Multi-specific binding proteins for activating natural killer cells and therapeutic uses thereof for treating cancer

Abstract

To provide multi-specific binding proteins for activating natural killer cells and therapeutic uses thereof for treating cancer.SOLUTION: Disclosed herein are a multi-specific binding protein that binds to a tumor-associated antigen, an NKG2D receptor and CD16 as well as pharmaceutical compositions and therapeutic methods useful for cancer treatment.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 456,535, filed February 8, 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 entirety of which is incorporated herein by reference. The ASCII copy was created on February 6, 2018, is named DFY-001PC_SL.txt, and is 71,169 bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to a multispecific binding protein that binds to a tumor-associated antigen, the NKG2D receptor, and CD16. [Background technology]

[0004] background Cancer remains a major 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 the destruction of tumor cells. Antibodies that bind to certain tumor-associated antigens and certain immune cells have been described in the literature. See, for example, WO2016 / 134371 and WO2015 / 095412.

[0006] Natural killer (NK) cells are components of the innate immune system and constitute 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 inflammatory cytokines such as IFN-gamma and chemokines that promote the recruitment of other leukocytes to target tissues. NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated through their activating receptors (e.g., NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of some immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. [Prior art documents] [Patent documents]

[0007] [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]

[0008] Abstract The present invention provides multispecific binding proteins that bind to tumor-associated antigens in cancer cells and NKG2D and CD16 receptors in natural killer cells to activate 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. Such proteins can associate with two or more NK activating receptors and can 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.

[0009] In some embodiments, a multispecific binding protein can incorporate a first antigen-binding site that binds NKG2D, a second antigen-binding site that binds a tumor-associated antigen, and an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

[0010] In some embodiments, the multispecific binding protein is trivalent and comprises a first and a second antigen-binding site that both bind to the same tumor-associated antigen, a third antigen-binding site that binds to NKG2D, and an antibody Fc domain, or portion thereof, sufficient to bind CD16.

[0011] In some embodiments, the multispecific binding protein is tetravalent and comprises a first and a second antigen binding site that both bind to the same tumor-associated antigen, a third and a fourth antigen binding site that both bind to NKG2D, and an antibody Fc domain, or a portion thereof, sufficient to bind CD16.

[0012] The antigen-binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged like an antibody or fused together to form an scFv), or one or more of the antigen-binding sites may be a V-chain variable domain, such as a camelid antibody. H H antibodies or V antibodies such as those found in cartilaginous fishes NAR In some examples, the tumor-associated antigen may be selected from the group consisting of HER2, CD20, CD33, B-cell maturation antigen (BCMA), EpCAM, CD2, CD19, 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.

[0013] Another aspect of the present invention provides a method of treating cancer in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein as described herein to treat the cancer. Exemplary cancers for treatment using the multispecific binding protein include, for example, HER2-expressing carcinomas. [Brief description of the drawings]

[0014] [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.

[0015] [Diagram 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.

[0016] [Diagram 3] Figure 3 is a diagram of TriNKET in the 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 of a rat antibody and one half of a mouse antibody.

[0017] [Figure 4] Figure 4 is a diagram of TriNKET in KiH common light chain (LC) format using knobs-into-holes (KIH) technology. KiH is a heterodimer containing two Fabs that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation. TriNKET in KiH format can be a heterodimeric construct with two fabs that bind target 1 and target 2, containing two different heavy chains and a common light chain that pairs with both HCs.

[0018] [Diagram 5] Figure 5 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. 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.

[0019] [Figure 6] Figure 6 is a diagram of TriNKET in orthogonal Fab interface (ortho-Fab) form, a heterodimeric construct containing two Fabs that bind target 1 and target 2 fused to Fc. LC-HC pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

[0020] [Figure 7] FIG. 7 is a diagram of TrinKET in the 2in1 Ig format.

[0021] [Figure 8] Figure 8 is a diagram of TriNKET in ES format, a heterodimeric construct containing two different Fabs that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

[0022] [Figure 9] 9 is a diagram of TriNKET in a Fab arm exchange form, an antibody that has had its Fab arms exchanged by swapping its heavy chain (HC) and associated light chain (LC) (half molecule) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm exchange form (cFae) is a heterodimer that contains two Fabs that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0023] [Figure 10] FIG. 10 is a diagram of TriNKET in the SEED Body format, a heterodimer containing two Fabs that bind targets 1 and 2, and an Fc stabilized by a heterodimerization mutation.

[0024] [Figure 11] Figure 11 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 targets 1 and 2 fused to Fc. Heterodimerization is ensured by the leucine zipper motif fused to the C-terminus of Fc.

[0025] [Figure 12] FIG. 12 is a diagram of TriNKET in Cov-X-Body format.

[0026] [Figure 13] Figures 13A and 13B 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, which targets antigen 1, contains a kappa LC, while the second Fab, which targets antigen 2, contains a lambda LC. Figure 13A is an exemplary diagram of one form of κλ-Body, and Figure 13B is an exemplary diagram of another κλ-Body.

[0027] [Figure 14] FIG. 14 is a graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

[0028] [Figure 15] FIG. 15 is a graph showing the binding affinity of NKG2D binding domains (listed as clones) to cynomolgus monkey recombinant NKG2D in an ELISA assay.

[0029] [Figure 16] FIG. 16 is a graph showing the binding affinity of NKG2D binding domains (listed as clones) to mouse recombinant NKG2D in an ELISA assay.

[0030] [Figure 17] FIG. 17 is a graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D by flow cytometry showing fold mean fluorescence intensity (MFI) over background.

[0031] [Figure 18]FIG. 18 is a graph showing binding of NKG2D binding domains (listed as clones) to EL4 cells expressing mouse NKG2D by flow cytometry showing fold mean fluorescence intensity (MFI) over background.

[0032] [Figure 19] FIG. 19 is a 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.

[0033] [Figure 20] FIG. 20 is a 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.

[0034] [Figure 21] FIG. 21 is a 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.

[0035] [Figure 22] FIG. 22 is a graph showing activation of human NKG2D by NKG2D binding domains (listed as clones) by quantitating the percentage of TNF-alpha positive cells expressing human NKG2D-CD3 zeta fusion protein.

[0036] [Figure 23] FIG. 23 is a graph showing activation of mouse NKG2D by NKG2D binding domains (listed as clones) by quantitating the percentage of TNF-alpha positive cells expressing mouse NKG2D-CD3 zeta fusion protein.

[0037] [Figure 24] FIG. 24 is a graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0038] [Diagram 25] FIG. 25 is a graph showing activation of human NK cells by NKG2D binding domains (listed as clones).

[0039] [Figure 26] FIG. 26 is a graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0040] [Figure 27] FIG. 27 is a graph showing activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0041] [Figure 28] FIG. 28 is a graph showing the cytotoxic effect of NKG2D binding domains (listed as clones) on tumor cells.

[0042] [Figure 29] FIG. 29 is a graph showing the melting temperatures of NKG2D binding domains (listed as clones) as measured by differential scanning fluorimetry.

[0043] [Diagram 30] FIG. 30 is a graph showing enhanced activation of human NK cells by multispecific binding proteins.

[0044] [Diagram 31] FIG. 31 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity by human NK cells against tumor target cells.

[0045] [Diagram 32]FIG. 32 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by human NK cells.

[0046] [Diagram 33] FIG. 33 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by human NK cells.

[0047] [Diagram 34] FIG. 34 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by human NK cells.

[0048] [Diagram 35] FIG. 35 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by mouse NK cells.

[0049] [Diagram 36] FIG. 36 is a graph showing multispecific binding proteins that induced higher levels of cytotoxicity against tumor target cells by mouse NK cells.

[0050] [Figure 37] Figure 37 shows the binding profile of TriNKET targeting CD33 to NKG2D expressed in EL4 cells. Figure 37 shows the binding of two TriNKETs when the CD33 binding domain is used as the second targeting arm.

