A protein that binds to Her2, NKG2D, and CD16

Abstract

Multispecific binding proteins that bind to HER2, NKG2D receptor, and CD16, as well as pharmaceutical compositions and therapeutic methods useful for treating cancer, are described. Patients with HER2-overexpressing breast cancer have substantially lower overall survival rates and shorter disease-free intervals than patients whose cancer does not overexpress HER2. Overexpression of HER2 is also known to cause many other types of cancer, including breast cancer, ovarian cancer, esophageal cancer, bladder cancer, and gastric cancer, salivary duct cancer, lung adenocarcinoma, and aggressive forms of uterine cancer, such as uterine serous endometrial cancer. The multispecific binding proteins disclosed herein provide a means for treating such cancers.

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 / 461,146, filed February 20, 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, the entire contents of which are incorporated herein by reference. The ASCII copy was created on February 16, 2018, is named DFY-008PC_SL.txt, and is 92,807 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to multispecific binding proteins that bind to human epidermal growth factor receptor 2 (HER2 or ErbB2), the NKG2D receptor, and CD16. [Background technology]

[0004] background Cancer remains a significant health problem, despite substantial research efforts and scientific advances reported in the literature to treat this disease. Among the most frequently diagnosed cancers are prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Current treatment options for these cancers are not effective for all patients and / or may have substantial adverse side effects. Other types of cancer also remain difficult to treat using existing treatment 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 engagers, are cancer immunotherapies described in the literature that bind to tumor cells and T cells to promote tumor cell destruction. Antibodies that bind to certain tumor-associated antigens and 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 were initially 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., via cytotoxic granules containing perforin and granzymes, as well as via 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 via various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited by the activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated via their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of several immunoglobulins via the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. HER2 (ErbB2) is a transmembrane glycoprotein belonging to the epidermal growth factor receptor family. HER2 (ErbB2) is a receptor tyrosine kinase that regulates cell survival, proliferation, and growth. HER2 plays an important role in human malignancies. The erbB2 gene is amplified or overexpressed in approximately 30% of human breast cancers. Patients with HER2-overexpressing breast cancer have substantially lower overall survival rates and shorter disease-free intervals than patients whose cancers do not overexpress HER2. Furthermore, overexpression of HER2 results in increased breast cancer metastasis. Overexpression of HER2 is also known to cause numerous other types of cancer, including breast cancer, ovarian cancer, esophageal cancer, bladder cancer, and gastric cancer, salivary duct cancer, lung adenocarcinoma, and aggressive forms of uterine cancer, such as uterine serous endometrial cancer. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2015 / 095412 Summary of the Invention [Means for solving the problem]

[0009] Abstract The present invention provides multispecific binding proteins that bind to HER2 on cancer cells and to the NKG2D and CD16 receptors on natural killer cells. Such proteins can associate with two or more NK activating receptors and block the binding of natural ligands to NKG2D. In certain embodiments, the proteins can stimulate NK cells in humans and other species, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in further detail below.

[0010] Thus, one aspect of the invention provides a protein incorporating a first antigen-binding site that binds to NKG2D, a second antigen-binding site that binds to HER2, and an antibody Fc domain, or portion thereof, sufficient to bind to CD 16, or a third antigen-binding site that binds to CD 16. The antigen-binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged like an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may incorporate a V-chain variable domain, such as a camelid antibody. H H antibodies or V antibodies such as those found in cartilaginous fish NAR It may also be a single domain antibody such as an antibody.

[0011] A first antigen-binding site that binds to NKG2D may, in one embodiment, incorporate a heavy chain variable domain related to SEQ ID NO: 1, e.g., 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 by incorporating amino acid sequences identical to the CDR1 (SEQ ID NO: 62), CDR2 (SEQ ID NO: 63), and CDR3 (SEQ ID NO: 64) sequences of SEQ ID NO: 1. 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 incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 65), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 67) 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 incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 68), CDR2 (SEQ ID NO: 69), and CDR3 (SEQ ID NO: 70) sequences of SEQ ID NO: 42. In other embodiments, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 43 and a light chain variable domain related to SEQ ID NO: 44. 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: 43 and / or 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: 43.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:44 and / or 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:44.

[0012] Alternatively, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 45 and a light chain variable domain related to SEQ ID NO: 46, e.g., 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: 45 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: 46, respectively. In another embodiment, the first antigen-binding site may incorporate a heavy chain variable domain related to SEQ ID NO: 47 and a light chain variable domain related to SEQ ID NO: 48, e.g., 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: 47 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: 48, respectively.

[0013] The second antigen-binding site may optionally incorporate a heavy chain variable domain related to SEQ ID NO: 49 and a light chain variable domain related to SEQ ID NO: 53. 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: 49 and / or incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 52) 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: 53 and / or 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: 53.

[0014] Alternatively, the second 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. 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: 57 and / or incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 77), CDR2 (SEQ ID NO: 78), and CDR3 (SEQ ID NO: 79) sequences of SEQ ID NO: 57. 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:58 and / or incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:80), CDR2 (SEQ ID NO:81), and CDR3 (SEQ ID NO:82) sequences of SEQ ID NO:58.

[0015] In another embodiment, the second 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 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: 59 and / or incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 83), CDR2 (SEQ ID NO: 84), and CDR3 (SEQ ID NO: 85) sequences of SEQ ID NO: 59. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:60 and / or incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:86), CDR2 (SEQ ID NO:87), and CDR3 (SEQ ID NO:88) sequences of SEQ ID NO:60.

[0016] 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.

[0017] In some embodiments, the protein incorporates a portion of an antibody Fc domain sufficient 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.

[0018] Also provided are formulations containing one of these proteins, cells containing one or more nucleic acids that express these proteins, and methods of using these proteins to enhance tumor cell death.

[0019] Another aspect of the present invention relates to 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 proteins include, for example, breast, ovarian, esophageal, bladder, and gastric cancers, salivary duct cancer, adenocarcinoma of the lung, and aggressive forms of uterine cancer such as uterine serous endometrial cancer. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram of a multispecific binding protein containing an NKG2D-binding domain (right arm), a tumor-associated antigen-binding domain (left arm), and an Fc domain or portion thereof that binds to CD16.

[0021] [Figure 2] FIG. 2 is a diagram of a multispecific binding protein containing an NKG2D-binding domain (right arm), a tumor-associated antigen-binding domain (left arm) and an Fc domain or portion thereof that binds to CD16 in scFv format.

[0022] [Figure 3] FIG. 3 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

[0023] [Figure 4] FIG. 4 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D in an ELISA assay.

[0024] [Figure 5] FIG. 5 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to murine recombinant NKG2D in an ELISA assay.

[0025] [Figure 6] FIG. 6 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D by flow cytometry showing mean fluorescence intensity (MFI) fold over background.

[0026] [Figure 7] FIG. 7 is a bar graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing murine NKG2D by flow cytometry showing fold mean fluorescence intensity (MFI) over background.

[0027] [Figure 8] FIG. 8 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.

[0028] [Figure 9] FIG. 9 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.

[0029] [Figure 10] FIG. 10 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.

[0030] [Figure 11] FIG. 11 is a bar graph showing activation of human NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells expressing human NKG2D-CD3 zeta fusion protein.

[0031] [Figure 12] FIG. 12 is a bar graph showing activation of murine NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells expressing murine NKG2D-CD3 zeta fusion protein.

[0032] [Figure 13] FIG. 13 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0033] [Figure 14] FIG. 14 is a bar graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0034] [Figure 15] FIG. 15 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0035] [Figure 16] FIG. 16 is a bar graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0036] [Figure 17] FIG. 17 is a bar graph showing the cytotoxic effect of NKG2D binding domains (listed as clones) on tumor cells.

[0037] [Figure 18] FIG. 18 is a bar graph showing the melting temperatures of NKG2D binding domains (listed as clones) as determined by differential scanning fluorimetry.

[0038] [Figure 19] FIG. 19 is a graph showing enhanced activation of human NK cells by multispecific binding proteins.

[0039] [Figure 20] FIG. 20 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity by human NK cells against tumor target cells.

[0040] [Figure 21] FIG. 21 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity by human NK cells against tumor target cells.

[0041] [Figure 22] FIG. 22 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity by human NK cells against tumor target cells.

[0042] [Figure 23] FIG. 23 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity by human NK cells against tumor target cells.

[0043] [Figure 24] FIG. 24 is a graph showing that multispecific binding proteins induced higher levels of cytotoxicity against tumor target cells by mouse NK cells.

[0044] [Figure 25] FIG. 25 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by mouse NK cells.

[0045] [Figure 26] Figure 26 shows the binding profile of HER2-targeted TriNKET to NKG2D expressed in EL4 cells, showing that the same two NKG2D binding domains are now paired with the HER2 second targeting arm.

[0046] [Figure 27-1] Figure 27A shows the binding profile of HER2-targeted TriNKET to HER2 expressed in human 786-O renal cell carcinoma cells. Figure 27B shows that TriNKET-containing NKG2D-binding clone C26 binds to RMA cells transduced with human HER2, and Figure 27C shows that TriNKET-containing NKG2D-binding clone F04 binds to RMA cells transduced with human HER2. [Figure 27-2] Figure 27A shows the binding profile of HER2-targeted TriNKET to HER2 expressed in human 786-O renal cell carcinoma cells. Figure 27B shows that TriNKET-containing NKG2D-binding clone C26 binds to RMA cells transduced with human HER2, and Figure 27C shows that TriNKET-containing NKG2D-binding clone F04 binds to RMA cells transduced with human HER2.

[0047] [Figure 28-1] Figures 28A-28C are bar graphs showing that TriNKET and trastuzumab were able to activate primary human NK cells in coculture with HER2-positive human tumor cells, as indicated by increased CD107a degranulation and IFNγ cytokine production. Compared to the monoclonal antibody trastuzumab, both TriNKETs demonstrated superior activation of human NK cells with various human HER2 cancer cells. Figure 28A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. Figure 28B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. Figure 28C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells. [Figure 28-2]Figures 28A-28C are bar graphs showing that TriNKET and trastuzumab were able to activate primary human NK cells in coculture with HER2-positive human tumor cells, as indicated by increased CD107a degranulation and IFNγ cytokine production. Compared to the monoclonal antibody trastuzumab, both TriNKETs demonstrated superior activation of human NK cells with various human HER2 cancer cells. Figure 28A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. Figure 28B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. Figure 28C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells.

[0048] [Figure 29] Figures 29A and 29B are graphs showing that TriNKET provides a greater benefit to HER2-moderate and -low cancers than trastuzumab. Figure 29A shows activated human NK cell killing of HER2-high SkBr-3 tumor cells. Figure 29B shows human NK cell killing of HER2-low 786-O tumor cells. TriNKET provides a greater benefit to cancer cells with low HER2 expression than trastuzumab.

[0049] [Figure 30] Figures 30A-30C are bar graphs of synergistic activation of NK cells using CD16 and NKG2D. Figure 30A shows levels of CD107a, Figure 30B shows levels of IFNγ, and Figure 30C shows levels of CD107a and IFNγ. Graphs show mean (n=2) ± SD. Data are representative of five independent experiments using five different healthy donors.

[0050] [Figure 31]Figure 31 is a bar graph showing NK cell activation using TriNKET targeting NKG2D and CD16. The antibody tested is a human IgG1 isotype. The graph shows the mean (n=2) ± SD.

[0051] [Figure 32-1] Figures 32A-32C are graphs showing TriNKET enhancement of cytotoxic activity using human IL-2-activated NK cells and human resting NK cells. Figure 32A shows the percent specific lysis of SkBr-3 tumor cells by human resting NK cells. Figure 32B shows the percent specific lysis of SkBr-3 tumor cells by human IL-2-activated NK cells. Figure 32C shows the percent specific lysis of NCI-H661 lung cancer cells by human IL-2-activated NK cells. [Figure 32-2] Figures 32A-32C are graphs showing TriNKET enhancement of cytotoxic activity using human IL-2-activated NK cells and human resting NK cells. Figure 32A shows the percent specific lysis of SkBr-3 tumor cells by human resting NK cells. Figure 32B shows the percent specific lysis of SkBr-3 tumor cells by human IL-2-activated NK cells. Figure 32C shows the percent specific lysis of NCI-H661 lung cancer cells by human IL-2-activated NK cells.

