similar affinity to human (Figure 3) and cynomolgus monkey (Figure 4) recombinant NKG2D-Fc, but with lower affinity to mouse (Figure 5) recombinant NKG2D-Fc.

[0198] The NKG2D-binding domain binds to cells expressing NKG2D. The EL4 mouse lymphoma cell line was engineered to express human or mouse NKG2D-CD3 zeta signaling domain chimeric antigen receptors. NKG2D-binding clones, isotype controls, or positive controls were used at 100 nM concentrations to stain extracellular NKG2D expressed on EL4 cells. Antibody binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and fold over background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[0199] The NKG2D-binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. Positive control antibodies (anti-mouse NKG2D clones MI-6 and CX-5 containing heavy and light chain variable domains selected from SEQ ID NOs: 101-104, or available from eBioscience) produced the best FOB binding signals. NKG2D binding affinity for each clone was similar between cells expressing human NKG2D (Figure 6) and mouse NKG2D (Figure 7).

[0200] Example 2 - NKG2D binding domain blocks natural ligand binding to NKG2D Competition with ULBP-6 Recombinant human NKG2D-Fc protein was adsorbed to microplate wells, which were then blocked with bovine serum albumin to reduce nonspecific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by the addition of the NKG2D-binding domain clone. After a 2-hour incubation, the wells were washed, and ULBP-6-His-biotin that remained bound to the NKG2D-Fc-coated wells was detected with horseradish peroxidase-conjugated streptavidin and TMB substrate. Absorbance was measured at 450 nM and corrected to 540 nM. After background subtraction, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin blocked from binding to the NKG2D-Fc protein in the wells. A positive control antibody (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D-binding domains blocked ULBP-6 binding to NKG2D, while an isotype control showed little competition with ULBP-6 (Figure 8).

[0201] The ULBP-6 sequence is represented by SEQ ID NO:108. [ka]

[0202] Competition with MICA Recombinant human MICA-Fc protein was adsorbed to microplate wells, which were then blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the NKG2D-binding domain. After incubation and washing, NKG2D-Fc-biotin that remained bound to the MICA-Fc-coated wells was detected using streptavidin-HRP and TMB substrate. Absorbance was measured at 450 nM and corrected to 540 nM. After background subtraction, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin blocked from binding to the MICA-Fc-coated wells. A positive control antibody (comprising heavy and light chain variable domains selected from SEQ ID NOs: 101-104) and various NKG2D-binding domains blocked MICA binding to NKG2D, while an isotype control showed little competition with MICA (Figure 9).

[0203] Conflict with Rae-1 Delta Recombinant mouse Rae-1 delta-Fc (purchased from R&D Systems) was adsorbed to 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 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 background subtraction, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin blocked from binding to the Rae-1 delta-Fc-coated wells. Positive controls (anti-mouse NKG2D clones MI-6 and CX-5 containing heavy and light chain variable domains selected from SEQ ID NOs: 101-104 or 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).

[0204] Example 3 - NKG2D binding domain clones activate NKG2D The nucleic acid sequences of human and mouse NKG2D were fused to a nucleic acid sequence encoding the CD3 zeta signaling domain to obtain chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into retroviral vectors using Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with virus containing NKG2D-CAR together with 8 μg / mL of polybrene. 24 hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0205] To determine whether the NKG2D-binding domains activate NKG2D, they were adsorbed to microplate wells, and NKG2D-CAR EL4 cells were cultured on the antibody fragment-coated wells for 4 hours 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).

[0206] Example 4 - NKG2D binding domain activates NK cells Primary human NK cells Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated from PBMCs using negative selection with magnetic beads, and the purity of the isolated NK cells was typically >95%. The isolated NK cells were then cultured for 24–48 hours in medium containing 100 ng / mL IL-2, after which they were transferred to wells of a microplate adsorbed with the NKG2D binding domain and cultured in medium containing a fluorophore-conjugated anti-CD107a antibody, brefeldin-A, and monensin. After culture, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-γ. CD107a and IFN-γ staining was observed for CD3. - CD56 + NK cell activation was assessed by analyzing CD107a / IFN-γ double-positive cells. An increase in CD107a / IFN-γ double-positive cells indicates successful NK cell activation due to the binding of two activating receptors, rather than one. The NKG2D binding domain and positive control (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) showed a higher percentage of CD107a / IFN-γ double-positive cells than the isotype control. +and IFN-γ + (FIGS. 13 and 14 represent data from two independent experiments, each using PBMCs from a different donor for NK cell preparation.)