[0051] [Figure 38] Figure 38 shows the binding profile of TriNKET targeting HER2 to NKG2D expressed in EL4 cells, showing that the same two NKG2D binding domains were now paired with the second targeting arm of HER2.

[0052] [Figure 39]FIG. 39 is the binding profile of TriNKET targeting BCMA to NKG2D expressed in EL4 cells.

[0053] [Diagram 40] Figure 40 is a histogram of TriNKET targeting CD20 binding to NKG2D expressed in EL4 cells. Unstained EL4 cells were used as a negative control for the fluorescent signal. Unstained: filled; CD20-TriNKET-F04: solid line; CD20-TriNKET-C26: dashed line.

[0054] [Diagram 41] FIG. 41 is the binding profile of CD33-targeted TriNKET to CD33 expressed on MV4-11 human AML cells.

[0055] [Diagram 42] FIG. 42 is the binding profile of HER2-targeting TriNKET to HER2 expressed in human 786-O renal cell carcinoma cells.

[0056] [Diagram 43] FIG. 43 is the binding profile of BCMA-targeted TriNKET to BCMA expressed in MM.1S human myeloma cells.

[0057] [Diagram 44] Figure 44 is a histogram of CD20-targeted TriNKET binding to CD20 expressed in Raji human lymphoma cells. Unstained cells were used as a negative control for the fluorescent signal. Unstained: filled; CD20-TriNKET-F04: solid line; CD20-TriNKET-C26: dashed line.

[0058] [Diagram 45]Figures 45A-45C are bar graphs of synergistic activation of NK cells using CD16 and NKG2D. Figure 45A shows the levels of CD107a, Figure 45B shows the levels of IFNγ, and Figure 45C shows the levels of CD107a and IFNγ. Graphs show the mean (n=2) ± SD. Data are representative of 5 independent experiments using 5 different healthy donors.

[0059] [Diagram 46] Figure 46 is a bar graph showing activation of NK cells using TriNKET targeting NKG2D and CD16. The antibody tested was of human IgG1 isotype. Graph shows mean (n=2)±SD.

[0060] [Figure 47-1] Figures 47A-47C are bar graphs showing that TriNKET and trastuzumab were able to activate primary human NK cells in co-culture with HER2-positive human tumor cells, as indicated by increased CD107a degranulation and IFNγ cytokine production. Compared to the monoclonal antibody trastuzumab, both TriNKETs showed superior activation of human NK cells with various human HER2 cancer cells. Figure 47A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. Figure 47B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. [Figure 47-2] FIG. 47C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells.

[0061] [Figure 48]Figures 48A and 48B are line graphs showing TriNKET-mediated activation of resting human NK cells or IL-2-activated human NK cells in co-culture with the CD33-expressing human AML cell line MV4-11. Figure 48A shows TriNKET-mediated activation of resting human NK cells. Figure 48B shows TriNKET-mediated activation of IL-2-activated human NK cells from the same donor.

[0062] [Figure 49] Figures 49A and 49B are graphs showing TriNKET enhancement of cytotoxic activity using IL-2-activated human NK cells and resting human NK cells. Figure 49A shows the percentage of specific lysis of SkBr-3 tumor cells by resting human NK cells. Figure 49B shows the percentage of specific lysis of SkBr-3 tumor cells by IL-2-activated human NK cells.

[0063] [Figure 50] Figures 50A and 50B are graphs showing that TriNKET provides a greater advantage to HER2-moderate and low cancers compared to trastuzumab. Figure 50A shows activated human NK cell killing of HER2-high SkBr-3 tumor cells. Figure 50B shows human NK cell killing of HER2-low 786-O tumor cells. TriNKET provides a greater advantage to cancer cells with low HER2 expression compared to trastuzumab.

[0064] [Figure 51] Figures 51A-51C are histograms showing expression of high affinity FcRγI (CD64) in three human AML cell lines, the Molm-13 cell line (Figure 51A), the Mv4-11 cell line (Figure 51B), and the THP-1 cell line (Figure 51C).

[0065] [Figure 52] Figures 52A and 52B are line graphs of monoclonal antibody or TriNKET mediated activation of human NK cells in co-culture with either Molm-13 cells (Figure 52B) or THP-1 cells (Figure 52A).

[0066] [Figure 53-1] Figures 53A-53C are line graphs of human NK cytotoxicity assays using three human AML cell lines as targets. Figure 53A shows that Mv4-11 cells, which express CD64 but at lower levels than THP-1, showed reduced efficacy with monoclonal anti-CD33. Figure 53B shows that monoclonal antibodies against CD33 show good efficacy against Molm-13 cells, which do not express CD64. [Figure 53-2] Figure 53C shows that THP-1 cells showed no effect with monoclonal anti-CD33 alone. The same line graphs shown in Figure 53C are also applicable to the line graphs in Figures 53A and 53B.

[0067] [Figure 54-1] Figures 54A and 54B are bar graphs showing that B cells from healthy donors are susceptible to TriNKET-mediated lysis.

[0068] [Figure 54-2] Figures 54C and 54D are bar graphs showing that bone marrow cells are resistant to TriNKET-mediated lysis.

[0069] [Figure 55] FIG. 55 is a line graph of TriNKET-mediated hPBMC killing of SkBr-3 tumor cells in long-term co-culture.

[0070] [Figure 56] FIG. 56 is a flow chart of the efficacy study design of RMA / S-HER2 subcutaneous SC2.2.

[0071] [Figure 57] FIG. 57 is a line graph showing that SC2.2 does not affect subcutaneous RMA / S-HER2 tumor growth.

[0072] [Figure 58] Figures 58A and 58B are graphs showing in vitro binding by mcFAE-C26.99 TriNKET. 4-fold dilutions of the indicated antibodies at 60 μg / mL were added to 2×105 B16F10 tumor cells (Figure 58A) or EL4-mNKG2D cells (Figure 58B). Binding was assessed using a goat anti-mouse PE secondary antibody followed by flow cytometry analysis.

[0073] [Figure 59] FIG. 59 is a graph showing increased NK cytotoxicity mediated by mcFAE-C26.99 TriNKET.

[0074] [Figure 60] Figures 60A and 60B show the antitumor effect of mcFAE-C26.99 TriNKET in the B16F10 sc model. Mice were treated intraperitoneally with (Figure 60A) isotype control mouse IgG2a mab C1.18.4 and mouse anti-Tyrp-1 monoclonal antibody or (Figure 60B) isotype control mouse IgG2a mab C1.18.4 and mcFAE-C26.99 TriNKET, injected at a dose of 150 μg (days 6, 8, 10, 12, 14, 16 and 21). Tumor growth was evaluated for 28 days. Graphs show tumor growth curves of individual mice.

[0075] [Figure 61] Figures 61A and 61B show the antitumor effect of mcFAE-C26.99 TriNKET in the B16F10 iv model. Figure 61A shows the tumor burden when the antibody was administered at a dose of 150 μg (days 4, 6, 8, 11, 13, 15). Figure 61B shows the tumor burden when the antibody was administered at a dose of 150 μg (days 7, 9, 11, 13, 15). 18 days after tumor challenge, mice were euthanized and surface lung metastases were scored.

[0076] [Figure 62]FIG. 62 is a bar graph showing that human NK cells are activated by TriNKET when cultured with CD20+ Raji cells.

[0077] [Figure 63] FIG. 63 is a bar graph showing human NK activation in culture with BCMA-positive MM.1S human myeloma cells.

[0078] [Figure 64] FIG. 64 is a graph showing that TriNKET enhances human NK cell lysis of KMS12-PE myeloma cells.

[0079] [Figure 65] FIG. 65 is a graph showing that TriNKET targeting BCMA with a distinct NKG2D binding domain enhances human NK cell lysis of KMS12-PE myeloma cells.

[0080] [Figure 66] FIG. 66 is a line graph showing that trispecific binding in one molecule is important for maximal NK cell activity.