[0052] [Figure 33-1] Figures 33A and 33B are bar graphs showing that B cells from healthy donors are susceptible to TriNKET-mediated lysis. [Figure 33-2] Figures 33C and 33D are bar graphs showing that bone marrow cells are resistant to TriNKET-mediated lysis.

[0053] [Figure 34] FIG. 34 is a line graph of TriNKET-mediated hPBMC killing of SkBr-3 tumor cells in long-term co-culture.

[0054] [Figure 35]FIG. 35 is a line graph showing that trispecific binding in one molecule is important for maximal NK cell activity.

[0055] [Figure 36] Figure 36 is a flow chart of the RMA / S-HER2 subcutaneous SC2.2 efficacy study design.

[0056] [Figure 37] FIG. 37 is a line graph showing that SC2.2 has no effect on subcutaneous RMA / S-HER2 tumor growth.

[0057] [Figure 38] Figure 38A shows that HER2-TriNKET-C26 crosslinks hNKG2D-Fc to RMA-HER2 cells. Figure 38B shows that HER2-TriNKET-F04 crosslinks hNKG2D-Fc to RMA-HER2 cells. The dotted line represents the isotype control. The open solid line represents the unstained control. The solid solid line represents TriNKET.

[0058] [Figure 39] Figure 39 is a diagram of TriNKET in a triomab format, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies derived from two parent antibodies, each half antibody having one light chain and one heavy chain. The triomab format can be a heterodimeric construct containing one half rat antibody and one half mouse antibody.

[0059] [Figure 40]Figure 40 is a diagram of TriNKET in a KiH common light chain (LC) format using knobs-into-holes (KIH) technology. KiH is a heterodimer containing two Fabs that bind to targets 1 and 2, and an Fc stabilized by heterodimerization mutations. TriNKET in the KiH format can be a heterodimeric construct containing 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.

[0060] [Figure 41] Figure 41 is a diagram of TriNKET in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies via a naturally occurring flexible linker, resulting in a tetravalent IgG-like molecule. A 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 construct contains a conventional Fc.

[0061] [Figure 42] Figure 42 is a diagram of TriNKET in an orthogonal Fab interface (ortho-Fab) format, a heterodimeric construct containing two Fabs that bind target 1 and target 2 fused to an Fc. LC-HC pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0062] [Figure 43] FIG. 43 is a diagram of TrinKET in a 2-in-1 Ig format.

[0063] [Figure 44] Figure 44 is a diagram of TriNKET in ES format, 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.

[0064] [Figure 45] Figure 45 is a diagram of TriNKET in Fab arm exchanged form, i.e., an antibody that has had its Fab arms exchanged by swapping the 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 exchanged form (cFae) is a heterodimer containing two Fabs that bind to targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0065] [Figure 46] FIG. 46 is a diagram of TriNKET in its SEED Body form, a heterodimer containing two Fabs that bind to targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0066] [Figure 47] Figure 47 is a diagram of TriNKET in the LuZ-Y format, which uses a leucine zipper to induce heterodimerization of two different HCs. The LuZ-Y format 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.

[0067] [Figure 48] FIG. 48 is a diagram of TriNKET in Cov-X-Body form.

[0068] [Figure 49] Figures 49A and 49B are diagrams of TriNKET in the κλ-Body format, 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 49A is an exemplary diagram of one form of κλ-Body, and Figure 49B is an exemplary diagram of another κλ-Body.

[0069] [Figure 50] Figure 50 is an Oasc-Fab heterodimer construct containing a Fab that binds target 1 and an scFab that binds target 2 fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0070] [Figure 51] Figure 51 shows DuetMab, a heterodimeric construct containing two different Fabs that bind antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure correct pairing of the LC and HC.

[0071] [Figure 52] Figure 52 is a CrossmAb, 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 are switched with the Vh and VL domains, e.g., CH1 is fused in-line with the VL, while CL is fused in-line with the VH.

[0072] [Figure 53] Figure 53 shows Fit-Ig, 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. This construct contains wild-type Fc. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description The present invention provides multispecific binding proteins that bind to HER2 in cancer cells and to the NKG2D and CD16 receptors on natural killer cells to activate the natural killer cells, pharmaceutical compositions comprising such multispecific binding proteins, and therapeutic methods using such multispecific proteins and pharmaceutical compositions, such as for the treatment of cancer. Various aspects of the invention are described below in multiple sections. However, an aspect of the invention described in one particular section is not limited to any particular section.

[0074] To facilitate the understanding of this invention, several terms and phrases are defined below.

[0075] As used herein, the terms "a" and "an" mean "one or more" and include plurals unless the context is inappropriate. 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 highly divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions," which are interposed between adjacent, more conserved stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are 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, providing a "single domain antibody." The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, where a peptide linker may be used to link the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0076] As used herein, the term "tumor-associated antigen" 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 in tumor-associated vasculature, extracellular matrix, mesenchymal stroma, or immune infiltrate.

[0077] 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 include humans.

[0078] 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 "treat" includes any effect, e.g., improving, reducing, reducing, modulating, improving, or eliminating a condition, disease, disorder, etc., or ameliorating the symptoms thereof.

[0079] 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.

[0080] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain 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] .

[0081] 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 that, upon administration to a subject, can provide the 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, can be utilized in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0082] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxide, alkaline earth metal (e.g., magnesium) hydroxide, ammonia, and bases of formula NW4 + (Wherein W is C 1~4 This includes compounds in which the aryl group is alkyl.

[0083] 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, and the like. Other examples of salts include Na + , NH4+ and NW4 + (Wherein W is C 1~4 The anions of the compounds of the present invention combined with suitable cations such as alkyl groups.

[0084] For therapeutic use, the salts of the compounds of the invention are intended 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.

[0085] Throughout the description where compositions are described as having, including, or comprising certain ingredients, or processes and methods are described as having, including, or comprising certain steps, it is further intended that there are compositions of the invention that consist essentially of, or consist of, the recited ingredients, and that there are processes and methods of the invention that consist essentially of, or consist of, the recited process steps.

[0086] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the variable's prior definition takes precedence. I. Protein

[0087] The present invention provides multispecific binding proteins that bind to HER2 on cancer cells and the NKG2D and CD16 receptors on natural killer cells, activating the natural killer cells. The multispecific binding proteins are useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of the multispecific binding proteins to the NKG2D and CD16 receptors on natural killer cells enhances the activity of the natural killer cells in their destruction of cancer cells. Binding of the multispecific binding protein to HER2 on cancer cells brings the cancer cells into close proximity with the natural killer cells, facilitating their direct and indirect destruction. Further description of exemplary multispecific binding proteins is provided below.

[0088] The first component of the multispecific binding protein binds to NK cells, γδ T cells, and CD8 + The multispecific binding protein binds to NKG2D receptor-expressing cells, which may include, but are not limited to, αβ T cells. Upon binding to NKG2D, the multispecific binding protein can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating the NKG2D receptor.

[0089] The second component of the multispecific binding protein binds to HER2-expressing cells, which may include, but are not limited to, breast, ovarian, esophageal, bladder and gastric cancer, salivary duct cancer, adenocarcinoma of the lung and aggressive forms of uterine cancer such as uterine serous endometrial cancer.

[0090] The third component of the multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0091] The multispecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric multispecific antibody (FIG. 1) comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and a first light chain constant domain. 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 HER2. The first Fc domain and the second Fc domain can bind to CD16 together (Figure 1). In some embodiments, the first immunoglobulin light chain may be identical to the second immunoglobulin light chain.

[0092] Another exemplary format relates to a heterodimeric multispecific antibody (Figure 2) comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused via either a linker or an antibody hinge to a single-chain variable fragment (scFv) composed of paired heavy and light chain variable domains that bind to NKG2D or HER2. 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 constant light chain domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to NKG2D or HER2. The first and second Fc domains can together bind to CD16 (Figure 2).

[0093] One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the antigen-binding site may be a single chain or a disulfide-stabilized variable region (ScFv), or may form a tetravalent or trivalent molecule.

[0094] In some embodiments, the multispecific binding protein is in the form of a Triomab, a trifunctional, bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies derived from two parent antibodies, each half antibody having one light chain and one heavy chain.

[0095] In some embodiments, the multispecific binding protein is a KiH common light chain (LC) format that uses knob-into-hole (KIH) technology. KIH is a C-terminal fragment of the LC to promote heterodimerization. HThis involves engineering three domains to create either a "knob" or a "hole" in each heavy chain. The concept behind "knob-into-hole (KiH)" Fc technology is to create a "knob" (e.g., T366W in EU numbering) in one CH3 domain (CH3A) by replacing small residues with bulky residues. CH3A To accommodate this "knob," the neighboring residues closest to the knob in the other CH3 domain (CH3B) were replaced with smaller residues (i.e., T366S / L368A / Y407V). CH3B) to generate complementary "hole" surfaces. The "hole" mutations were optimized by structurally 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 FcgammaRs. Mol Immunol (2014) 58(1):132-8 showed that at the core interface between the CH3 domains, hydrophobic interactions driven by steric complementarity thermodynamically favor heterodimerization, whereas knob-knob and hole-hole interfaces do not favor homodimerization due to steric hindrance and interference with favorable interactions, respectively.

[0096] In some embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies via a naturally occurring flexible linker, resulting in a tetravalent IgG-like molecule.

[0097] In some embodiments, the multispecific binding protein is in the form of 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 domain design allows for the LC and HC of one Fab to be separated. VH-CH1 Complementary mutations are introduced only at the interface between the two Fabs, leaving the other Fab unchanged.

[0098] In some embodiments, the multispecific binding protein is in a 2in1 Ig format. In some embodiments, the multispecific binding protein is in an ES configuration, 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. In some embodiments, the multispecific binding protein is in a κλ-Body configuration, which is a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations. Fab1, which targets antigen 1, contains a kappa LC, while the second Fab, which targets antigen 2, contains a lambda LC. Figure 49A is an exemplary diagram of one configuration of a κλ-Body, and Figure 49B is an exemplary diagram of another κλ-Body.

[0099] In some embodiments, the multispecific binding protein is in Fab arm exchange form (an antibody in which the Fab arms have been exchanged by swapping the heavy chain and associated light chain (half molecule) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody). In some embodiments, the multispecific binding protein is in SEED Body form. The SEED (strand-exchange engineered domain) platform was designed to generate asymmetric and bispecific antibody-like molecules that may broaden the therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related sequences of immunoglobulins in the conserved CH3 domain. The SEED design allows for the efficient generation of AG / GA heterodimers while avoiding homodimerization of the AG and GA SEED CH3 domains (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)). In some embodiments, the multispecific binding protein is in SEED Body form. The bispecific binding protein is in the LuZ-Y form, which uses a leucine zipper to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0100] In some embodiments, the multispecific binding protein is in the form of a Cov-X-Body. In bispecific CovX-Body, two different peptides are linked together using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. While the pharmacophore is responsible for functional activity, the antibody scaffold provides a long half-life and Ig-like distribution. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with another pharmacophore (Doppalapudi VR et al., PNAS (2010) 107(52):22611-22616).

[0101] In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer, comprising a Fab that binds target 1 and an scFab that binds target 2, fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0102] In some embodiments, the multispecific binding protein is in the form of a DuetMab, a heterodimeric construct containing two different Fabs that bind to antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fabs 1 and 2 contain unique S-S bridges that ensure correct pairing of the LC and HC.

[0103] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, 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 are switched with the VH and VL domains, e.g., CH1 is fused in-line with the VL and CL is fused in-line with the VH.

[0104] In some embodiments, the multispecific binding protein is in the Fit-Ig format, 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. This construct contains a wild-type Fc.

[0105] Additional multispecific binding protein formats can be devised by combining the various formats of NKG2D-binding fragments and HER2-binding fragments described herein.

[0106] Table 1 lists peptide sequences of heavy and light chain variable domains that can be combined to bind to NKG2D. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0107] Alternatively, as described in US Pat. No. 9,273,136, a heavy chain variable domain defined by SEQ ID NO: 45 may be paired with a light chain variable domain defined by SEQ ID NO: 46 to form an antigen-binding site capable of binding to NKG2D. [ka]

[0108] Alternatively, as described in US Pat. No. 7,879,985, a heavy chain variable domain defined by SEQ ID NO: 47 may be paired with a light chain variable domain defined by SEQ ID NO: 48 to form an antigen-binding site capable of binding to NKG2D. [ka]

[0109] Table 2 lists peptide sequences of heavy and light chain variable domains that can combine to bind to HER2. [Table 2-1] [Table 2-2]

[0110] Alternatively, novel antigen binding sites capable of binding to HER2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO:61. [ka]

[0111] Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, within human IgG1, interaction with CD16 is primarily focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, vol. 406 (6793): pp. 267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0112] The construction of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the construction of homodimers and heterodimers of each antibody heavy chain. The selective construction of heterodimers can be promoted by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480 and US14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1, incorporating different pairs of amino acid substitutions into the first and second polypeptides, which allows these two chains to selectively heterodimerize with each other. The positions of the amino acid substitutions exemplified below are all numbered according to the EU index as in Kabat.