[0207] Primary mouse NK cells Spleens were obtained from C57Bl / 6 mice and disrupted through a 70 μm cell strainer to obtain a single cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (#A1049201, purchased from Thermo Fisher Scientific, 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove red blood cells. The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours before being harvested and prepared for NK cell isolation. NK cells (CD3 - NK1.1 + ) were isolated from splenocytes using a negative depletion technique with magnetic beads and typically reached >90% purity. Purified NK cells were cultured for 48 hours in medium containing 100 ng / mL mIL-15, after which they were transferred to wells of microplates adsorbed with NKG2D-binding domains and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin A, and monensin. After culture in the NKG2D-binding domain-coated wells, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-γ. CD107a and IFN-γ staining were performed using CD3 - NK1.1 + NK cell activation was assessed by analyzing CD107a / IFN-γ double-positive cells. The increase in CD107a / IFN-γ double-positive cells indicates successful NK cell activation by binding two activating receptors, rather than one. The NKG2D binding domain and positive controls (selected from anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) showed a higher percentage of CD107a binding than the isotype control. + and IFN-γ +(FIGS. 15 and 16 represent data from two independent experiments, each using different mice for NK cell preparation.)

[0208] Example 5 - NKG2D binding domains enable cytotoxicity of target tumor cells Human and mouse primary NK cell activation assays demonstrated an increase in cytotoxicity markers on NK cells after incubation with the NKG2D-binding domain. To determine whether this translated into increased tumor cell lysis, a cell-based assay was utilized, with each NKG2D-binding domain acting as a monospecific antibody. The Fc region was used as one targeting arm, while the Fab region (NKG2D-binding domain) served as another targeting arm to activate NK cells. THP-1 cells are of human origin and express high levels of Fc receptors; they were used as tumor targets using the Perkin Elmer DELFIA cytotoxicity kit. THP-1 cells were labeled with BATDA reagent and incubated for 10 min. 5 The labeled THP-1 cells were resuspended in culture medium at 1000 μL / mL. The labeled THP-1 cells were then combined with an NKG2D antibody, and mouse NK cells were isolated in microtiter plate wells at 37°C for 3 hours. After incubation, 20 μL of culture supernatant was removed and mixed with 200 μL of europium solution. The mixture was incubated for 15 minutes in the dark with shaking. 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 was calculated according to the kit's instructions.

[0209] The positive control ULBP-6, a natural ligand for NKG2D, showed increased specific lysis of THP-1 target cells by mouse NK cells. NKG2D antibodies also increased specific lysis of THP-1 target cells, while an isotype control antibody showed decreased specific lysis. The dotted line indicates specific lysis of THP-1 cells by mouse NK cells without added antibody (Figure 17).

[0210] Example 6 - NKG2D antibodies exhibit high thermal stability The melting temperature of the NKG2D binding domain was assayed using differential scanning fluorimetry. The extrapolated apparent melting temperatures are higher compared to typical IgG1 antibodies (Figure 18).