[0081] [Figure 67] Figure 67 is an Oasc-Fab heterodimer construct containing a Fab that binds target 1 and a scFab that binds target 2 fused to Fc. Heterodimerization is ensured by mutations in the Fc.

[0082] [Figure 68] Figure 68 is a DuetMab, a heterodimeric construct containing two different Fabs that bind antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fabs 1 and 2 contain differential S-S bridges that ensure correct pairing of the LC and HC.

[0083] [Figure 69]Figure 69 is a CrossmAb, a heterodimeric construct with two different Fabs that bind 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.

[0084] [Figure 70] Figure 70 is 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 PREFERRED EMBODIMENTS

[0085] Detailed Description The present invention provides multispecific binding proteins that bind to tumor-associated antigens in cancer cells and NKG2D and CD16 receptors in natural killer cells to activate 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 sections. However, an aspect of the invention described in one particular section is not limited to any particular section.

[0086] To facilitate the understanding of this invention, several terms and phrases are defined below.

[0087] As used herein, the terms "a" and "an" mean "one or more" and include plurals unless the context is inappropriate.

[0088] As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly branched stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" that are interposed between more conserved adjacent 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 positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "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 an 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.

[0089] As used herein, the term "tumor-associated antigen" refers to any antigen, including but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, 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.

[0090] As used herein, the terms "subject" and "patient" refer to an organism that is 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.

[0091] 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, such as improving, decreasing, reducing, modulating, improving, or eliminating a condition, disease, disorder, or the like, or improving symptoms thereof.

[0092] 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 in vivo or ex vivo diagnostic or therapeutic uses.

[0093] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] .

[0094] As used herein, the term "pharmaceutical acceptable salt" refers to any pharmaceutical acceptable salt (e.g., acid or base) of the compound of the present invention that can provide the compound of the present invention or its active metabolite or residue upon administration to a subject. As known to those skilled in the art, the "salt" of the compound 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, are not themselves pharmaceutical acceptable, but can be utilized in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutical acceptable acid addition salts.

[0095] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of the formula NW4 + (Wherein, W is C 1~4 The compounds include those in which the aryl group is an alkyl group.

[0096] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include Na + , NH4+ and NW4 + (Wherein, W is C 1~4 The anions of the compounds of the present invention include those combined with suitable cations such as alkyl groups.

[0097] For therapeutic use, the salts of the compounds of the invention are intended to be pharma- ceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharma- ceutically acceptable compound.

[0098] Throughout the description where compositions are described as having, including, or comprising certain components, 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 components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the recited process steps.

[0099] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of the variable takes precedence. I. Protein

[0100] The present invention provides a multispecific binding protein that binds to tumor-associated antigens on cancer cells and NKG2D and CD16 receptors on natural killer cells to activate the natural killer cells. The multispecific binding protein is useful in the pharmaceutical compositions and treatment methods described herein. Binding of the multispecific binding protein to the NKG2D and CD16 receptors on natural killer cells enhances the activity of the natural killer cells to destroy the cancer cells. Binding of the multispecific binding protein to the tumor-associated antigens on the cancer cells brings the cancer cells into close proximity with the natural killer cells, facilitating direct and indirect destruction of the cancer cells by the natural killer cells. Further description of exemplary multispecific binding proteins is provided below.

[0101] The first component of the multispecific binding protein binds to NK cells, γδ T cells and CD8 + The multispecific binding protein binds to cells expressing the NKG2D receptor, which may include, but is not limited to, αβT cells. Upon binding to NKG2D, the multispecific binding protein may block natural ligands, such as ULBP6 and MICA, from binding to NKG2D.

[0102] The second component of the multispecific binding protein binds to one or more tumor-associated antigens, which may include, but are not limited to, HER2, CD20, CD33, BCMA, EpCAM, CD2, CD19, 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.

[0103] 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.

[0104] The multispecific binding protein can take several formats, including but not limited to those shown in the following examples. One format is a heterodimeric multispecific antibody, 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, a first heavy chain variable domain and, optionally, a first CH1 heavy chain domain. The immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. The 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, which can pair with an immunoglobulin light chain identical to that paired with the first immunoglobulin heavy chain, except that when the immunoglobulin light chain is paired with the second immunoglobulin heavy chain, the resulting antigen-binding site binds to a tumor antigen. The first Fc domain and the second Fc domain together can bind to CD16 (Figure 1).

[0105] Another exemplary format relates to a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, an immunoglobulin light chain and a second immunoglobulin heavy chain. The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, which is fused to a single chain Fv (scFv) that binds to NKG2D, either via a linker or an antibody hinge. Various linkers can be used to link the scFv to the first Fc domain or within the scFv itself. Additionally, the scFv may incorporate mutations that allow for disulfide bond formation to stabilize the overall scFv structure. The scFv may also incorporate mutations to modify the isoelectric point of the entire first immunoglobulin heavy chain and / or to allow easier downstream purification. 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 immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds to tumor antigens. The first Fc domain and the second Fc domain can bind to CD16 together (Figure 2).

[0106] An alternative format of the heterodimeric multispecific protein comprises a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, a first immunoglobulin light 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 light chain variable domain and a light chain constant domain. The first immunoglobulin light chain forms an antigen binding site that binds to the tumor antigen together with the first immunoglobulin heavy chain. 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 light chain variable domain and a light chain constant domain. The immunoglobulin light chain and the second immunoglobulin heavy chain form an antigen binding site that binds to the same tumor antigen. The second immunoglobulin heavy chain can be paired with the immunoglobulin light chain, and this immunoglobulin light chain can be identical to the immunoglobulin light chain that is paired with the first immunoglobulin heavy chain, except that when the immunoglobulin light chain is paired with the second immunoglobulin heavy chain, the resulting antigen binding site is a second antigen binding site that binds to the tumor antigen. In certain embodiments, the first Fc domain and the second Fc domain can be combined to CD16 (Figure 1).

[0107] 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.

[0108] 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.

[0109] In some embodiments, the multispecific binding protein is in the form of a KiH common light chain (LC) that uses knob-into-hole (KIH) technology. KIH is a C-terminal fragment of the C-terminal end ... H The technique involves engineering three domains to create either a "knob" or a "hole" in each heavy chain. The concept behind the "knob-into-hole (KiH)" Fc technology is to engineer a "knob" (i.e., T366W in EU numbering) into 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 a complementary "hole" surface. The "hole" mutations were optimized by structurally-informed 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.

[0110] 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.

[0111] In some embodiments, the multispecific binding proteins are 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 is used to engineer the LC and HC domains in one Fab. VH-CH1 Complementary mutations are introduced only at the interface of Fab, leaving the other Fab unchanged.

[0112] In some embodiments, the multispecific binding protein is in a 2in1 Ig format. In some embodiments, the multispecific binding protein is in an ES form, which is a heterodimeric construct containing two different Fabs that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc. In some embodiments, the multispecific binding protein is in a κλ-Body form, which is a heterodimeric construct with two different Fabs fused to 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 13A is an exemplary diagram of one form of a κλ-Body, and Figure 13B is an exemplary diagram of another κλ-Body.

[0113] In some embodiments, the multispecific binding protein is in Fab arm exchange form (an antibody that has had its Fab arms exchanged by swapping its 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 the LuZ-Y format, which uses a leucine zipper to induce heterodimerization of two different HCs (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

[0114] In some embodiments, the multispecific binding protein is in the form of a Cov-X-Body. In the bispecific CovX-Body, two different peptides are linked together using a branched azetidinone linker and fused to the scaffold antibody in a site-specific manner under mild conditions. The antibody scaffold provides a long half-life and Ig-like distribution, while the pharmacophore is responsible for the functional activity. To generate optimized or unique bispecific antibodies, the pharmacophore can be chemically optimized or replaced with other pharmacophores (Doppalapudi VR et al., PNAS (2010), vol. 107(52); pp. 22611-22616).