[0113] In one situation, 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 with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (the protrusion) fits into the surface of the smaller amino acid substitution (the cavity). For example, one polypeptide can incorporate a T366W substitution, and the other polypeptide can incorporate three substitutions including T366S, L368A, and Y407V.

[0114] The antibody heavy chain variable domains of the present invention can optionally be linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region comprising a hinge, CH2, and CH3 domain, with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region relative to the human IgG1 constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, These include 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.

[0115] In certain embodiments, mutations that may be incorporated into CH1 of the human IgG1 constant region may be 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 amino acids E123, F116, S176, V163, S174, and / or T164.

[0116] The amino acid substitutions may be selected from the set of substitutions set out in Table 3 below. [Table 3]

[0117] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 4 below. [Table 4]

[0118] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 5 below. [Table 5]

[0119] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6. [Table 6]

[0120] Alternatively, at least one amino acid substitution may be selected from the set of substitutions in Table 7 below, where the position indicated in the "First Polypeptide" column is replaced with any known negatively charged amino acid and the position indicated in the "Second Polypeptide" column is replaced with any known positively charged amino acid. [Table 7]

[0121] Alternatively, at least one amino acid substitution may be selected from the set in Table 8 below, where the position indicated in the "First Polypeptide" column is replaced with any known negatively charged amino acid and the position indicated in the "Second Polypeptide" column is replaced with any known negatively charged amino acid. [Table 8]

[0122] Alternatively, the amino acid substitutions may be selected from the set in Table 9 below. [Table 9]

[0123] Alternatively or additionally, the structural stability of a heteromultimeric protein can be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, which results in the formation of an artificial disulfide bridge within the interface of the two polypeptides.

[0124] The above multispecific proteins can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, 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.

[0125] To achieve the highest yield of multispecific proteins, different ratios of the first, second, and third expression vectors can be tested 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.

[0126] Clones can be cultured under conditions suitable for bioreactor scale-up and can maintain expression of the multispecific protein. The multispecific protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. II. Characteristics of Multispecific Proteins

[0127] In certain embodiments, the multispecific binding proteins described herein comprising an NKG2D binding domain and an HER2 binding domain bind to cells expressing human NKG2D. In certain embodiments, the multispecific binding proteins comprising an NKG2D binding domain and an HER2 binding domain bind to HER2 at a level comparable to that of a monoclonal antibody having the same HER2 binding domain. For example, a multispecific binding protein comprising an NKG2D binding domain and a trastuzumab-derived HER2 binding domain can bind to HER2 expressed in cells at a level comparable to that of trastuzumab.

[0128] However, the multispecific binding proteins described herein are more effective at reducing tumor growth and killing cancer cells. For example, the multispecific binding proteins of the present disclosure targeting HER2-expressing tumors / cancer cells are more effective than SC2.2, a single-chain bispecific molecule constructed from an scFv derived from trastuzumab linked to ULBP-6, a ligand for NKG2D. SC2.2 simultaneously binds to HER2+ cancer cells and NKG2D+ NK cells. Therefore, the effectiveness of SC2.2 in reducing the number of HER2+ cancer cells was investigated. In vitro activation and cytotoxicity assays demonstrated that SC2.2 was effective in activating and killing NK cells. However, SC2.2 failed to demonstrate efficacy in an RMA / S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using an RMA / S-HER2-overexpressing syngeneic mouse model (Figure 36). In this mouse model, SC2.2 failed to demonstrate tumor growth control compared to vehicle controls (Figure 37). Thus, although SC2.2 could activate and kill NK cells and bind to HER2+ cancer cells, these properties were insufficient to effectively control HER2+ tumor growth.

[0129] In certain embodiments, the multispecific binding proteins described herein, which comprise an NKG2D-binding domain and a tumor-associated antigen-binding domain, activate primary human NK cells when cultured with tumor cells expressing the antigen. NK cell activation is indicated by CD107a degranulation and increased IFNγ cytokine production. Furthermore, compared to monoclonal antibodies comprising a tumor-associated antigen-binding domain, the multispecific binding proteins exhibit superior activation of human NK cells in the presence of tumor cells expressing the antigen. For example, compared to the monoclonal antibody trastuzumab, the multispecific binding proteins of the present disclosure, which comprise a HER2-binding domain, exhibit superior activation of human NK cells in the presence of HER2-expressing cancer cells.

[0130] In certain embodiments, the multispecific binding proteins described herein, comprising an NKG2D-binding domain and a binding domain for a tumor-associated antigen, enhance the activity of resting human NK cells and IL-2-activated human NK cells in the presence of tumor cells expressing the antigen. Resting NK cells exhibited less background IFNγ production and CD107a degranulation than IL-2-activated NK cells. In certain embodiments, resting NK cells exhibit greater changes in IFNγ production and CD107a degranulation compared to IL-2-activated NK cells. In certain embodiments, IL-2-activated NK cells exhibit a greater percentage of cells becoming IFNγ+;CD107a+ after stimulation with TriNKET.

[0131] In certain embodiments, multispecific binding proteins described herein that contain an NKG2D-binding domain and a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) enhance the cytotoxic activity of resting human NK cells and IL-2-activated human NK cells in the presence of tumor cells expressing the antigen. Furthermore, multispecific binding proteins that contain a binding domain for HER2 (e.g., A40-multispecific binding protein, A44-multispecific binding protein, A49-multispecific binding protein, C26-multispecific binding protein, F04-multispecific binding protein, F43-multispecific binding protein, F47-multispecific binding protein, and F63-multispecific binding protein) more strongly direct activated and resting NK cell responses against tumor cells compared to monoclonal antibodies that contain HER2. In certain embodiments, multispecific binding proteins offer advantages against tumor cells that express moderate and low levels of HER2 compared to monoclonal antibodies that contain HER2 binding sites. Thus, treatments involving multispecific binding proteins may be superior to monoclonal antibody treatments.

[0132] In certain embodiments, the multispecific binding proteins described herein (e.g., A40-multispecific binding protein, A49-multispecific binding protein, C26-multispecific binding protein, F04-multispecific binding protein, F43-multispecific binding protein, F47-multispecific binding protein, and E79-multispecific binding protein) comprising a binding domain for HER2 are advantageous compared to monoclonal antibodies for treating cancers that highly express Fc receptors (FcRs) or that reside in tumor microenvironments with high levels of FcRs. Monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blockade, among others. Among FcγRs, CD16 has the lowest affinity for IgG Fc, and FcγRI (CD64) is a high-affinity FcR that binds IgG Fc approximately 1,000-fold more strongly than CD16. CD64 is normally expressed in many hematopoietic lineages, including the myeloid lineage, and may be expressed in tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, are also known to express CD64 and infiltrate the tumor microenvironment. Expression of CD64 by tumors or in the tumor microenvironment can have adverse effects on monoclonal antibody therapy. Because antibodies preferentially bind to high-affinity receptors, CD64 expression in the tumor microenvironment makes it difficult for these antibodies to associate with CD16 on the surface of NK cells. Multispecific binding proteins can overcome the adverse effects of CD64 expression (whether in the tumor or the tumor microenvironment) on monoclonal antibody therapy by targeting two activating receptors on the surface of NK cells. Dual targeting of two activating receptors on NK cells results in stronger specific binding to NK cells, allowing multispecific binding proteins to mediate human NK cell responses against any tumor cells, regardless of CD64 expression on the tumor cells.

[0133] In some embodiments, the multispecific binding proteins described herein (e.g., A40-multispecific binding protein, A49-multispecific binding protein, C26-multispecific binding protein, F04-multispecific binding protein, F43-multispecific binding protein, F47-multispecific binding protein, and E79-multispecific binding protein) comprising a binding domain for HER2 offer a better safety profile due to reduced on-target off-tumor side effects. Although natural killer cells and CD8 T cells recognize normal self from tumor cells by different mechanisms, both NK cells and CD8 T cells can directly lyse tumor cells. NK cell activity is regulated by the balance of signals from activating receptors (e.g., NCRs, NKG2D, CD16) and inhibitory receptors (e.g., KIRs, NKG2A). The balance of these activating and inhibitory signals enables NK cells to distinguish healthy autologous cells from stressed, virus-infected, or transformed autologous cells. This "built-in" self-tolerance mechanism helps protect normal, healthy tissues from NK cell responses. Extending this principle, NK cell self-tolerance allows multispecific binding proteins to target antigens expressed on both the self and tumor without extratumoral side effects or with an increased therapeutic window. Unlike natural killer cells, T cells require recognition of specific peptides presented by MHC molecules for activation and effector function. T cells are the primary target of immunotherapy, and many strategies have been developed to redirect T cell responses against tumors. T cell bispecifics, checkpoint inhibitors, and CAR-T cells are all FDA-approved but often have dose-limiting toxicities. T cell bispecifics and CAR-T cells circumvent the TCR-MHC recognition system by using binding domains to target antigens on the surface of tumor cells and engineered signaling domains to transmit activation signals to effector cells.Although these therapies are effective in eliciting antitumor immune responses, they are often associated with cytokine release syndrome (CRS) and on-target and off-tumor side effects. In this context, multispecific binding proteins are unique because they do not "disable" the natural systems of NK cell activation and inhibition. Rather, multispecific binding proteins are designed to tip this balance, providing additional activation signals to NK cells while maintaining NK tolerance to healthy self.

[0134] In some embodiments, multispecific binding proteins described herein comprising an NKG2D binding domain (e.g., A40-multispecific binding protein, A49-multispecific binding protein, C26-multispecific binding protein, F04-multispecific binding protein, F43-multispecific binding protein, F47-multispecific binding protein, and E79-multispecific binding protein) that comprise a binding domain for HER2 delay tumor progression more effectively than monoclonal antibodies comprising the same tumor antigen binding domain. In some embodiments, multispecific binding proteins comprising an NKG2D binding domain and a tumor antigen binding domain are more effective against cancer metastasis than monoclonal antibodies comprising the same tumor antigen binding domain. III. Therapeutic applications

[0135] The present invention provides methods for treating cancer using the multispecific binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat a variety of HER2-expressing cancers by administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein.

[0136] The therapeutic method may be characterized by the cancer being treated. For example, in certain embodiments, the cancer is breast cancer, ovarian cancer, esophageal cancer, bladder cancer, and stomach cancer, salivary duct cancer, adenocarcinoma of the lung, and aggressive forms of uterine cancer such as uterine serous endometrial cancer.

[0137] Therapeutic methods can be characterized by the cancer to be treated.For example, in certain embodiments, the cancer is a solid tumor.In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate 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 cancer, parotid gland cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bile duct carcinoma, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma, and the like. papilloma / cytoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, yolk sac 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, cancer of the gastric cardia, cancer of the gastric fundus, gastrinomas, glioblastoma, glucagonoma, cardiac cancer, hemangiblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasianeoplasia), intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, serous papillary adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cell 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, rhabdomyosarcoma, 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 melanoma, VIP-secreting tumor, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0138] In certain other embodiments, the cancer is a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0139] The cancer to be treated may be characterized by the presence of specific antigens expressed on the surface of cancer cells. In certain embodiments, the cancer cells may express one or more of the following in addition to HER2: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, and PD1. IV. Combination Therapy

[0140] Another aspect of the present invention provides combination therapy: the multispecific binding proteins described herein are used in combination with additional therapeutic agents to treat cancer.

[0141] Exemplary therapeutic agents that may be used as part of a combination therapy in treating 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, amsacrile, flucloxone, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon-alpha, interferon-alpha These include interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variants of the above agents that may exhibit differential binding to their cognate receptors and increased or decreased serum half-lives.

[0142] Another class of drugs that can be used as part of the combined therapy in treating cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include the drug that inhibits 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.CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for treating melanoma.

[0143] Still other agents that can be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0144] Further categories of anti-cancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors + 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors (ii) an inhibitor selected from an 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; (ii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0145] The proteins of the present invention may also be used as an adjunct to surgical removal of the primary lesion.