[0211] Example 7 - Synergistic activation of human NK cells by cross-linking NKG2D and CD16 Primary human NK cell activation assay Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells were purified from PBMCs using negative magnetic beads (StemCell #17955). NK cells were >90% CD3 as determined by flow cytometry. - CD56 + The cells were then grown for 48 hours in medium containing 100 ng / mL hIL-2 (Peprotech #200-02) before use in activation assays. Antibodies were coated onto 96-well flat-bottom plates at concentrations of 2 μg / mL (anti-CD16, Biolegend #302013) and 5 μg / mL (anti-NKG2D, R&D #MAB139) in 100 μL of sterile PBS overnight at 4°C, followed by thorough washing of the wells to remove excess antibody. For assessment of degranulation, 5 × 10 IL-2-activated NK cells were cultured in PBS. 5 The cells were resuspended at 1 x 10 cells / mL in culture medium supplemented with 100 ng / mL human IL-2 (hIL2) and 1 μg / mL APC-conjugated anti-CD107a mAb (Biolegend #328619). 5Cells / well were added to the antibody-coated plates. Protein transport inhibitors brefeldin A (BFA, Biolegend #420601) and monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively. The plated cells were incubated for 4 hours at 37°C in 5% CO2. For intracellular staining of IFN-γ, NK cells were labeled with anti-CD3 (Biolegend #300452) and anti-CD56 mAb (Biolegend #318328), then fixed, permeabilized, and labeled with anti-IFN-γ mAb (Biolegend #506507). Raw CD56 + CD3 - After gating on the cells, NK cells were analyzed by flow cytometry for expression of CD107a and IFN-γ.

[0212] To investigate the relative potency of receptor combinations, cross-linking of NKG2D or CD16 and co-cross-linking of both receptors by plate-bound stimulation were performed. As shown in Figure 19 (Figures 19A-19C), combined stimulation of CD16 and NKG2D resulted in significantly increased levels of CD107a (degranulation) (Figure 19A) and / or IFN-γ production (Figure 19B). The dotted lines represent the additive effect of stimulation of each receptor individually.

[0213] CD107a levels and intracellular IFN-γ production of IL-2-activated NK cells were analyzed 4 hours after plate-bound 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 IFN-γ levels, and Figure 19C shows CD107a and IFN-γ levels. Data shown in Figures 19A-19C are representative of five independent experiments using five different healthy donors.

[0214] Example 8 - Evaluation of TriNKET binding to human NKG2D expressed on cells The EL4 mouse lymphoma cell line was engineered to express human NKG2D. Multispecific binding proteins, such as the trispecific binding protein (TriNKET), each containing an NKG2D-binding domain, an FLT3-binding domain, and an Fc domain that binds to CD16, were tested for their affinity to bind 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 mean fluorescence intensity (MFI) values ​​were normalized to a secondary antibody control to obtain fold-over-background (FOB) values.

[0215] The TriNKETs tested were A44-TriNKET-FLT3-IMCEB10 (the NKG2D-binding domain from clone ADI-27744 and the FLT3-binding domain obtained by combining the heavy chain variable region of SEQ ID NO: 109 and the light chain variable region of SEQ ID NO: 113 from the FLT3 monoclonal antibody IMCEB10), A44-TriNKET-FLT3-4G8 (the NKG2D-binding domain from clone ADI-27744 and the FLT3-binding domain obtained by combining the heavy chain variable region of SEQ ID NO: 117 and the light chain variable region of SEQ ID NO: 121 from the monoclonal antibody 4G8), A49-TriNKET-FLT3-IMCEB10 (the NKG2D-binding domain from clone ADI-27749 and the FLT3 The FLT3-binding domains were: A49-TriNKET-FLT3-4G8 (the NKG2D-binding domain from clone ADI-27749 and the FLT3-binding domain from monoclonal antibody 4G8, for example, by combining the heavy chain variable region of SEQ ID NO: 117 and the light chain variable region of SEQ ID NO: 121); and C26-TriNKET-FLT3-4G8 (the NKG2D-binding domain from clone ADI-28226 and the FLT3-binding domain from monoclonal antibody 4G8, for example, by combining the heavy chain variable region of SEQ ID NO: 117 and the light chain variable region of SEQ ID NO: 121). The FLT3 monoclonal antibody IMCEB10 was used as a control. TriNKETs containing the NKG2D-binding domain (A44, A49, or C26) were shown to bind to NKG2D expressed on the cell surface. TriNKETs containing the same NKG2D-binding domain but different FLT3-binding domains showed similar binding affinity to NKG2D on the cell surface (Figure 35).