[0115] In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer, comprising a Fab that binds target 1 and a scFab that binds target 2, fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

[0116] In some embodiments, the multispecific binding protein is in the DuetMab format, a heterodimeric construct containing two different Fabs that bind antigens 1 and 2, and an Fc stabilized by a heterodimerization mutation. Fab1 and 2 contain unique S-S bridges that ensure correct pairing of the LC and HC.

[0117] In some embodiments, the multispecific binding protein is in a CrossmAb format, which is a heterodimeric construct with two different Fabs that bind 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.

[0118] 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.

[0119] Table 1 lists peptide sequences of heavy and light chain variable domains that can combine to bind to NKG2D. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0120] Alternatively, as described in US 9,273,136, a heavy chain variable domain defined by SEQ ID NO:69 may be paired with a light chain variable domain defined by SEQ ID NO:70 to form an antigen-binding site capable of binding to NKG2D. [ka] [ka]

[0121] Alternatively, as described in US 7,879,985, a heavy chain variable domain defined by SEQ ID NO: 71 may be paired with a light chain variable domain defined by SEQ ID NO: 72 to form an antigen-binding site capable of binding to NKG2D. [ka]

[0122] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly focused on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, vol. 406 (no. 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.

[0123] 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 in 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, US14 / 830336.For example, mutations can be made in the CH3 domain based on human IgG1, incorporating different pairs of amino acid substitutions in the first and second polypeptides, which allow these two chains to selectively heterodimerize with each other. The positions of the exemplified amino acid substitutions below are all numbered according to the EU index, as in Kabat.

[0124] 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 (bulge) fits into the surface of the smaller amino acid substitution (cavity). For example, one polypeptide can incorporate a T366W substitution and the other polypeptide can incorporate three substitutions including T366S, L368A, and Y407V.

[0125] The antibody heavy chain variable domain of the present invention may 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.

[0126] In certain embodiments, mutations that may be incorporated into the 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 the Cκ of the human IgG1 constant region may be amino acids E123, F116, S176, V163, S174, and / or T164.

[0127] Alternatively, the amino acid substitutions may be selected from the set of substitutions set out in Table 2 below. [Table 2]

[0128] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 3 below. [Table 3]

[0129] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 4 below. [Table 4-1] [Table 4-2]

[0130] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 5. [Table 5]

[0131] Alternatively, at least one amino acid substitution may be selected from the set of substitutions in Table 6 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 6]

[0132] Alternatively, at least one amino acid substitution may be selected from the set in Table 7 below, where the position shown in the "First Polypeptide" column is replaced with any known negatively charged amino acid and the position shown in the "Second Polypeptide" column is replaced with any known negatively charged amino acid. [Table 7]

[0133] Alternatively, amino acid substitutions may be selected from the set in Table 8 below. [Table 8]

[0134] 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, resulting in the formation of an artificial disulfide bridge within the interface of the two polypeptides.

[0135] The above multispecific protein can be produced using recombinant DNA technology well known to those skilled in the art.For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector, a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector, and the first, second and third expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0136] In order to achieve the highest yield of multispecific protein, different ratios of the first, second and third expression vectors can be examined 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.

[0137] Clones can be cultured under conditions suitable for bioreactor scale-up and can maintain expression of the multispecific proteins. The multispecific proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. II. Features of TriNKET

[0138] In certain embodiments, the TriNKET described herein, which comprises NKG2D binding domain and binding domain for tumor-associated antigen, binds to the cells expressing human NKG2D.In certain embodiments, the TriNKET, which comprises NKG2D binding domain and binding domain for tumor-associated antigen, binds to tumor-associated antigen at a level comparable to that of monoclonal antibody with the same tumor-associated antigen binding domain.For example, the TriNKET, which comprises NKG2D binding domain and HER2 binding domain from trastuzumab, can bind to HER2 expressed in cells at a level comparable to that of trastuzumab.

[0139] However, TriNKET as described herein is more effective in reducing tumor growth and killing cancer cells. For example, TriNKET of the present disclosure targeting HER2-expressing tumor / cancer cells is more effective than SC2.2, a single-chain bispecific molecule constructed from scFv derived from trastuzumab linked to ULBP-6, a ligand for NKG2D. SC2.2 binds simultaneously to HER2+ cancer cells and NKG2D+ NK cells. Therefore, the efficacy of SC2.2 in reducing the number of HER2+ cancer cells was examined. In vitro activation and cytotoxicity assays showed that SC2.2 is effective in activating and killing NK cells. However, SC2.2 failed to show efficacy in RMA / S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using a RMA / S-HER2 overexpressing syngeneic mouse model. In this mouse model, SC2.2 failed to show tumor growth control compared to vehicle control. 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.

[0140] In certain embodiments, the TriNKET described herein, which comprises an NKG2D binding domain and a binding domain for a tumor-associated antigen, activates primary human NK cells when cultured with tumor cells expressing the antigen. The activation of NK cells is indicated by degranulation of CD107a and increased IFNγ cytokine production. Furthermore, compared to monoclonal antibodies comprising a tumor-associated antigen binding domain, TriNKET exhibits superior activation of human NK cells in the presence of tumor cells expressing the antigen. For example, compared to monoclonal antibody trastuzumab, TriNKET of the present disclosure, which has a HER2 binding domain, has superior activation of human NK cells in the presence of HER2-expressing cancer cells.

[0141] In certain embodiments, the TriNKET described herein, which comprises an NKG2D binding domain and a binding domain for a tumor-associated antigen, enhances 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 exhibit 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.

[0142] In certain embodiments, TriNKETs described herein that contain a binding domain for an NKG2D binding domain and 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, TriNKETs (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that contain a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) more strongly direct activated and resting NK cell responses against tumor cells compared to monoclonal antibodies that contain the same tumor-associated antigen binding site. In certain embodiments, TriNKET presents advantages against tumor cells expressing moderate and low tumor antigens compared with monoclonal antibodies that contain the same tumor antigen binding site.Therefore, treatments that include TriNKET may be superior to monoclonal antibody treatments.

[0143] In certain embodiments, the TriNKETs described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that contain binding domains for tumor-associated antigens (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) are advantageous for treating cancers that highly express Fc receptors (FcRs) or that reside in tumor microenvironments with high levels of FcRs, compared to monoclonal antibodies. Monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blocking, 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 1000-fold stronger than CD16. CD64 is normally expressed in many hematopoietic lineages, such as myeloid lineages, 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, expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to associate with CD16 on the surface of NK cells. TriNKET can overcome the adverse effects of CD64 expression (whether in the tumor or in 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, so TriNKET can mediate human NK cell responses against any tumor cell, regardless of CD64 expression on the tumor cell.

[0144] In some embodiments, TriNKETs described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that contain binding domains for tumor-associated antigens (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) provide a better safety profile due to reduced on-target off-tumor side effects. Although the mechanisms by which natural killer cells and CD8 T cells recognize normal self from tumor cells are different, 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., NCR, NKG2D, CD16) and inhibitory receptors (e.g., KIR, NKG2A). The balance of these activating and inhibitory signals allows NK cells to discriminate healthy self cells from stressed, virus-infected, or transformed self cells. This “built-in” self-tolerance mechanism helps protect normal, healthy tissues from NK cell responses. Extending this principle, NK cell self-tolerance allows TriNKET to target antigens expressed in both self and tumors 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 deliver 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, TriNKET is unique because it does not "disable" the natural systems 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.

[0145] In some embodiments, TriNKETs described herein that contain an NKG2D binding domain (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that contain a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) slow tumor progression more effectively than monoclonal antibodies that contain the same tumor antigen binding domain. In some embodiments, TriNKETs that contain an NKG2D binding domain and a tumor antigen binding domain are more effective against cancer metastasis than monoclonal antibodies that contain the same tumor antigen binding domain.