[0146] The amounts and relative timing of administration of the multispecific binding protein and additional therapeutic agent can 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 one or more pharmaceutical compositions containing the therapeutic agents, can be administered in any order, for example, sequentially, concomitantly, together, simultaneously, etc. Furthermore, for example, the multispecific binding protein can be administered for the time during which the additional therapeutic agent exerts its preventive or therapeutic effect, or vice versa. V. Pharmaceutical Compositions

[0147] The present disclosure also features pharmaceutical compositions containing a therapeutically effective amount of the proteins described herein. The compositions can be formulated for use in various drug delivery systems. To create a suitable formulation, one or more physiologically acceptable excipients or carriers can also be included in the composition. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th Edition, 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science, Vol. 249:1527-1533, 1990).

[0148] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing 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 freeze-dried (lyophilized) and may be contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulations from 12, 27, or 45 vials may be combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg per vial.

[0149] The present disclosure can be present in a liquid, aqueous pharmaceutical formulation that includes a therapeutically effective amount of protein in a buffer solution that forms the formulation.

[0150] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, e.g., 7 to 7.5. The resulting solid composition may be packaged in a plurality of single-dose units, each containing a fixed amount of one or more of the above-mentioned agents. The solid composition may also be packaged in flexible-quantity containers.

[0151] In certain embodiments, the present disclosure provides formulations having an extended shelf life 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 oxide.

[0152] In certain embodiments, aqueous formulations are prepared containing proteins of the present disclosure in a pH buffer solution. The buffers of the present invention may have a pH ranging from about 4 to about 8, e.g., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH ranging from about 5.0 to about 5.2. pH ranges intermediate to those listed above are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination of the values ​​listed above as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0153] 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 may be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or about pH 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 comprises 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 comprises 1-1.5 mg / ml citric acid, 0.25-0.5 mg / ml sodium citrate, 1.25-1.75 mg / ml disodium phosphate dihydrate, 0.7-1.1 mg / ml sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / ml sodium chloride. In certain embodiments, the pH of the formulation is adjusted using sodium hydroxide.

[0154] Polyols, which can act as tonicifiers and stabilize antibodies, can also be included in the formulation. The polyol is added to the formulation in an amount that can vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added can also vary depending on the molecular weight of the polyol. For example, a small amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that can be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / ml. In certain embodiments, the mannitol concentration can be about 7.5 to 15 mg / ml. In certain embodiments, the mannitol concentration can be about 10 to 14 mg / ml. In certain embodiments, the mannitol concentration can be about 12 mg / ml. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0155] 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 refer to polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0156] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be provided at a concentration of 10 mg / mL in a USP / Ph Eur Type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of a USP and Ph Eur compliant elastomer. In certain embodiments, the vial may be filled with 61.2 mL of protein product solution to allow for a draw volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline.

[0157] In certain embodiments, the liquid formulations of the present disclosure can be prepared as a 10 mg / mL solution combined with a sugar at a stabilized level. In certain embodiments, the liquid formulations can be prepared in an aqueous carrier. In certain embodiments, the stabilizer can be added in an amount that is not more than the amount that would result in an undesirable or inappropriate viscosity for intravenous administration. In certain embodiments, the sugar may be a disaccharide, e.g., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0158] In certain embodiments, the pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0159] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from proteins to form succinimide intermediates that can undergo hydrolysis. The succinimide intermediate results in a 17-dalton mass loss from the parent peptide. Subsequent hydrolysis results in an 18-dalton mass gain. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Therefore, deamidation is typically detectable as a 1-dalton mass gain. Deamidation of asparagine produces 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 deamidation rate. Gly and Ser following Asn in the protein sequence are more susceptible to deamidation.

[0160] 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.

[0161] 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 buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0162] Preservatives can be added to the formulations herein, if desired, to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0163] Intravenous (IV) formulations may be the preferred route of administration in certain cases, such as when a patient is hospitalized after transplant and receives all medications via the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0164] In certain embodiments, salts or buffer components can 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 can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions can serve as counterions.

[0165] Preservatives can be added to the formulations herein, if desired, to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0166] Aqueous carriers of interest herein are pharmaceutically acceptable (safe and non-toxic for human administration). and are useful in the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0167] The present disclosure can also be present as a lyophilized formulation comprising a protein and a lyoprotectant. The lyoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0168] The amount of sucrose or maltose useful for stabilizing a lyophilized drug product can be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be 1:2 to 1:5.

[0169] In certain embodiments, the pH of the formulation before lyophilization can be set by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

[0170] Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range for the lyophilized drug product may be 7-8.

[0171] In certain embodiments, salts or buffer components can 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 can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions can serve as counterions.

[0172] In certain embodiments, a "bulking agent" can 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 formulation of the present invention can contain such a bulking agent.

[0173] Preservatives can be added to the formulations herein, if desired, to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0174] In certain embodiments, the lyophilized drug 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 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.

[0175] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

[0176] 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 solution (sodium chloride solution).

[0177] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient.

[0178] A specific dose may be a uniform dose for each patient, e.g., 50-5000 mg of protein. Alternatively, a patient's dose 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 medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment can be routinely made by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages can also be determined by the use of known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted as disease progression is monitored. Blood levels of the targetable construct or complex in the patient may be measured to determine whether dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or compounds, and their dosages, are 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).

[0179] Generally, dosages based on body weight are 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 1 00 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 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, and about 50 mg to about 100 mg / kg body weight.

[0180] Doses may be given one or more times daily, one or more times weekly, one or more times monthly, or one or more times annually, or even once every 2 to 20 years. One skilled in the art can easily estimate repetition rates for dosing based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. The administration of the present invention can be performed in a variety of ways. Administration may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by perfusion via a catheter, or by direct intralesional injection. It may be administered one or more times daily, one or more times weekly, one or more times monthly, and one or more times yearly.

[0181] The above description describes multiple aspects and embodiments of the invention. The present application specifically contemplates all combinations and permutations of aspects and embodiments. [Example]

[0182] The invention generally described herein will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention. Example 1 The NKG2D-binding domain binds to NKG2D The NKG2D-binding domain binds to purified recombinant NKG2D

[0183] The nucleic acid sequence of the human, mouse, or cynomolgus monkey NKG2D extracellular domain was fused to a nucleic acid sequence encoding a human IgG1 Fc domain and transfected into mammalian cells for expression. 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 wells pre-adsorbed with the NKG2D-Fc fusion protein. 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. Binding signals were visualized by adding 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, to the wells, and the absorbance was measured at 450 nM and corrected at 540 nM. NKG2D binding domain clones, isotype controls, or positive controls (selected from SEQ ID NOs: 45-48, or anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) were added to each well.

[0184] The isotype control showed little binding to the recombinant NKG2D-Fc protein, whereas the positive control showed the strongest binding to the recombinant antigen. Although the affinity varied among clones, the NKG2D-binding domains produced by all clones showed binding to all human, mouse, and cynomolgus monkey recombinant NKG2D-Fc proteins. Overall, each anti-NKG2D clone bound with similar affinity to human (Figure 3) and cynomolgus monkey (Figure 4) recombinant NKG2D-Fc, but showed relatively low affinity to mouse (Figure 5) recombinant NKG2D-Fc. The NKG2D-binding domain binds to cells expressing NKG2D

[0185] 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 in 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.

[0186] The NKG2D-binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. Positive control antibodies (selected from SEQ ID NOs: 45-48, or anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) provided the best FOB binding signals. NKG2D binding affinity was similar between cells expressing human NKG2D (FIG. 6) and cells expressing mouse NKG2D (FIG. 7). Example 2 The NKG2D-binding domain blocks the binding of natural ligands to NKG2D Competition with ULBP-6

[0187] 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 streptavidin conjugated to horseradish peroxidase and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin in the wells that was blocked from binding to the NKG2D-Fc protein. A positive control antibody (selected from SEQ ID NOs: 45-48) and various NKG2D-binding domains blocked ULBP-6 binding to NKG2D, whereas the isotype control showed little competition with ULBP-6 (Figure 8). Competition with MICA

[0188] Recombinant human MICA-Fc protein was adsorbed to microplate wells, which were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the NKG2D-binding domain. After incubation and washing, NKG2D-Fc-biotin 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 (selected from SEQ ID NOs: 45-48) and various NKG2D binding domains blocked MICA binding to NKG2D, while an isotype control showed little competition with MICA (FIG. 9). Conflict with Rae-1 Delta

[0189] 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, the specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of NKG2D-Fc-biotin blocked from binding to the Rae-1 delta-Fc-coated wells. Positive control antibodies (selected from SEQ ID NOs: 45-48, or anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) and various NKG2D-binding domain clones blocked Rae-1 delta binding to mouse NKG2D, whereas isotype control antibodies showed little competition with Rae-1 delta (Figure 10). Example 3 NKG2D-binding domain clones activate NKG2D

[0190] The nucleic acid sequences of human and mouse NKG2D were fused to the nucleic acid sequence encoding the CD3 zeta signaling domain to generate chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into retroviral vectors using Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with the virus containing NKG2D-CAR along with 8 μg / mL of polybrene. 24 hours after infection, the expression levels of NKG2D-CAR in EL4 cells were analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0191] To determine whether the NKG2D-binding domains activate NKG2D, they were adsorbed to microplate wells, and NKG2D-CAR EL4 cells were cultured in the antibody fragment-coated wells for 4 hours in the presence of brefeldin-A and monensin. Intracellular TNFα production, an indicator of NKG2D activation, was assayed by flow cytometry. The percentage of TNFα-positive cells was normalized to cells treated with a positive control. All NKG2D-binding domains activated both human NKG2D (FIG. 11) and mouse NKG2D (FIG. 12). Example 4 The NKG2D-binding domain activates NK cells Primary human NK cells

[0192] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated. 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. They were then 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, the NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFNγ. CD3 - CD56 + NK cell activation was assessed by analyzing the staining of CD107a and IFNγ in the cells. An increase in CD107a / IFNγ double-positive cells indicates better NK cell activation due to engagement of two activating receptors rather than one receptor. The NKG2D binding domain and positive control (selected from SEQ ID NOs: 45-48) showed that a higher percentage of NK cells express CD107a than the isotype control. + and IFγ +(FIGS. 13 and 14 represent two independent experiments, each using PBMCs from a different donor for the preparation of NK cells). Primary mouse NK cells

[0193] Spleens were obtained from C57Bl / 6 mice and crushed through a 70 μm cell strainer to obtain a single-cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific, #A1049201; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove red blood cells. The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours, then harvested and prepared for NK cell isolation. NK cells (CD3+) were then isolated from the spleen cells using a negative depletion technique using magnetic beads, typically at a purity of >90%. - NK1.1 + Purified NK cells were cultured for 48 hours in medium containing 100 ng / mL mIL-15, then transferred to wells of a microplate onto which the NKG2D-binding domain had been adsorbed, and cultured in medium containing a fluorophore-conjugated anti-CD107a antibody, brefeldin A, and monensin. After culturing in wells coated with the binding domains, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFNγ. - NK1.1 + NK cell activation was assessed by analyzing CD107a and IFNγ staining in cells. An increase in CD107a / IFNγ double-positive cells indicates better NK cell activation due to engagement of two activating receptors rather than one. NKG2D binding domains and positive controls (selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) showed a higher percentage of NK cells expressing CD107a than the isotype control. + and IFNγ +(FIGS. 15 and 16 represent two independent experiments, each using a different mouse for NK cell preparation). Example 5 The NKG2D-binding domain enables cytotoxicity of target tumor cells

[0194] Human and mouse primary NK cell activation assays demonstrate increased cytotoxicity markers on NK cells after incubation with the NKG2D-binding domain. To address whether this translates to increased tumor cell lysis, we utilized a cell-based assay in which each NKG2D-binding domain represents a monospecific antibody. The Fc region was used as one targeting arm, while the Fab region (NKG2D-binding domain) acted as another targeting arm to activate NK cells. THP-1 cells, which are of human origin and express high levels of Fc receptors, 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 μg / mL. The labeled THP-1 cells were then combined with NKG2D antibodies and incubated in a microtiter plate well at 37°C for 3 hours to isolate murine NK cells. After incubation, 20 μl of the culture supernatant was removed, mixed with 200 μl of europium solution, and incubated with shaking in the dark for 15 minutes. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (excitation 337 nm, emission 620 nm), and the specific lysis rate was calculated according to the kit instructions.