[0216] Example 9 - Evaluation of TriNKET binding to FLT3 expressed on cancer cells Binding of FLT3-targeted TriNKET to tumor-associated antigens was assayed using FLT3-expressing human AML cell lines (Molm-13 and EOL-1). TriNKET was incubated with the cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and mean fluorescence intensity (MFI) values ​​were normalized to a secondary antibody control to obtain fold-over-background (FOB) values.

[0217] FLT3-targeted TriNKETs containing the FLT3-binding domain of IMCEB10 or 4G8 showed positive binding to Molm-13 and EOL-1 cells (Figures 36A and 36B). The binding of TriNKETs containing either the FLT3-binding domain of IMCEB10 or 4G8 to human AML cell lines was independent of the NKG2D-binding domain. TriNKETs containing the FLT3-binding domain of 4G8 showed higher maximum binding and EC 50 The values ​​were shown.

[0218] Example 10 - Internalization of FLT3-targeted TriNKET on FLT3-expressing cells The human AML cell lines Molm-13 and EOL-1 were used to assess the internalization of FLT3-targeted TriNKET after binding to FLT3 expressed on the cell surface. TriNKET or the monoclonal antibody lintuzumab was diluted to 20 μg / mL and used to stain the cells. After staining, two-thirds of the samples were placed at 37°C overnight to promote internalization, and the remaining one-third of the samples were detected using a fluorophore-conjugated anti-human IgG secondary antibody to obtain baseline MFI. After 2 and 20 hours of incubation at 37°C, the samples were removed from the incubator, and bound antibody on the cell surface was detected using a fluorophore-conjugated anti-human IgG secondary antibody to obtain sample MFI. Internalization was calculated as follows: % internalization = (1 - (sample MFI / baseline MFI)) * 100%.

[0219] Figures 37A and 37B show the internalization of FLT3-targeted TriNKET after incubation with EOL-1 and Molm-13 cells, respectively. Because CD33 is expressed on both Molm-13 and EOL-1 cell lines, the anti-CD33 monoclonal antibody lintuzumab was used as a positive control for internalization. Lintuzumab showed high levels of internalization on both cell lines, and internalization increased over time. TriNKET containing the FLT3-binding domain of IMCEB10 showed higher levels of internalization after 2 hours of incubation compared to TriNKET containing the FLT3-binding domain of 4G8. After 20 hours of incubation, TriNKET containing the FLT3-binding domain of 4G8 and TriNKET containing the FLT3-binding domain of IMCEB10 showed similar levels of internalization on cells.

[0220] Example 11 - TriNKET enhances human NK cell cytotoxicity against cancer cells To test the ability of human NK cells to lyse FLT3-expressing cancer cells in the presence of FLT3-targeting TriNKET, peripheral blood mononuclear cells (PBMCs) were isolated and prepared for NK cell isolation. PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. The isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection technique using magnetic beads, and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were cultured in medium containing 100 ng / mL IL-2 or were left overnight without cytokines. IL-2-activated or left overnight NK cells were used in cytotoxicity assays the following day.

[0221] All cytotoxicity assays were prepared as follows: FLT3-positive tumor cells EOL-1 were harvested from culture, washed with PBS, and cytotoxicity assayed with BATDA reagent (Perkin Elmer) was performed. For labeling with C136-100, 10 6The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with HBS and resuspended at 0.5–1.0 × 10 5 The cells were resuspended in culture medium at 100 μL / mL. To prepare background wells, an aliquot of labeled cells was set aside and the cells were separated from the medium. 100 μL of medium was carefully added to triplicate wells to avoid disturbing cell pelleting. 100 μL of BATDA-labeled cells were added to each well of a 96-well plate. Wells were reserved for spontaneous release from target cells, and wells were prepared for maximum lysis of target cells by adding 1% Triton-X. FLT3 monoclonal antibody or FLT3-targeting TriNKET was diluted in culture medium, and 50 μL of diluted monoclonal antibody or TriNKET was added to each well. The plated and / or activated NK cells were harvested from the culture, washed, and incubated for 10 min. 5 ~2.0×10 6 NK cells were resuspended in culture medium at 1 / mL depending on the desired effector-to-target cell ratio. 50 μL of NK cells were added to each well of the plate for a total culture volume of 200 μL. Plates were incubated at 37°C with 5% CO2 for 2-3 hours before proceeding with the assay.