[0146] The above description describes multiple aspects and embodiments of the invention. The application specifically contemplates all combinations and permutations of the aspects and embodiments. III. Therapeutic applications

[0147] The present invention provides methods for treating cancer using the multispecific binding proteins described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) and / or pharmaceutical compositions described herein that include binding domains for tumor-associated antigens (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2). The methods can be used to treat a variety of cancers, including solid tumors, lymphomas, and leukemias. The type of cancer to be treated desirably matches the type of cancer cells to which the multispecific binding protein binds. For example, treatment of cancers that express epithelial cell adhesion molecule (EpCAM), such as colon cancers that express EpCAM, which bind to the multispecific binding protein, is desirably treated using the multispecific binding proteins described herein. Further aspects and embodiments of the therapeutic methods are described below.

[0148] Thus, one aspect of the present invention provides a method of treating cancer in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) comprising a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2) to treat the cancer. Exemplary cancers for treatment include solid tumors, leukemias, and lymphomas.

[0149] Therapeutic methods may be characterized by the cancer being 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, renal cancer, gastric cancer, testicular cancer, or uterine cancer. In still other embodiments, the cancer is angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary tract cancer, thyroid cancer, or thyroid cancer. Acral lentiginous melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal cancer, anal Cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma papilloma / cell tumor, 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, eye and orbit cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric cardia cancer, gastric fundus cancer, gastrinoma, 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 neoplasia neoplasia), intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, serous papillary adenocarcinoma, penile cancer, pharyngeal cancer, pituitary adenoma tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cell carcinoma, paranasal 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 carcinoma, VIP-secreting tumor, vulvar cancer, well-differentiated carcinoma, or Wilms tumor.

[0150] In certain other embodiments, the cancer is a non-Hodgkin's lymphoma, such as B-cell lymphoma or 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.

[0151] The cancer to be treated may be characterized by the presence of certain antigens expressed on the surface of cancer cells. In certain embodiments, cancer cells express one or more of the following: HER2, CD20, CD33, BCMA, EpCAM, CD2, CD19, 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.

[0152] In certain embodiments, the proteins of the disclosure are used in the manufacture of a medicament for use in a method of directly or indirectly enhancing tumor cell death (synonymous with lysis, killing, ablation, survival or reduced cell proliferation, etc.) or a method of directly or indirectly enhancing tumor cell death (synonymous with lysis, killing, ablation, survival or reduced cell proliferation, etc.) by exposing tumor or cancer cells and natural killer cells to a protein of the disclosure (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that comprises a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2). As described above, tumor cells that respond to the protein express the tumor-associated antigen to which the second antigen-binding site of the protein binds. For example, in an exemplary embodiment, C26-TriNKET-CD20 is used to target CD20-expressing tumor or cancer cells (either resting or activated), and in another exemplary embodiment, C26-TriNKET-BMCA is used to target BMCA-expressing tumor or cancer cells (either resting or activated).

[0153] In certain embodiments, the proteins of the present disclosure are used in a method of treating cancer in a subject in need thereof, or in the manufacture of a medicament for use in a method of treating cancer in a subject in need thereof, comprising administering to the subject a protein or formulation comprising a protein of the present disclosure (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that comprises a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2). As described above, cancer cells that respond to the protein express the tumor-associated antigen to which the second antigen-binding site of the protein binds. For example, in an exemplary embodiment, C26-TriNKET-CD20 is used to target CD20-expressing cancer cells (either resting or activated), and in another exemplary embodiment, C26-TriNKET-BMCA is used to target BMCA-expressing tumor or cancer cells (either resting or activated). IV. Combination Therapy

[0154] Another aspect of the invention provides combination therapy: the multispecific binding proteins described herein are used in combination with additional therapeutic agents to treat cancer.

[0155] 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, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, Included are medicaments such as drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor, and modifications of the above-mentioned agents which may exhibit differential binding to their cognate receptors and increased or decreased serum half-lives.

[0156] Another class of drugs that can be used as part of combination therapy in treating cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include drugs that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3.Ipilimumab, a CTLA4 inhibitor, has been approved by the US Food and Drug Administration to treat melanoma.

[0157] Still other agents that may 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).

[0158] 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 agents, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) inhibitors selected from OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS agonists; and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0159] The proteins of the present invention may also be used as an adjunct to surgical removal of the primary lesion.

[0160] The amount of multispecific binding protein and additional therapeutic agent and the relative timing of administration can be selected to achieve the desired combined therapeutic effect.For example, when administering combined therapy to a patient who needs such administration, the combined therapeutic agent or one or more pharmaceutical compositions containing the therapeutic agent can be administered in any order, such as sequentially, jointly, together, simultaneously, etc.Furthermore, for example, the multispecific binding protein can be administered during the time that the additional therapeutic agent exerts its preventive or therapeutic effect, or vice versa. V. Pharmaceutical Compositions

[0161] The present disclosure also features pharmaceutical compositions containing a therapeutically effective amount of a protein described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET) that includes a binding domain for a tumor-associated antigen (non-limiting examples of tumor-associated antigens include CD20, BCMA, and HER2). The compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition to create a suitable formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th Edition, 1985. For a brief review of methods for drug delivery, see, eg, Langer (Science 249:1527-1533, 1990).

[0162] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing a tube and / or 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 as about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored as about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial.

[0163] 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.

[0164] 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 may be 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, most preferably between 7 and 8, e.g., 7 to 7.5. The resulting solid composition may be packaged in a number of single-dose units, each containing a fixed amount of one or more of the agents described above. The solid composition may also be packaged in flexible-quantity containers.

[0165] 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.

[0166] In certain embodiments, an aqueous formulation is prepared comprising the protein of the present disclosure in a pH buffer solution. The buffer of the present invention may have a pH in the range of about 4 to about 8, e.g., about 4.5 to about 6.0, or about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Intermediate pH ranges 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.

[0167] 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 from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or from 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), 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.

[0168] Polyols that can act as tonicifiers and stabilize the antibodies can also be included in the formulation. The polyol is added to the formulation in an amount that can vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also vary with respect to the molecular weight of the polyol. For example, a small amount of a monosaccharide (e.g., mannitol) can be added compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that can be used in the formulation as an isotonicity agent is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / ml. In certain embodiments, the mannitol concentration can be about 7.5 to 15 mg / ml. In certain embodiments, the mannitol concentration can be about 10 to 14 mg / ml. In certain embodiments, the mannitol concentration can be about 12 mg / ml. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0169] 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 the aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain 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.

[0170] 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 an elastomer conforming to USP and Ph Eur. 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.

[0171] In certain embodiments, the liquid formulation of the present disclosure can be prepared as a 10mg / mL concentration solution combined with sugar at a stabilizing level.In certain embodiments, the liquid formulation 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 can cause undesirable or inappropriate viscosity for intravenous administration.In certain embodiments, the sugar can be a disaccharide, for example, sucrose.In certain embodiments, the liquid formulation can also include one or more of a buffer, a surfactant, and a preservative.

[0172] In certain embodiments, the pH of the liquid formulation can be set by adding a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0173] 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 during sample analysis. Deamidation is the loss of NH3 from a protein forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a mass loss of 17 Daltons of the parent peptide. Subsequent hydrolysis results in a mass gain of 18 Daltons. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Thus, deamidation is typically detectable as a mass gain of 1 Dalton. 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 rate of deamidation. Gly and Ser following Asn in the protein sequence result in higher susceptibility to deamidation.

[0174] 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.

[0175] The aqueous carrier of interest herein is pharma- ceutically acceptable (safe and non-toxic for human administration) and is useful for preparing liquid preparations.Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.

[0176] Preservatives can be added to the formulations herein, if necessary, to reduce bacterial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multiple dose) formulation.

[0177] 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 prior to administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0178] In certain embodiments, salts or buffer components can be added in amounts between 10 mM and 200 mM. The salts and / or buffers are pharma- ceutically 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.

[0179] Preservatives can be added to the formulations herein, if necessary, to reduce bacterial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multiple dose) formulation.