[0195] The positive control ULBP-6, a natural ligand for NKG2D, increased the specific lysis rate of THP-1 target cells by mouse NK cells. NKG2D antibodies also increased the specific lysis rate of THP-1 target cells, whereas an isotype control antibody reduced the specific lysis rate. The dotted line indicates the specific lysis rate of THP-1 cells by mouse NK cells without added antibody (Figure 17). Example 6 NKG2D antibodies exhibit high thermal stability

[0196] The melting temperature of the NKG2D-binding domain was assayed using differential scanning fluorimetry, and the extrapolated apparent melting temperature is higher compared to typical IgG1 antibodies (FIG. 18). Example 7 Multispecific binding proteins exhibit enhanced ability to activate NK cells

[0197] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated. 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. They were then transferred to wells of microplates pre-adsorbed with polyspecific and bispecific binding proteins, respectively, 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 IFNγ. CD3 - CD56 + NK cell activation was assessed by analyzing CD107a and IFNγ staining in the cells. An increase in CD107a / IFNγ double-positive cells indicates better NK cell activation. AL2.2 is a multispecific binding protein containing a HER2-binding domain (trastuzumab), an NKG2D-binding domain (ULBP-6), and a human IgG1 Fc domain. It was generated by controlled Fab arm exchange (cFAE) starting from trastuzumab homodimer and ULBP-6-Fc homodimer (see Labrijn et al., Nature Protocols, Vol. 9, pp. 2450-2463). SC2.2 is a single-chain protein containing an scFv derived from trastuzumab and ULBP-6 (SEQ ID NO: 93). [ka]

[0198] Analysis of CD107a and IFNγ staining showed that isotype control IgG did not activate NK cells, but after stimulation with the multispecific binding protein, a higher percentage of NK cells expressed CD107a compared with the bispecific protein. + and IFNγ + These results demonstrate that engagement of two activating receptors (NKG2D and CD16) rather than just one (NKG2D) results in more potent NK cell activation (Figure 19). This increase in NK cell activation is expected to translate into more potent tumor cell killing. Example 8 Multispecific binding proteins exhibit enhanced cytotoxicity against target tumor cells Primary human NK cytotoxicity assay

[0199] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated. The purity of the isolated NK cells was typically >95%. NK cells were then cultured overnight in medium containing 100 ng / mL IL-2 and then used in cytotoxicity assays. The following day, NK cells were cultured at a concentration of 5 × 10 5 Human breast cancer cells SkBr-3 cells were labeled with BATDA reagent according to the Perkin Elmer DELFIA cytotoxicity kit and resuspended in fresh culture medium at 5 × 10 4The cells were resuspended in culture medium at 1000µL / mL. Various dilutions of the multispecific binding protein were made in the culture medium. NK cells, labeled SkBr-3 cells, and the multispecific binding protein were then combined in wells of a microtiter plate and incubated at 37°C for 3 hours. After incubation, 20µl of the culture supernatant was removed, mixed with 200µl of europium solution, and incubated in the dark for 15 minutes with shaking. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (excitation 337nm, emission 620nm), and the specific lysis rate was calculated according to the kit's instructions. AL0.2 is a multispecific binding protein containing a HER2-binding domain (trastuzumab), an NKG2D-binding domain (selected from SEQ ID NOs: 1-44), and a human IgG1 Fc domain. It was produced by controlled Fab arm exchange (cFAE) starting with trastuzumab homodimer and anti-NKG2D homodimer. AL0.2si is based on AL0.2 and contains an additional D265A mutation in the Fc domain that abolishes CD16 binding. Trastuzumab-si is based on trastuzumab and contains an additional D265A mutation in the Fc domain that abolishes CD16 binding. AL2.2 is a multispecific binding protein containing a HER2 binding domain (trastuzumab), an NKG2D binding domain (ULBP-6), and a human IgG1 Fc domain. SC2.2 is a single-chain protein containing an scFv derived from trastuzumab and ULBP-6.

[0200] AL0.2 demonstrated enhanced lysis of SkBr-3 target cells by human NK cells compared to trastuzumab in a dose-dependent manner, with a p-value of 0.0311 at EC50 (Figure 20). AL0.2si (Figure 21) and trastuzumab-si (Figure 22) demonstrated reduced potency and maximum specific lysis of SkBr-3 cells compared to AL0.2, with p-values ​​of 0.0002 and 0.0001 at EC50, respectively (Figures 21-22). Furthermore, AL0.2 demonstrated enhanced lysis of SkBr-3 cells compared to AL2.2 in a dose-dependent manner (Figure 23). Isotype control IgG did not demonstrate an increase in specific lysis at any of the concentrations tested. Together, the data indicate that a multispecific binding protein that associates with two activating receptors on NK cells and one tumor antigen induces more potent killing of tumor cells by human NK cells compared with a bispecific protein that associates with one activating receptor on NK cells and one tumor antigen. Cytotoxicity assay of primary murine NK cells

[0201] Spleens were obtained from C57Bl / 6 mice and crushed through a 70 μm cell strainer to obtain a single-cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific, #A1049201; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA) to remove red blood cells. The remaining cells were cultured with 100 ng / mL hIL-2 for 72 hours, then harvested and prepared for NK cell isolation. NK cells (CD3+) were then isolated from the spleen cells using a negative depletion technique using magnetic beads, typically at a purity of >90%. - NK1.1 + Purified NK cells were cultured in medium containing 100 ng / mL mIL-15 for 48 hours and then cultured at 10 6The cells were resuspended in culture medium at 2 × 10 / mL. RMA-HER2-dTomato, a mouse tumor cell line engineered to express HER2 and dTomato, and its control counterpart, RMA cells expressing zsGreen, were used as targets. 5 The cells were resuspended in culture medium at 1 / mL and seeded into microplate wells at a 1:1 ratio. Dilutions of the multispecific proteins were made in culture medium and added to the RMA cells along with the NK cells. After overnight incubation at 37°C and 5% CO2, the percentages of RMA-HER2-dTomato and RMA-zsGreen cells were determined by flow cytometry using a fluorescent reporter to identify the two cell types. Specific target cell death = (1-((% of RMA-Ca2T-dTomato cells in treatment group) * % of RMA-zsGreen cells in the control group / (% of RMA-Ca2T-dTomato cells in the control group * % of RMA-zsGreen cells in treatment groups * 100%.

[0202] AL2.2 is more potent at redirecting NK cell responses against tumor targets than SC2.2 (Figure 25) and trastuzumab (Figure 24). Control proteins showed little effect on specific target killing. These data indicate that multispecific binding proteins that associate with two activating receptors on NK cells and one tumor antigen induce more potent killing of tumor cells by mouse NK cells compared with bispecific proteins that associate with one activating receptor on NK cells and one tumor antigen. Example 9 Multispecific binding proteins bind to NKG2D

[0203] As shown in Figure 1, the EL4 mouse lymphoma cell line was engineered to express human NKG2D trispecific binding proteins (TriNKETs), each containing an NKG2D-binding domain, a HER2-binding domain, and an Fc domain that binds to CD16, and their affinity for extracellular NKG2D expressed in EL4 cells was tested. Binding of the multispecific binding proteins to NKG2D 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.

[0204] The TriNKETs tested included HER2-TriNKET-C26 (ADI-28226 and HER2-binding domain) and HER2-TriNKET-F04 (ADI-29404 and HER2-binding domain). The HER2-binding domain used in the tested molecules consisted of the heavy and light chain variable domains of trastuzumab.

[0205] The data show that the HER2-targeted TriNKET of the present disclosure binds to NKG2D (FIG. 26). Example 10 Multispecific binding proteins that bind to human tumor antigens Trispecific binding protein that binds to HER2

[0206] HER2-expressing human cancer cell lines were used to assay the binding of TriNKET to tumor-associated antigens. The renal cell carcinoma cell line 786-O expresses low levels of HER2. TriNKET and, where appropriate, a parent anti-HER2 monoclonal antibody (trastuzumab) were incubated with the cells, and 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) from TriNKET and trastuzumab normalized to the secondary antibody control. HER2-TriNKET-C26 and HER2-TriNKET-F04 show comparable levels of binding to HER2 expressed in 786-O cells compared to trastuzumab (Figure 27A).

[0207] RMA cells transduced with human HER2 were used to test the binding of HER2-targeted TriNKET to cell-expressed human HER2. TriNKET was diluted to 20 μg / mL, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and binding to cell-expressed HER2 was compared with isotype-stained and unstained cell populations. Figures 27B and 27C show the binding profiles of TriNKETs containing two different NKG2D-binding domains but with the same HER-binding domain (the binding profile of C26.2 TriNKET with a HER2-binding site is shown in Figure 27B, and the binding profile of F04.2 TriNKET with a HER2-binding site is shown in Figure 27C). Both TriNKETs show similar levels of binding to cell surface HER2 on RMA cells. Example 11 Multispecific binding protein-activated NK cells

[0208] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 +) were isolated. The purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. IL-2-activated NK cells were used within 24–48 h after activation.

[0209] Human cancer cells expressing tumor antigens were collected and cultured at 2 × 10 6 Activated NK cells were harvested, washed, and resuspended in culture medium at 2 × 10 / mL. Monoclonal antibodies targeting tumor antigens or TriNKET were diluted in culture medium. 6 The tumor cells were then resuspended at 1000 μg / mL. The tumor cells were then mixed with the monoclonal antibody TriNKET to activate NK cells in the presence of IL-2. Brefeldin-A and monensin were also added to the mixed cultures to block protein export from the cells for intracellular cytokine staining. Fluorophore-conjugated anti-CD107a was added to the mixed cultures, and the cultures were incubated for 4 hours before preparing samples for FACS analysis using fluorophore-conjugated antibodies against CD3, CD56, and IFNγ. CD107a and IFNγ staining was compared with CD3. - CD56 + NK cell activation was assessed by analyzing CD107a / IFNγ double positive cells, indicating better NK cell activation due to engagement of two activating receptors rather than one receptor.

[0210] TriNKET mediates the activation of human NK cells cocultured with HER2-expressing SkBr-3 cells (Figure 28A), Colo201 cells (Figure 28B), and HCC1954 cells (Figure 28C), as indicated by increased CD107a degranulation and IFNγ production. SkBr-3 and HCC1954 cells express high levels of surface HER2, while Colo201 cells express moderate levels of HER2. Compared to the monoclonal antibody trastuzumab, TriNKET demonstrates superior activation of human NK cells in the presence of human cancer cells. NK cells alone and NK cells + SkBr-3 cells were used as negative controls.

[0211] TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) mediated activation of human NK cells cocultured with CD33-expressing human AML Mv4-11 cells, demonstrating increased CD107a degranulation and IFNγ production. Compared with monoclonal anti-CD33 antibodies, TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) demonstrated superior activation of human NK cells in the presence of HER2-expressing human cancer cells (Figures 28A-28C). Primary human NK cells are activated by TriNKET in co-culture with target-expressing human cancer cell lines Example 12 Trispecific binding proteins enable cytotoxicity of targeted cancer cells

[0212] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated. The purity of the isolated NK cells was typically >90%. The isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. IL-2-activated or resting NK cells were used in cytotoxicity assays the following day.

[0213] To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, the CytoTox 96 non-radioactive cytotoxicity assay from Promega (G1780) was used according to the manufacturer's instructions. Briefly, human cancer cells expressing tumor antigens were harvested, washed, and then lysed at 1–2 × 10 5 Resting and / or activated NK cells were harvested, washed, and resuspended in the same culture medium as the cancer cells at 10 / mL. 5 ~2.0×10 6The cells were resuspended at 1000kJ / mL. In each well of a 96-well plate, 50 μl of the cancer cell suspension was mixed with 50 μl of the NK cell suspension with or without TriNKET, which targets a tumor antigen expressed on cancer cells. After 3 hours and 15 minutes of incubation at 37°C and 5% CO2, 10x lysis buffer was added to wells containing only cancer cells and to wells containing medium only for maximum lysis and negative reagent controls, respectively. The plate was then returned to the incubator for another 45 minutes to achieve a total incubation time of 4 hours. Next, the cells were pelleted, and the culture supernatant was transferred to a new 96-well plate and mixed with substrate for color development. The new plate was incubated at room temperature for 30 minutes, and absorbance was read at 492 nm on a SpectraMax i3x. The percentage specific lysis of cancer cells was calculated as follows: % specific lysis = ((experimental lysis - spontaneous lysis from NK cells alone - spontaneous lysis from cancer cells alone) / (maximum lysis - negative reagent control)) × 100%.