[0222] After 2-3 hours of incubation, plates were removed from the incubator and cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μL of culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μL of room temperature europium solution was added to each well. Plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. Plates were read using either a Victor3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)) * 100%.

[0223] FLT3-targeting TriNKET mediated human NK cell cytotoxicity against FLT3-positive EOL-1 cancer cells. As shown in Figure 38A, both TriNKETs (A49-TriNKET-IMCEB10 and A44-TriNKET-IMCEB10) were able to enhance the cytotoxic activity of NK cells against cancer cells in a dose-responsive manner. Both TriNKETs were significantly more potent than the corresponding FLT3 monoclonal antibody IMCEB10. As shown in Figure 38B, TriNKETs (A49-TriNKET-4G8, A44-TriNKET-4G8, and C26-TriNKET-4G8) were able to enhance the cytotoxic activity of NK cells against cancer cells in a dose-responsive manner. TriNKETs were significantly more potent than the corresponding FLT3 monoclonal antibody 4G8. Furthermore, TriNKET containing the FLT3-binding domain of 4G8 was more potent in mediating human NK cell cytotoxicity than TriNKET containing the FLT3-binding domain of IMCEB10.

[0224] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0225] Doctrine of Equivalents The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A protein, (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to FLT3; and (c) an antibody Fc domain or a sufficient portion thereof that binds to CD16, or a third antigen-binding site that binds to CD16.

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

3. The protein of 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 of claim 3 , wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide.

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

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

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

93.

9. 8. The protein of claim 1, 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. 8. The protein of claim 1, 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 of any one of claims 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 57 and a light chain variable domain at least 90% identical to SEQ ID NO:

58.

12. 8. The protein of any one of claims 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 59 and a light chain variable domain at least 90% identical to SEQ ID NO:

60.

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

70.

15. The protein of any one of claims 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 77 and a light chain variable domain at least 90% identical to SEQ ID NO:

78.

16. The protein of any one of claims 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 85 and a light chain variable domain at least 90% identical to SEQ ID NO:

86.

17. 8. The protein of claim 1, 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. 8. The protein of claim 1, 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. 8. The protein of claim 1, 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 of claim 1 or 2, wherein the first antigen-binding site is a single domain antibody.

21. The single domain antibody is Ｈ H fragment or V ＮＡＲ 21. The protein of claim 20, which is a fragment.

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

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

24. The protein of any one of claims 1 to 23, wherein the second antigen-binding site binds to FLT3, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 109, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

113.

25. The protein of any one of claims 1 to 23, wherein the second antigen-binding site binds to FLT3, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 117, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

121.

26. The protein of any one of claims 1 to 23, wherein the second antigen-binding site binds to FLT3, the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 125, and the light chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

129.

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

28. the second antigen-binding site is V Ｈ H fragment or V ＮＡＲ 28. The protein of claim 27, which is a fragment.

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

30. 30. The protein of claim 29, wherein the antibody Fc domain comprises the hinge and CH2 domains of a human IgG1 antibody.

31. 31. The protein of claim 29 or 30, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody.

32. 32. The protein of claim 31 , wherein the Fc domain comprises an amino acid sequence at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, K439.

33. 10. A formulation comprising a protein according to any one of the preceding claims and a pharmaceutically acceptable carrier.

34. A cell comprising one or more nucleic acids that express a protein according to any one of claims 1 to 32.

35. 33. A method for enhancing tumor cell death, comprising exposing tumor cells and natural killer cells to an effective amount of the protein of any one of claims 1 to 32, wherein the tumor cells express FLT3.

36. 34. A method for treating cancer, said method comprising administering to a patient an effective amount of the protein of any one of claims 1 to 32 or the formulation of claim 33.

37. 37. The method of claim 36, wherein the cancer is leukemia.

38. 38. The method of treating cancer of claim 37, wherein the leukemia is selected from the group consisting of acute myeloid leukemia, T-cell leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

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