[0180] The aqueous carrier of interest herein is pharma- ceutically acceptable (safe and non-toxic for human administration) and is useful for preparing liquid preparations.Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.

[0181] The present disclosure can also be present as a lyophilized formulation comprising a protein and a lycoprotectant. The lycoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the lycoprotectant can be sucrose or maltose. The lyophilized formulation can include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0182] The amount of sucrose or maltose useful for stabilizing a lyophilized drug product can be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be between 1:2 and 1:5.

[0183] In certain embodiments, the pH of the formulation before lyophilization can be set by adding a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharma-ceutically acceptable base can be sodium hydroxide.

[0184] Prior to lyophilization, the pH of the solution containing the protein of the disclosure may be adjusted to between 6 and 8. In certain embodiments, the pH range for the lyophilized drug product may be 7-8.

[0185] In certain embodiments, salts or buffer components can be added in amounts between 10 mM and 200 mM. The salts and / or buffers are pharma- ceutically 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.

[0186] 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., facilitating the production of an essentially uniform lyophilized cake that maintains an open-pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulation of the present invention can contain such a bulking agent.

[0187] Preservatives can be added to the formulations herein, if necessary, to reduce bacterial action. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multiple dose) formulation.

[0188] In certain embodiments, the lyophilized drug product may be composed of an aqueous carrier.The aqueous carrier of interest herein is one that is pharma- ceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing liquid formulations 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.

[0189] 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.

[0190] 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).

[0191] 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.

[0192] The specific dose may be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, the patient's dose may be adjusted to the approximate weight or surface area of ​​the patient. 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 age, sex, and medical condition of the patient. Further refinement of the calculations required to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially 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 see if 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).

[0193] In general, 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, about 50 mg to about 100 mg / kg body weight.

[0194] Doses may be given once or more per day, once or more per week, once or more per month, or once or more per year, or even once every 2-20 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. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavity, by perfusion via a catheter, or by direct intralesional injection. It may be administered once or more per day, once or more per week, once or more per month, and once or more per year.

[0195] The above description describes multiple aspects and embodiments of the invention. The application specifically contemplates all combinations and permutations of the aspects and embodiments. EXAMPLES

[0196] 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

[0197] Nucleic acid sequences of human, mouse or cynomolgus NKG2D extracellular domain were fused to a nucleic acid sequence encoding a human IgG1 Fc domain and introduced into mammalian cells for expression. After purification, the NKG2D-Fc fusion protein was adsorbed to the wells of a microplate. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D binding domain was titrated and 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 and specifically recognizing the human kappa light chain to avoid Fc cross-reaction. 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.

[0198] The isotype control showed little binding to recombinant NKG2D-Fc protein, while the positive control bound most strongly to the recombinant antigen. Although the affinity varied from clone to clone, the NKG2D binding domains produced by all clones showed binding to all human (Figure 14), mouse (Figure 16), and cynomolgus (Figure 15) recombinant NKG2D-Fc proteins. In general, each anti-NKG2D clone bound with similar affinity to human (Figure 14) and cynomolgus (Figure 15) recombinant NKG2D-Fc, but relatively low affinity to mouse (Figure 16) recombinant NKG2D-Fc. The NKG2D-binding domain binds to cells expressing NKG2D

[0199] EL4 mouse lymphoma cell lines were 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 concentration 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.

[0200] The NKG2D binding domains produced by all clones bound to EL4 cells expressing human and mouse NKG2D. The positive control antibody (selected from SEQ ID NOs: 45-48, or anti-mouse NKG2D clones MI-6 and CX-5 available at eBioscience) gave the best FOB binding signal. The NKG2D binding affinity of each clone was similar between cells expressing human NKG2D (Figure 17) and cells expressing mouse NKG2D (Figure 18) (Figures 17-18, respectively). Example 2 The NKG2D-binding domain blocks binding of natural ligands to NKG2D Competition with ULBP-6

[0201] Recombinant human NKG2D-Fc protein was adsorbed to the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by the addition of the NKG2D-binding domain clone. After 2 hours of incubation, the wells were washed, and ULBP-6-His-biotin that remained bound to the NKG2D-Fc-coated wells was detected by streptavidin conjugated with horseradish peroxidase and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, the specific binding of the NKG2D-binding domain to the NKG2D-Fc protein was calculated from the percentage of 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, while an isotype control showed little competition with ULBP-6 (Figure 19). Conflict with MICA

[0202] Recombinant human MICA-Fc protein was adsorbed to the wells of a microplate, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the NKG2D binding domain. After incubation and washing, NKG2D-Fc-biotin 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 that was 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. 20). Conflict with Rae-1 Delta

[0203] Recombinant mouse Rae-1 delta-Fc (purchased from R&D Systems) was adsorbed to the wells of the microplate, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the addition of the NKG2D binding domain. After incubation and washing, the NKG2D-Fc-biotin that remained bound to the Rae-1 delta-Fc-coated wells was detected 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 that was 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 an isotype control antibody showed little competition with Rae-1 delta (Figure 21). Example 3 NKG2D-binding domain clones activate NKG2D

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

[0205] To determine whether the NKG2D binding domains activate NKG2D, they were adsorbed to microplate wells and NKG2D-CAR EL4 cells were cultured in the presence of brefeldin-A and monensin in the antibody fragment-coated wells for 4 hours. Intracellular TNF-alpha production, an indicator of NKG2D activation, was assayed by flow cytometry. The percentage of TNF-alpha positive cells was normalized to cells treated with a positive control. All NKG2D binding domains activated both human NKG2D (Figure 22) and mouse NKG2D (Figure 23). Example 4 The NKG2D-binding domain activates NK cells Primary human NK cells

[0206] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated from the NK cells. The purity of isolated NK cells was typically >95%. The isolated NK cells were then cultured for 24–48 h in medium containing 100 ng / mL IL-2, after which they were transferred to wells of a microplate adsorbed with NKG2D binding domain and cultured in medium containing fluorophore-conjugated anti-CD107a antibody, brefeldin-A, and monensin. After culture, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD3 - CD56 + NK cell activation was assessed by analyzing the staining of CD107a and IFN-gamma in the cells. An increase in CD107a / IFN-gamma double positive cells indicates better NK cell activation due to engagement of two activating receptors instead of 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 IFN-gamma + (FIG. 24 and FIG. 25 represent two independent experiments, each using PBMCs from a different donor for the preparation of NK cells). Primary mouse NK cells

[0207] 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 and then harvested and prepared for NK cell isolation. NK cells (CD3 - NK1.1 + ) were isolated. Purified NK cells were cultured for 48 hours in medium containing 100 ng / mL mIL-15, then transferred to wells of microplates adsorbed with NKG2D binding domains and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin-A, and monensin. After culture in wells coated with NKG2D binding domains, NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-gamma. CD3 - NK1.1 + NK cell activation was assessed by analyzing the staining of CD107a and IFN-gamma in the cells. An increase in CD107a / IFN-gamma double positive cells indicates better NK cell activation due to engagement of two activating receptors instead of one. The NKG2D binding domain and positive control (selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) showed that a higher percentage of NK cells express CD107a than the isotype control. + and IFN-gamma +(FIG. 26 and FIG. 27 are representative of two independent experiments, each using a different mouse for the preparation of NK cells). Example 5 The NKG2D-binding domain enables cytotoxicity of targeted tumor cells

[0208] Human and mouse primary NK cell activation assays show increased cytotoxic 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 is 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 at 4°C. 5 The labeled THP-1 cells were then combined with NKG2D antibody and mouse NK cells were isolated in the wells of a microtiter plate at 37°C for 3 hours. After incubation, 20 μl of 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 by 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.