[0214] TriNKET enhances the cytotoxicity of NK cells against targets with low surface expression compared to the cytotoxic activity of the anti-HER2 monoclonal antibody trastuzumab. Resting human NK cells were mixed with high-HER2-expressing SkBr tumor cells and low-HER2-expressing 786-O cancer cells to assay the ability of TriNKET to enhance the cytotoxic activity of resting human NK cells against high- and low-HER2-expressing cancer cells in a dose-responsive manner. The dotted lines in Figures 29A and 29B indicate the cytotoxic activity of resting NK cells against cancer cells in the absence of TriNKET. As shown in Figure 29B, when activated human NK cells were mixed with low-HER2-expressing 786-O cells and TriNKET containing a HER2-binding domain (e.g., CD26-TriNKET and F04-TriNKET), dose-responsive cytotoxic activity of activated human NK cells against cancer cells was observed. Example 13 Synergistic activation of human NK cells by cross-linking of NKG2D and CD16 Primary human NK cell activation assay

[0215] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral human blood buffy coats using density gradient centrifugation. NK cells were purified from PBMCs using negative 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 overnight at 4°C 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, followed by extensive washing of the wells to remove excess antibody. For assessment of degranulation, IL-2-activated NK cells were cultured at 5 × 10 in culture medium supplemented with 100 ng / mL hIL-2 and 1 μg / mL APC-conjugated anti-CD107a mAb (Biolegend #328619). 5 The cells were resuspended at 1 x 10 cells / mL. 5 Cells / well were added onto 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 at 37°C in 5% CO2 for 4 hours. For intracellular staining of IFN-γ, NK cells were labeled with anti-CD3 (Biolegend #300452) and anti-CD56 mAb (Biolegend #318328), followed by fixation, permeabilization, and labeling with anti-IFN-γ mAb (Biolegend #506507). NK cells were then stained for live CD56 + CD3 - After gating on cells, they were analyzed for CD107a and IFN-γ expression by flow cytometry.

[0216] To investigate the relative potency of receptor combinations, cross-linking of NKG2D or CD16 and co-cross-linking of both receptors was performed by plate-bound stimulation. As shown in Figure 30 (Figures 30A-30C), combined stimulation of CD16 and NKG2D resulted in a significant increase in CD107a (degranulation) levels (Figure 30A) and / or IFN-γ production levels (Figure 30B). The dotted lines represent the additive effect of individual stimulation of each receptor.

[0217] After 4 hours of plate-bound stimulation with anti-CD16, anti-NKG2D, or a combination of both monoclonal antibodies, IL-2-activated NK cells were analyzed for CD107a levels and intracellular IFN-γ production. Graphs show mean (n=2) ± SD. Figure 19A shows CD107a levels, Figure 19B shows IFN-γ levels, and Figure 30C shows CD107a and IFN-γ levels. Data shown in Figures 30A-30C are representative of five independent experiments using five different healthy donors.

[0218] After 4 hours of plate-bound stimulation with trastuzumab, anti-NKG2D, or TriNKET, derived from the binding domains of trastuzumab and anti-NKG2D antibodies, IL-2-activated NK cells were analyzed for CD107a degranulation and intracellular IFN-γ production (FIG. 31). In all cases, the antibodies tested were of the human IgG1 isotype. Graphs show the mean (n=2) ± SD. Example 14 Assessment of TriNKET binding to human NKG2D expressed by cells

[0219] EL4 cells transduced with human NKG2D were used to test binding to human NKG2D expressed by the cells. TriNKET was diluted to 20 μg / mL and then serially diluted. Cells were stained using mAb or TriNKET dilutions, and binding of TriNKET or mAb was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and binding MFI was normalized to the secondary antibody control to obtain fold over background values. Assessment of TriNKET binding to human cancer antigens expressed by cells

[0220] Tumor antigen binding of TriNKET derived from different NKG2D-targeting clones was evaluated using HER2-expressing human cancer cell lines. The human renal cell carcinoma cell line 786-O expresses low levels of HER2 and was used to evaluate TriNKET binding to cell-expressed HER2. TriNKET was diluted to 20 μg / mL and incubated with each cell line. TriNKET binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and the MFI of binding to cell-expressed HER2 was normalized to the secondary antibody control to obtain fold-over-background values. Determination of antibody binding capacity of human HER2-positive cancer cell lines

[0221] Antibody binding capacity (ABC) of HER2-positive human cancer cell lines was measured. Antibody-labeled beads were prepared using the Quantum Simply Cellular kit (#815) from Bangs Lab according to the manufacturer's instructions. Briefly, each of the four bead populations was stained with a saturating amount of anti-HER2 antibody, and the cell population was stained with a saturating amount of the same antibody. Sample data were acquired for each bead population and cell population. The QuickCal worksheet provided with the kit was used to generate standard curves and extrapolate ABC values ​​for each cell line. Activation of primary NK cells by TriNKET

[0222] 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. The purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were cultured in medium containing 100 ng / mL IL-2 for activation or rested overnight without cytokines. IL-2-activated NK cells were used after 24–48 hours. Resting NK cells were always used the day after purification.

[0223] Human cancer cell lines expressing the cancer target of interest were harvested from culture and cultured at 2 × 10 cells. 6 The monoclonal antibody or TriNKET targeting the cancer target of interest was diluted in culture medium. Resting and / or activated NK cells were harvested from the culture, the cells were washed, and 2 × 10 6 The NK cells were resuspended in culture medium at 100 μl / mL. IL-2 and fluorophore-conjugated anti-CD107a were added to the NK cells for activation culture. Brefeldin-A and monensin were diluted in culture medium to block protein export from the cells for intracellular cytokine staining. 50 μl of tumor target, mAb / TriNKET, BFA / monensin, and NK cells were added to a 96-well plate in a total culture volume of 200 μl. The plates were cultured for 4 hours, after which samples were prepared for FACS analysis.

[0224] After a 4-hour activation culture, cells were prepared for analysis by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFNγ. CD107a and IFNγ staining was analyzed in the CD3-CD56+ population to assess NK cell activation. Primary human NK cell cytotoxicity assay

[0225] PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using negative selection techniques using magnetic beads. 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 rested overnight without cytokines. The following day, IL-2-activated or resting NK cells were used in cytotoxicity assays. Cyto Tox96 LHD Release Assay:

[0226] The ability of human NK cells to lyse tumor cells was measured with or without the addition of TriNKET using the Cyto Tox96 non-radioactive cytotoxicity assay from Promega (G1780). Human cancer cell lines expressing the cancer target of interest were harvested from culture, washed with PBS, and 1–2 × 10 cells were harvested for use as target cells. 5 The cells were resuspended in growth medium at 10 / mL. 50 μl of the target cell suspension was added to each well. Monoclonal antibodies or TriNKET targeting the cancer antigen of interest were diluted in culture medium, and 50 μl of the diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the culture, the cells were washed, and 10 μl of the diluted mAb or TriNKET was added to each well in culture medium depending on the desired E:T ratio. 5 ~2.0×10 6 The NK cells were resuspended at 100 / mL. 50 μl of NK cells were added to each well of the plate for a total culture volume of 150 μl. The plates were incubated at 37°C with 5% CO2 for 3 hours and 15 minutes. After incubation, 10x lysis buffer was added to the target cell-only wells and to wells containing medium only for maximum lysis and volume adjustment. The plates were then returned to the incubator for an additional 45 minutes, for a total incubation of 4 hours before color development.

[0227] After incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 50 μl of culture supernatant was transferred to a clean microplate and 50 μl of substrate solution was added to each well. The plate was protected from light and incubated at room temperature for 30 minutes. 50 μl of stop solution was added to each well and absorbance was read at 492 nm on a SpectraMax i3x. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release from effector - spontaneous release from target) / (maximum release - spontaneous release)). * 100%. DELFIA Cytotoxicity Assay:

[0228] Human cancer cell lines expressing the target of interest were harvested from culture, washed with PBS, and incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were resuspended in growth medium at 0.5–1.0 × 10 / mL. Target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with PBS 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 the labeled cells was set aside and spun out of the medium. 100 μl of medium was carefully added to triplicate wells without disturbing the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. One well was reserved for spontaneous release from the target cells, and one well was prepared for maximum lysis of the target cells by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target of interest were diluted in culture medium, and 50 μl of diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the culture, the cells were washed, and 10 μl of the diluted mAb or TriNKET was added to each well, depending on the desired E:T ratio. 5 ~2.0×10 6 NK cells were resuspended at 100 / mL. 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 and 5% CO for 2-3 hours before the assay developed.

[0229] After 2-3 hours of incubation, the plates were removed from the incubator and the 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. The plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. The plates were read using either a Victor 3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)). * 100%. Long-term human PBMC cytotoxicity assay:

[0230] SkBr-3 target cells were labeled with BacMam 3.0 NucLight Green (#4622) to enable target cell tracking. SkBr-3 target cells were labeled according to the manufacturer's protocol. Annexin V Red (Essen Bioscience #4641) was diluted and prepared according to the manufacturer's instructions. Monoclonal antibodies or TriNKET were diluted in culture medium. 50 μl of mAb or TriNKET, Annexin V, and resting NK cells were added to wells of a 96-well plate containing previously labeled SkBr-3 cells. 50 μl of complete culture medium was added for a total culture volume of 200 μl.

[0231] Image collection was set up in an IncuCyte S3. Images for the phase, green, and red channels were collected every hour, with two images per well. Image analysis was performed using the IncuCyte S3 software. Masks were created for the green and red channels to count the number of tumor cells and Annexin V-positive cells, respectively. The following formula was used to calculate the % of Annexin V-positive Mv4-11 target cells: % of Annexin V-positive SkBr-3 cells = ((number of duplicates) / (number of green objects)). * 100%. Comparison of TriNKET and SC2.2 targeting HER+ cancer cells

[0232] TriNKET targeting HER2 was more effective than trastuzumab in reducing SkBr-3 cell numbers, with only 60% of the cells from time zero remaining after 60 hours. The disclosed TriNKET targeting HER2-expressing tumor / cancer cells was more effective than SC2.2, a single-chain bispecific molecule constructed from an scFv derived from trastuzumab linked to ULBP-6, a ligand for NKG2D. SC2.2 simultaneously binds to HER2+ cancer cells and NKG2D+ NK cells. Therefore, the efficacy of SC2.2 in reducing HER2+ cancer cell numbers was investigated. In vitro activation and cytotoxicity assays demonstrated that SC2.2 was effective in activating and killing NK cells. However, SC2.2 failed to demonstrate efficacy in an RMA / S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using an RMA / S-HER2-overexpressing syngeneic mouse model. In this mouse model, SC2.2 failed to show tumor growth control compared with vehicle controls. Thus, although SC2.2 can activate and kill NK cells and bind to HER2+ cancer cells, these properties are insufficient to effectively control HER2+ tumor growth. Evaluation of SC2.2 serum half-life in C57Bl / 6 mice

[0233] To determine the serum half-life of SC2.2 in C57Bl / 6 mice, SC2.2 was labeled with a fluorescent tag to track its concentration in vivo. SC2.2 was labeled with IRDye 800CW (Licor #929-70020). The labeled protein was intravenously injected into three C57Bl / 6 mice, and blood was collected from each mouse at the indicated time points. After collection, the blood was centrifuged at 1000g for 15 minutes, and serum was collected from each sample and stored at 4C until all time points had been collected.

[0234] Serum was imaged using an Odyssey CLx infrared imaging system, and the fluorescent signal from the 800 channel was quantified using Image J software. Image intensity was normalized to the first time point, and the data were fitted to a biphasic decay equation. In this experimental system, the beta half-life of SC2.2 was calculated to be approximately 7 hours. In vivo testing of SC2.2 against RMA / S-HER2 subcutaneous tumors

[0235] To test the efficacy of SC2.2 against subcutaneous RMA / S-HER2 tumors, an in vivo study was designed according to Figure 37. 6 RMA / S cells were injected subcutaneously into the flanks of 20 C57B1 / 6 mice. Starting 2 days after tumor innoculation, SC2.2 was administered daily by IP injection. SC2.2 was administered at high and low concentrations along with a vehicle control. Starting 4 days after tumor innoculation, tumors were measured on Mondays, Wednesdays, and Fridays throughout the study. Tumor volume was calculated using the following formula: tumor volume = length x width x height. Antibody binding capacity of human HER2-positive cancer cell lines

[0236] Table 10 shows the results of HER2 surface quantification. SkBr-3 and HCC1954 cells were confirmed to have high (+++) levels of surface HER2. ZR-75-1 and Colo201 showed intermediate (++) levels of surface HER2, and 786-O showed the lowest level of HER2 (+).

[0237] [Table 10] Primary human NK cells are activated by TriNKET in co-culture with human cancer lines expressing various levels of HER2

[0238] Figures 28A-28C show that TriNKET and trastuzumab were able to activate primary human NK cells in coculture with HER2-positive human tumor cells, as indicated by increased CD107a degranulation and IFNγ cytokine production. Compared with the monoclonal antibody trastuzumab, both TriNKETs (HER2-TriNKET-C26 and HER2-TriNKET-F04) demonstrated superior activation of human NK cells with various human HER2 cancer cells.

[0239] Figure 28A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells, Figure 28B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells, and Figure 28C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells. TriNKET enhances the cytotoxicity of resting and IL-2-activated human NK cells

[0240] Figures 32A-32B show TriNKET enhancement of cytotoxic activity using IL-2-activated and resting human NK cells. Figure 32A shows the percent specific lysis of SkBr-3 tumor cells by resting human NK cells. Figure 32B shows the percent specific lysis of SkBr-3 tumor cells by IL-2-activated human NK cells. The IL-2-activated and resting NK cell populations were derived from the same donor. Compared with trastuzumab, TriNKET more strongly directs the response against SkBr-3 cells by either activated or resting NK cell populations. Figure 32C shows the percent specific lysis of HER2-expressing NCI-H661 lung cancer cells by human resting NK cells. Two TriNKETs with different NKG2D-binding domains can induce even higher maximal lysis of NCI-H661 HER2+ cancer cells compared with the monoclonal antibody trastuzumab. TriNKET enhances NK cell cytotoxicity against targets with low surface expression

[0241] The effect of TriNKET on target cells with low HER2 surface expression was investigated. Figures 29A-29B show that TriNKET provides a greater benefit to HER2-moderate and -low cancers compared to trastuzumab. Figure 29A shows activated human NK cell killing of HER2-high SkBr-3 tumor cells. Figure 29B shows human NK cell killing of HER2-low 786-O tumor cells. The benefits of TriNKET in treating cancers with high FcR expression or in tumor microenvironments with high levels of FcR

[0242] Monoclonal antibody therapy has been approved for the treatment of many cancer types, including both hematological and solid tumors. While the use of monoclonal antibodies in cancer treatment has improved patient outcomes, limitations remain. Mechanistic studies have shown that monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blockade, among others.

[0243] Notably, ADCC is believed to be the primary mechanism by which monoclonal antibodies exert their effects. ADCC relies on antibody-Fc engagement with the low-affinity FcγRIII (CD16) on the surface of natural killer cells, which mediates direct lysis of tumor cells. Among FcγRs, CD16 has the lowest affinity for IgG Fc, while FcγRI (CD64) is a high-affinity FcR that binds IgG Fc approximately 1000-fold more strongly than CD16.

[0244] CD64 is normally expressed in many hematopoietic lineages, such as myeloid lineages, and can be expressed in tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, also express CD64 and are known to infiltrate the tumor microenvironment. Expression of CD64 by tumors or in the tumor microenvironment can have a detrimental effect on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to associate with CD16 on the surface of NK cells, as antibodies prefer to bind to high-affinity receptors. By targeting two activating receptors on the surface of NK cells, TriNKET can overcome the detrimental effects of CD64 expression on monoclonal antibody therapy. Killing of normal myeloid and normal B cells in PBMC cultures: TriNKET offers a better safety profile with fewer on-target and off-tumor side effects

[0245] Although both natural killer cells and CD8 T cells can directly lyse tumor cells, the mechanisms by which NK cells and CD8 T cells recognize normal self from tumor cells are distinct. NK cell activity is regulated by the balance of signals from activating receptors (e.g., NCR, NKG2D, CD16) and inhibitory receptors (e.g., KIR, NKG2A). The balance of these activating and inhibitory signals allows NK cells to distinguish healthy self cells from stressed, virus-infected, or transformed self cells. This "built-in" self-tolerance mechanism helps protect normal, healthy tissue from NK cell responses. Extending this principle, NK cell self-tolerance allows TriNKET to target antigens expressed on both self and tumors without extratumoral side effects or with an increased therapeutic window.

[0246] Unlike natural killer cells, T cells require recognition of specific peptides presented by MHC molecules for activation and effector function. T cells are a primary target of immunotherapy, and many strategies have been developed to redirect T cell responses against tumors. T cell bispecifics, checkpoint inhibitors, and CAR-T cells are all FDA-approved but often have dose-limiting toxicities. T cell bispecifics and CAR-T cells circumvent the TCR-MHC recognition system by using their binding domains to target antigens on the surface of tumor cells and engineered signaling domains to deliver activation signals to effector cells. While effective in eliciting antitumor immune responses, these therapies often suffer from cytokine release syndrome (CRS) and on-target and off-tumor side effects. In this context, TriNKET is unique because it does not "disable" the natural system of NK cell activation and inhibition. Rather, TriNKET is designed to tip this balance and provide additional activation signals to NK cells while maintaining NK tolerance to healthy self.

[0247] PBMCs were isolated from whole blood by density gradient centrifugation. Any contaminating red blood cells were lysed by incubation in ACK lysis buffer. PBMCs were washed three times in PBS and total PBMCs were counted. PBMCs were cultured at 100 ml / min in primary cell culture medium. 6 The PBMCs were adjusted to 1 / mL. 1 mL of PBMCs was seeded into a well of a 24-well plate, and the indicated TriNKET or mAb was added to the PBMC culture at 10 μg / mL. Cells were cultured overnight at 37°C with 5% CO2. The next day (24 hours later), PBMCs were harvested from the culture and prepared for FACS analysis. The percentages of CD45+;CD19+ B cells and CD45+;CD33+;CD11b+ myeloid cells were analyzed across the different treatment groups.

[0248] Figures 33A and 33B show that B cells from healthy donors are sensitive to TriNKET-mediated lysis, while Figures 33C and 33D show that autologous myeloid cells are protected from TriNKET-mediated NK cell responses and are therefore resistant to TriNKET lysis. PBMCs treated with TriNKET targeting CD20 showed a decrease in the frequency of CD19+ B cells in the CD45+ lymphocyte population (Figure 33A), but no effect on the CD45+, CD3-, CD56- lymphocyte population (Figure 33B). These cultures did not change the frequency of CD45+, CD33+, CD11b+ myeloid cells (Figure 33C) or the frequency of CD45+, CD33+, CD11b+ myeloid cells (Figure 33D). TriNKET mediates hPBMC killing of SkBr-3 tumor cells in long-term co-culture Primary human PBMC cytotoxicity assay

[0249] Figure 34 shows the long-term killing of SkBr-3 cells in culture with human PBMCs. When cultured alone, SkBr-3 cells proliferate, nearly doubling in size in 60 hours. Addition of human PBMCs to SkBr-3 cells in culture slows the proliferation rate, and to a lesser extent, addition of the isotype control TriNKET, which targets CD33. When cultures are treated with trastuzumab, SkBr-3 no longer proliferate; after 60 hours, only 80% of the cells from time zero remain. Because SkBr-3 cells are sensitive to HER2 signal blockade, the effect on SkBr-3 cell growth may be mediated by HER2 signal blockade or through Fc effector functions such as ADCC. Example 15 Cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies, or bispecific antibodies against HER2-positive cells

[0250] PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection technique using magnetic beads. 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 rested overnight without cytokines. IL-2-activated or resting NK cells were used in cytotoxicity assays the following day. DELFIA Cytotoxicity Assay:

[0251] Human cancer cell lines expressing the target of interest were harvested from culture, the cells were washed with HBS, and the cells were incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were resuspended in growth medium at 0.5–1.0 × 10 / mL. 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 the labeled cells was set aside and the cells were spun out of the medium. 100 μl of medium was carefully added to triplicate wells without disturbing the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. One well was reserved for spontaneous release from the target cells, and one well was prepared for maximum lysis of the target cells by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target of interest were diluted in culture medium, and 50 μl of diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the culture, the cells were washed, and 10 μl of BATDA-labeled cells were added to culture medium depending on the desired E:T ratio. 5 ~2.0×10 6 NK cells were resuspended at 100 / mL. 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 and 5% CO for 2-3 hours before the assay developed.

[0252] After 2-3 hours of incubation, the plates were removed from the incubator and the 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. The plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. The plates were read using either a Victor 3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (maximum release - spontaneous release)). * 100%. Combination of monoclonal antibody and bispecific NK cell engager does not reproduce TriNKET activity:

[0253] Figure 35 shows the cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies, or bispecific antibodies against the HER2-positive Colo-201 cell line. TriNKET (ADI-29404 (F04)), which targets HER2, induced maximum lysis of Colo-201 cells by resting human NK cells. A D265A mutation was introduced into the CH2 domain of TriNKET to abolish FcR binding. HER2-TriNKET (ADI-29404 (F04))-D265A was unable to mediate lysis of Colo-201 cells, demonstrating the importance of dual targeting of CD16 and NKG2D to NK cells. To further demonstrate the importance of dual targeting of NK cells, the monoclonal antibody trastuzumab was used to target HER2 and mediate ADCC by NK cells. Although trastuzumab alone was able to increase NK cell lysis of Colo-201 cells, the maximal lysis achieved by trastuzumab alone was approximately fourfold lower than that achieved by TriNKET. To understand the importance of targeting CD16 and NKG2D to the same molecule, the activity of TriNKET (ADI-29404 (F04)) was compared to that of a bispecific antibody targeting HER2 and NKG2D in combination with trastuzumab. When used at equimolar concentrations, the combination of the bispecific and trastuzumab failed to mediate maximal lysis of Colo-201 cells by resting human NK cells. The failure of the trastuzumab + bispecific combination demonstrates the importance of containing the trispecific binding domain of TriNKET in a single molecule. Incorporation by Reference Example 16 Cross-linking assay

[0254] RMA cells transduced with human HER2 were used to test simultaneous binding of HER2-targeted TriNKET to HER2 and NKG2D. TriNKET was used at 20 μg / mL to stain surface HER2. TriNKET binding was then detected using biotinylated recombinant human NKG2D-Fc. Bound NKG2D-Fc was then detected using streptavidin-APC. Cells were analyzed by flow cytometry, and TriNKET crosslinking was compared to isotype-stained and unstained cell populations. Figure 38A shows that TriNKET-C26, which contains the HER2 binding domain, crosslinks hNKG2D-Fc to RMA-HER2 cells, and Figure 38B shows that TriNKET-F04, which contains the HER2 binding domain, crosslinks hNKG2D-Fc to RMA-HER2 cells.