[0209] The positive control ULBP-6, a natural ligand for NKG2D, showed an increase in the rate of specific lysis of THP-1 target cells by mouse NK cells. NKG2D antibodies also increased the rate of specific lysis of THP-1 target cells, whereas an isotype control antibody showed a decrease in the rate of specific lysis. The dotted line shows the rate of specific lysis of THP-1 cells by mouse NK cells without added antibody (Figure 28). Example 6 NKG2D antibodies exhibit high thermal stability

[0210] The melting temperature of the NKG2D binding domain was assayed using differential scanning fluorimetry, and the extrapolated apparent melting temperature is higher compared to a typical IgG1 antibody (FIG. 29). Example 7 Multispecific binding proteins exhibit enhanced ability to activate NK cells

[0211] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood buffy coats using density gradient centrifugation. NK cells (CD3 - CD56 + ) were isolated from the NK cells. The purity of the isolated NK cells was typically >95%. The isolated NK cells were then cultured for 24–48 h in medium containing 100 ng / mL IL-2, after which they were transferred to wells of microplates adsorbed with polyspecific and bispecific binding proteins, respectively, and cultured in medium containing fluorophore-conjugated anti-CD107a antibodies, brefeldin-A, and monensin. After culture, the NK cells were assayed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD3 - CD56 + The staining of CD107a and IFN-gamma in cells was analyzed to evaluate the activation of NK cells. An increase in CD107a / IFN-gamma double positive cells indicates better activation of NK cells. 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 reaction (cFAE) starting from trastuzumab homodimer and ULBP-6-Fc homodimer (see Labrijn et al., Nature Protocols 9:2450-2463). SC2.2 is a single chain protein containing scFv derived from trastuzumab and ULBP-6 (SEQ ID NO:73). [ka]

[0212] Analysis of CD107a and IFN-gamma staining demonstrated that isotype control IgG did not demonstrate NK cell activation, whereas after stimulation with the multispecific binding protein, a higher percentage of NK cells expressed CD107a compared with the bispecific protein. + and IFN-gamma + It has been shown that engagement of two activating receptors (NKG2D and CD16) rather than just one (NKG2D) results in more potent NK cell activation (Figure 30). 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

[0213] 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 >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 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 100 µL / mL. Various dilutions of the multispecific binding protein were made in culture medium. Then, the NK cells, the labeled SkBr-3 cells and the multispecific binding protein were combined in the wells of a microtiter plate and incubated at 37 °C for 3 h. 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 min. Fluorescence was measured over time by 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'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 generated by a controlled Fab arm exchange reaction (cFAE) starting from a trastuzumab homodimer and an 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 that contains a HER2 binding domain (trastuzumab), an NKG2D binding domain (ULBP-6) and a human IgG1 Fc domain. SC2.2 is a single-chain protein that contains the scFv derived from trastuzumab and ULBP-6.

[0214] AL0.2 showed enhanced lysis of SkBr-3 target cells by human NK cells over trastuzumab in a dose-dependent manner, with a p-value of 0.0311 at EC50 (Figure 31). AL0.2si (Figure 32) and trastuzumab-si (Figure 33) showed a reduction in both potency and maximum specific lysis rate of SkBr-3 cells compared to AL0.2, with p-values ​​of 0.0002 and 0.0001 at EC50, respectively (Figures 32-33). Furthermore, AL0.2 showed enhanced lysis of SkBr-3 cells over AL2.2 in a dose-dependent manner (Figure 34). Isotype control IgG did not show an increase in specific lysis rate at any of the concentrations tested. The combined data demonstrated 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 mouse NK cells

[0215] 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 and then harvested and prepared for NK cell isolation. NK cells (CD3 - NK1.1 + ) were isolated. Purified NK cells were cultured in medium containing 100 ng / mL mIL-15 for 48 h and then cultured at 10 6The mouse tumor cell line RMA-HER2-dTomato, engineered to express HER2 and dTomato, and its control counterpart, RMA cells expressing zsGreen, were used as targets. They were resuspended in culture medium at 2 × 10 5 The cells were resuspended in culture medium at 100 / mL and seeded in a microplate well 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 control group / (% of RMA-Ca2T-dTomato cells in control group) * % of RMA-zsGreen cells in treatment groups * 100%.

[0216] AL2.2 is more potent in redirecting NK cell responses against tumor targets than SC2.2 (Figure 36) and trastuzumab (Figure 35). Control proteins were shown to have little effect on specific target killing. These data show that multispecific binding proteins that associate with two activating receptors and one tumor antigen on NK cells induce more potent killing of tumor cells by mouse NK cells compared to bispecific proteins that associate with one activating receptor and one tumor antigen on NK cells. Example 9 Multispecific binding proteins bind to NKG2D

[0217] As shown in Figure 1, EL4 mouse lymphoma cell lines were engineered to express human NKG2D trispecific binding proteins (TriNKETs), each of which contains an NKG2D-binding domain, a tumor-associated antigen-binding domain (such as a CD33-binding domain, a HER2-binding domain, a CD20-binding domain, or a BCMA-binding domain), and an Fc domain that binds to CD16, and tested for their affinity to extracellular NKG2D expressed in EL4 cells. 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 the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells was used to calculate fold over background (FOB).

[0218] The TriNKETs tested included CD33-TriNKET-C26 (ADI-28226 and CD33 binding domain), CD33-TriNKET-F04 (ADI-29404 and CD33 binding domain), HER2-TriNKET-C26 (ADI-28226 and HER2 binding domain), HER2-TriNKET-F04 (ADI-29404 and HER2 binding domain), and CD20-TriNKET-C26 (ADI-28226 and CD20 binding domain). The HER2 binding domain used in the tested molecules consisted of the heavy and light chain variable domains of trastuzumab. The CD33 binding domain consisted of the heavy and light chain variable domains listed below. [ka] The CD20 binding domain used in the tested molecules was composed of a heavy chain variable domain and a light chain variable domain. The BCMA binding domain used in the tested molecules was composed of a heavy chain variable domain and a light chain variable domain as listed below. EM-801 Heavy Chain Variable Domain (SEQ ID NO:82): [ka] EM-801 Light Chain Variable Domain (SEQ ID NO:83): [ka] EM-901 Heavy Chain Variable Domain (SEQ ID NO:76) [ka] EM-901 Light Chain Variable Domain (SEQ ID NO:77) [ka]

[0219] The data show that TriNKET of the present disclosure binds to NKG2D when the protein contains a tumor antigen binding domain such as CD33, HER2, CD20, and BCMA. Example 10 Multispecific binding proteins bind to human tumor antigens Trispecific binding protein binds to CD33, HER2, CD20 and BCMA

[0220] The CD33-expressing human AML cell line MV4-11 was used to assay binding of TriNKET to tumor-associated antigens. TriNKET and parental anti-CD33 monoclonal antibodies 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 parental monoclonal anti-CD33 antibodies normalized to the secondary antibody control. CD33-TriNKET-C26, and CD33-TriNKET-F04 show comparable levels of binding to CD33 compared to the parental anti-CD33 antibody (Figure 41).

[0221] Binding of TriNKET to tumor-associated antigens was assayed using human cancer cell lines expressing HER2. 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 42).

[0222] Binding of TriNKET to the tumor-associated antigen BCMA was assayed using MM.1S human myeloma cells expressing BCMA. TriNKET and, where appropriate, a parent anti-BCMA monoclonal antibody (EM-801) 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 EM-801 normalized to the secondary antibody control. C26-TriNKET-BCMA, F04-TriNKET-BCMA, F43-TriNKET-BCMA, and F47-TriNKET-BCMA show comparable levels of binding to BCMA expressed in MM.1S cells compared to EM-801 (Figure 43).

[0223] The binding of TriNKET to the tumor-associated antigen CD20 was assayed using Raji human lymphoma cells expressing CD20. TriNKET was incubated with the cells and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. The cells were analyzed by flow cytometry and histograms were plotted. As shown in Figure 44, CD20-TriNKET-C26 and CD20-TriNKET-F04 bind equally well to CD20. Example 11 Multispecific binding proteins activate NK cells

[0224] 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 were rested overnight without cytokines. IL-2-activated NK cells were used within 24–48 h after activation.