[0255] The entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes. equivalent

[0256] 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 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. The present invention provides, for example, the following items. (Item 1) (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to HER2; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16; Protein containing. (Item 2) 2. The protein of item 1, wherein the first antigen-binding site binds to NKG2D of humans, non-human primates, and rodents. (Item 3) 3. The protein of item 1 or 2, wherein the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 4) 4. The protein of item 3, wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide. (Item 5) 5. The protein of item 3 or 4, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 6) 6. The protein of item 5, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are present on the same polypeptide. (Item 7) 7. The protein of item 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. (Item 8) 10. The protein of any one of the preceding items, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:1. (Item 9) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 41 and a light chain variable domain at least 90% identical to SEQ ID NO: 42. (Item 10) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 43 and a light chain variable domain at least 90% identical to SEQ ID NO: 44. (Item 11) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 45 and a light chain variable domain at least 90% identical to SEQ ID NO: 46. (Item 12) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 47 and a light chain variable domain at least 90% identical to SEQ ID NO: 48. (Item 13) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 94 and a light chain variable domain at least 90% identical to SEQ ID NO: 95. (Item 14) 8. The protein of any of items 1 to 7, wherein the first antigen-binding site comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 102 and a light chain variable domain at least 90% identical to SEQ ID NO: 103. (Item 15) 3. The protein of item 1 or 2, wherein the first antigen-binding site is a single domain antibody. (Item 16) The single domain antibody is V H H fragment or V NAR Item 16. The protein according to item 15, which is a fragment. (Item 17) 17. The protein of any one of items 1, 2, 15, or 16, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 18) 18. The protein of item 17, wherein the heavy chain variable domain and the light chain variable domain of the second antigen-binding site are present on the same polypeptide. (Item 19) 10. The protein of any of the preceding items, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 49 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: 53. (Item 20) the heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 50; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 51, and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 52 10. The protein of any of the preceding items, comprising an amino acid sequence comprising: (Item 21) the light chain variable domain of the second antigen-binding site comprises: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 54; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 55, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 56 21. The protein according to item 20, comprising an amino acid sequence comprising: (Item 22) 19. The protein of any one of items 1 to 18, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 57 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: 58. (Item 23) the heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 77; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 78, and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 79 23. The protein of any one of items 1 to 18 or 22, comprising an amino acid sequence comprising: (Item 24) the light chain variable domain of the second antigen-binding site comprises: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 80; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 81, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 82 24. The protein according to item 23, comprising an amino acid sequence comprising: (Item 25) 19. The protein of any one of items 1 to 18, wherein the heavy chain variable domain of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO: 59 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: 60. (Item 26) the heavy chain variable domain of the second antigen-binding site comprises: a heavy chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 83; a heavy chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 84, and A heavy chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 85 26. The protein of any one of items 1 to 18 or 25, comprising an amino acid sequence comprising: (Item 27) the light chain variable domain of the second antigen-binding site comprises: a light chain CDR1 sequence identical to the amino acid sequence of SEQ ID NO: 86; a light chain CDR2 sequence identical to the amino acid sequence of SEQ ID NO: 87, and A light chain CDR3 sequence identical to the amino acid sequence of SEQ ID NO: 88 27. The protein according to item 26, comprising an amino acid sequence comprising: (Item 28) 17. The protein of any one of items 1 to 4 or 8 to 16, wherein the second antigen-binding site is a single domain antibody. (Item 29) the second antigen-binding site is VH H fragment or V NAR 29. The protein according to item 28, which is a fragment. (Item 30) 10. The protein of any one of the preceding items, 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. (Item 31) 31. The protein of item 30, wherein the antibody Fc domain comprises the hinge and CH2 domains of a human IgG1 antibody. (Item 32) 32. The protein according to item 30 or 31, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 33) 33. The protein of any one of items 30 to 32, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, K439. (Item 34) A formulation comprising the protein of any one of the preceding items and a pharmaceutically acceptable carrier. (Item 35) 34. A cell comprising one or more nucleic acids expressing the protein of any one of items 1 to 33. (Item 36) 34. A method for directly and / or indirectly enhancing tumor cell death, comprising exposing tumor and natural killer cells to a protein according to any one of items 1 to 33. (Item 37) A method for treating cancer, comprising administering to a patient the protein of any one of items 1 to 33 or the formulation of item 34. (Item 38) 38. The method according to item 37, wherein the cancer is selected from the group consisting of breast, ovarian, esophageal, bladder and stomach cancer, salivary duct cancer, adenocarcinoma of the lung and aggressive forms of uterine cancer such as uterine serous endometrial cancer.

Claims

1. (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to HER2; (c) a first antibody Fc domain of human IgG1 or a portion thereof and a second antibody Fc domain of human IgG1 or a portion thereof that together are sufficient to bind to CD16, wherein the first antibody Fc domain or a portion thereof and the second antibody Fc domain or a portion thereof contain different amino acid mutations and promote heterodimerization, a first antibody Fc domain of human IgG1 or a portion thereof and a second antibody Fc domain of human IgG1 or a portion thereof; and a protein comprising the same.

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

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

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

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

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

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

8. The first antigen-binding site is (a) a heavy chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 102 and a light chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 103, wherein the heavy chain variable domain amino acid sequence comprises the heavy chain CDR1 amino acid sequence of SEQ ID NO: 104, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 105, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 106, and the light chain variable domain amino acid sequence comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 107, the light chain CDR2 amino acid sequence of SEQ ID NO: 108, and the light chain CDR3 amino acid sequence of SEQ ID NO: 109, a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence; (b) A heavy-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 41 and a light-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 42, wherein the heavy-chain variable domain amino acid sequence comprises the heavy-chain CDR1 amino acid sequence of SEQ ID NO: 65, the heavy-chain CDR2 amino acid sequence of SEQ ID NO: 66, and the heavy-chain CDR3 amino acid sequence of SEQ ID NO: 67, and the light-chain variable domain amino acid sequence comprises the light-chain CDR1 amino acid sequence of SEQ ID NO: 68, the light-chain CDR2 amino acid sequence of SEQ ID NO: 69, and the light-chain CDR3 amino acid sequence of SEQ ID NO: 70; a heavy-chain variable domain amino acid sequence and a light-chain variable domain amino acid sequence; (c) A heavy-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 43 and a light-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 44, wherein the heavy-chain variable domain amino acid sequence comprises the heavy-chain CDR1 amino acid sequence of SEQ ID NO: 71, the heavy-chain CDR2 amino acid sequence of SEQ ID NO: 72, and the heavy-chain CDR3 amino acid sequence of SEQ ID NO: 73, and the light-chain variable domain amino acid sequence comprises the light-chain CDR1 amino acid sequence of SEQ ID NO: 74, the light-chain CDR2 amino acid sequence of SEQ ID NO: 75, and the light-chain CDR3 amino acid sequence of SEQ ID NO: 76; a heavy-chain variable domain amino acid sequence and a light-chain variable domain amino acid sequence; (d) The heavy-chain variable domain amino acid sequence of SEQ ID NO: 45 and the light-chain variable domain amino acid sequence of SEQ ID NO: 46; (e) The heavy-chain variable domain amino acid sequence of SEQ ID NO: 47 and the light-chain variable domain amino acid sequence of SEQ ID NO: 48; (f) A heavy-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 94 and a light-chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 95, wherein the heavy-chain variable domain amino acid sequence comprises the heavy-chain CDR1 amino acid sequence of SEQ ID NO: 96, the heavy-chain CDR2 amino acid sequence of SEQ ID NO: 97, and the heavy-chain CDR3 amino acid sequence of SEQ ID NO: 98, and the light-chain variable domain amino acid sequence comprises the light-chain CDR1 amino acid sequence of SEQ ID NO: 99, the light-chain CDR2 amino acid sequence of 100, and the light-chain CDR3 amino acid sequence of SEQ ID NO: 101; or (g) A heavy chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 1, wherein the heavy chain variable domain amino acid sequence comprises the heavy chain CDR1 amino acid sequence of SEQ ID NO: 62, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 63, and the heavy chain CDR3 amino acid sequence of SEQ ID NO:

64. The protein according to any one of claims 1 to 7, comprising the same.

9. The first antigen-binding site is (a) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 104; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 105; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 106; The light chain CDR1 amino acid sequence of SEQ ID NO: 107; The light chain CDR2 amino acid sequence of SEQ ID NO: 108; and The light chain CDR3 amino acid sequence of SEQ ID NO: 109; (b) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 65; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 66; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 67; The light chain CDR1 amino acid sequence of SEQ ID NO: 68; The light chain CDR2 amino acid sequence of SEQ ID NO: 69; and The light chain CDR3 amino acid sequence of SEQ ID NO: 70; (c) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 71; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 72; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 73; The light chain CDR1 amino acid sequence of SEQ ID NO: 74; The light chain CDR2 amino acid sequence of SEQ ID NO: 75; and The light chain CDR3 amino acid sequence of SEQ ID NO: 76; or (d) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 96; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 97; The heavy chain CDR3 amino acid sequence of SEQ ID NO: 98; The light chain CDR1 amino acid sequence of SEQ ID NO: 99; The light chain CDR2 amino acid sequence of SEQ ID NO: 100; and The light chain CDR3 amino acid sequence of SEQ ID NO: 101; or (e) The heavy chain CDR1 amino acid sequence of SEQ ID NO: 62; The heavy chain CDR2 amino acid sequence of SEQ ID NO: 63; and The heavy chain CDR3 amino acid sequence of SEQ ID NO: 64 The protein according to any one of claims 1 to 7, comprising the same.

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

11. The single-domain antibody is a V Ｈ H fragment or a V ＮＡＲ fragment, the protein according to claim 10.

12. The protein according to any one of claims 1, 2, 10, and 11, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

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

14. wherein the second antigen-binding site is (a) a heavy chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 49 and a light chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 53, wherein the heavy chain variable domain amino acid sequence comprises the heavy chain CDR1 amino acid sequence of SEQ ID NO: 50, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 51, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 52, and the light chain variable domain amino acid sequence comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 54, the light chain CDR2 amino acid sequence of SEQ ID NO: 55, and the light chain CDR3 amino acid sequence of SEQ ID NO: 56, a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence; (b) a heavy chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 57 and a light chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 58, wherein the heavy chain variable domain amino acid sequence comprises the heavy chain CDR1 amino acid sequence of SEQ ID NO: 77, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 78, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 79, and the light chain variable domain amino acid sequence comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 80, the light chain CDR2 amino acid sequence of SEQ ID NO: 81, and the light chain CDR3 amino acid sequence of SEQ ID NO: 82, a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence; or (c) a heavy chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 59 and a light chain variable domain amino acid sequence that is at least 90% identical to SEQ ID NO: 60, wherein the heavy chain variable domain amino acid sequence comprises the heavy chain CDR1 amino acid sequence of SEQ ID NO: 83, the heavy chain CDR2 amino acid sequence of SEQ ID NO: 84, and the heavy chain CDR3 amino acid sequence of SEQ ID NO: 85, and the light chain variable domain amino acid sequence comprises the light chain CDR1 amino acid sequence of SEQ ID NO: 86, the light chain CDR2 amino acid sequence of SEQ ID NO: 87, and the light chain CDR3 amino acid sequence of SEQ ID NO: 88, a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence and is included in the protein according to any one of claims 1 to 13.

15. wherein the second antigen-binding site (a) the heavy chain CDR1 amino acid sequence of SEQ ID NO: 50; the heavy chain CDR2 amino acid sequence of SEQ ID NO: 51; the heavy chain CDR3 amino acid sequence of SEQ ID NO: 52; the light chain CDR1 amino acid sequence of SEQ ID NO: 54; the light chain CDR2 amino acid sequence of SEQ ID NO: 55; and Light chain CDR3 amino acid sequence of SEQ ID NO: 56; (b) Heavy chain CDR1 amino acid sequence of SEQ ID NO: 77; Heavy chain CDR2 amino acid sequence of SEQ ID NO: 78; Heavy chain CDR3 amino acid sequence of SEQ ID NO: 79; Light chain CDR1 amino acid sequence of SEQ ID NO: 80; Light chain CDR2 amino acid sequence of SEQ ID NO: 81; and Light chain CDR3 amino acid sequence of SEQ ID NO: 82; or (c) Heavy chain CDR1 amino acid sequence of SEQ ID NO: 83; Heavy chain CDR2 amino acid sequence of SEQ ID NO: 84; Heavy chain CDR3 amino acid sequence of SEQ ID NO: 85 Light chain CDR1 amino acid sequence of SEQ ID NO: 86; Light chain CDR2 amino acid sequence of SEQ ID NO: 87; and Light chain CDR3 amino acid sequence of SEQ ID NO: 88 The protein according to any one of claims 1 to 13, comprising

16. The protein according to any one of claims 1 to 4 and 8 to 11, wherein the second antigen-binding site is a single-domain antibody.

17. wherein the second antigen-binding site is a V Ｈ H fragment or a V ＮＡＲ fragment, the protein according to claim 16.

18. The protein according to any one of claims 1 to 17, wherein the first and second antibody Fc domains each comprise the hinge and CH2 domains of a human IgG1 antibody.

19. The protein according to claim 18, wherein the first and second antibody Fc domains each comprise an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

20. A formulation comprising the protein according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. A cell comprising one or more nucleic acids encoding the protein according to any one of claims 1 to 19.

22. A composition comprising the protein according to any one of claims 1 to 19 for use in a method of directly and / or indirectly enhancing tumor cell death, the method comprising exposing the composition to the tumor cells and natural killer cells.

23. A composition for use in therapy, comprising the protein according to any one of claims 1 to 19 or the formulation according to claim 20.

24. A composition for use in a method of treating cancer, comprising the protein according to any one of claims 1 to 19 or the formulation according to claim 20, wherein the method comprises administering the composition to a patient.

25. The composition or formulation according to claim 24, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, esophageal cancer, bladder cancer, gastric cancer, salivary duct carcinoma, adenocarcinoma of the lung, and invasive forms of uterine cancer and uterine serous endometrial cancer.

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