[0225] Human cancer cells expressing tumor antigens were harvested and cultured at 2 × 10 6 Activated NK cells were harvested, washed, and resuspended in culture medium at 2 × 10 6 The tumor cells were then mixed with 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 IFN-gamma. CD107a and IFN-gamma staining was performed using CD3 - CD56 + NK cell activation was assessed by analysis of cells. An increase in CD107a / IFN-gamma double positive cells indicates better NK cell activation due to engagement of two activating receptors rather than one receptor.

[0226] TriNKET mediates activation of human NK cells co-cultured with HER2-expressing SkBr-3 cells (Figure 47A), Colo201 cells (Figure 47B), and HCC1954 cells (Figure 47C), respectively, as shown by increased CD107a degranulation and IFN-gamma production. SkBr-3 and HCC1954 cells express high levels of surface HER2, while Colo201 expresses moderate levels of HER2. Compared to the monoclonal antibody trastuzumab, TriNKET shows superior activation of human NK cells in the presence of human cancer cells. NK cells alone and NK cells + SkBr-3 cells are used as negative controls.

[0227] TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) mediated activation of human NK cells co-cultured with CD33-expressing human AML Mv4-11 cells, which showed increased CD107a degranulation and IFN-gamma production. Compared to monoclonal anti-CD33 antibodies, TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) showed superior activation of human NK cells in the presence of HER2-expressing human cancer cells (Figures 47A-47C). Primary human NK cells are activated by TriNKET in co-culture with target-expressing human cancer cell lines

[0228] Co-culturing primary human NK cells with CD20-positive human cancer cells resulted in TriNKET-mediated activation of primary human NK cells (Figure 62). TriNKETs targeting CD20 (e.g., C26-TriNKET-CD20 and F04-TriNKET-CD20) mediated activation of human NK cells co-cultured with CD20-positive Raji cells, as shown by increased CD107a degranulation and IFNγ cytokine production (Figure 62). Compared to the monoclonal antibody rituximab, both TriNKETs (e.g., C26-TriNKET-CD20 and F04-TriNKET-CD20) showed superior activation of human NK cells (Figure 62). [ka]

[0229] Co-culture of primary human NK cells with BCMA-positive MM.1S myeloma cells resulted in TriNKET-mediated activation of primary human NK cells. TriNKET targeting BCMA (e.g., C26-TriNKET-BMCA and F04-TriNKET-BMCA) mediated activation of human NK cells co-cultured with MM.1S myeloma cells, as shown by increased CD107a degranulation and IFNγ cytokine production (Figure 63). Compared to isotype TriNKET, TriNKET targeting BCMA (e.g., A44-TriNKET-BMCA, A49-TriNKET-BMCA, C26-TriNKET-BMCA, F04-TriNKET-BMCA, F43-TriNKET-BMCA, F43-TriNKET-BMCA, F47-TriNKET-BMCA, and F63-TriNKET-BMCA) showed increased NK cell activity (Figure 63). Example 12 Trispecific binding proteins enable cytotoxicity of targeted cancer cells

[0230] 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 or resting NK cells were used in cytotoxicity assays the following day.

[0231] To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, the cyto Tox 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 incubated at 1–2 × 10 5Resting and / or activated NK cells were harvested, washed, and resuspended in the same culture medium as the cancer cells at 10 5 ~2.0×10 6 The cells were resuspended at 100 / 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 targeting tumor antigens expressed in the cancer cells. After 3 hours and 15 minutes of incubation at 37° C. and 5% CO2, 10× lysis buffer was added to wells containing only cancer cells and to wells containing only medium for maximum lysis and negative reagent control, respectively. The plate was then returned to the incubator for another 45 minutes to achieve a total incubation of 4 hours. The cells were then 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 the absorbance was read at 492 nm on a SpectraMax i3x. The percentage of 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)) x 100%.

[0232] TriNKET mediates the cytotoxicity of human NK cells against the CD33-positive Molm-13 human AML cell line. As shown in FIG. 53B, when resting human NK cells are mixed with Molm-13 cancer cells, TriNKET (e.g., C26-TriNKET-CD33 and F04-TriNKET-CD33) can enhance the cytotoxic activity of resting human NK cells against cancer cells in a dose-responsive manner. The dotted line shows the cytotoxic activity of resting NK cells without TriNKET. When activated human NK cells are mixed with Molm-13 cancer cells, TriNKET enhances the cytotoxic activity of activated human NK cells against cancer cells in a dose-responsive manner even more than anti-CD33 antibody (FIG. 53B).

[0233] TriNKET enhances the cytotoxicity of NK cells against targets with low surface expression compared to the cytotoxic activity of trastuzumab, an anti-HER2 monoclonal antibody. 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 Figure 50A and Figure 50B show the cytotoxic activity of resting NK cells against cancer cells in the absence of TriNKET. As shown in Figure 50B, when activated human NK cells were mixed with low HER2-expressing 786-O cells, and TriNKET (e.g., CD26-TriNKET-HER2 and F04-TriNKET-HER2), a dose-responsive cytotoxic activity of activated human NK cells against cancer cells was observed.

[0234] TriNKET-mediated lysis of BCMA-positive myeloma cells was assayed. Figure 64 shows TriNKET-mediated lysis of BCMA-positive KMS12-PE myeloma cells by resting human NK effector cells. Two TriNKETs (cFAE-A49.801 and cFAE-A49.901) using the same NKG2D binding domain (A49) but different BCMA-targeting domains were tested for efficacy in vitro. Both TriNKETs enhanced NK cell lysis of KMS12-PE cells to a similar extent, but TriNKET using the EM-901-targeting domain provided increased potency.

[0235] Figure 65 shows the cytotoxic activity of several TriNKETs using different NKG2D binding domains (A40, A44, A49, C26, and F47) but the same BCMA-targeting domain. Varying the NKG2D binding domain of the BCMA-targeting TriNKETs resulted in variation in the maximum killing and potency of the TriNKETs. All TriNKETs showed increased killing of KMS12-PE target cells compared to the EM-901 monoclonal antibody (Figure 65). Example 13

[0236] We investigated the synergistic activation of human NK cells by cross-linking NKG2D and CD16. Primary human NK cell activation assay

[0237] 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 - CD56 + The cells were then grown for 48 hours in medium containing 100 ng / mL hIL-2 (Peprotech #200-02) before being used for 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 wells to remove excess antibody. For assessment of degranulation, IL-2-activated NK cells were cultured at 5 × 10 in culture medium supplemented with 100 ng / mL hIL2 and 1 μg / mL APC-conjugated anti-CD107a mAb (Biolegend #328619). 5 The cells were then resuspended at 1 × 10 5 Cells / well were added onto the antibody-coated plates. Protein transport inhibitors brefeldin A (BFA, Biolegend #420601) and monensin (Biolegend #420701) were added at a final dilution of 1:1000 and 1:270, respectively. 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 stained with live CD56 + CD3 -After gating on cells, they were analyzed for CD107a and IFN-γ expression by flow cytometry.

[0238] 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 45 (Figures 45A-45C), combined stimulation of CD16 and NKG2D resulted in a large increase in CD107a (degranulation) levels (Figure 45A) and / or IFN-γ production levels (Figure 45B). The dotted lines represent the additive effect of individual stimulation of each receptor.

[0239] 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 45A shows the levels of CD107a, Figure 45B shows the levels of IFNγ, and Figure 45C shows the levels of CD107a and IFN-γ production. Data shown in Figures 45A-45C are representative of five independent experiments using five different healthy donors.

[0240] CD107a degranulation and intracellular IFN-γ production of IL-2-activated NK cells were analyzed after 4 hours of plate-bound stimulation with trastuzumab, anti-NKG2D, or TriNKET derived from the binding domains of trastuzumab and anti-NKG2D antibodies (Figure 46). In all cases, the antibodies tested were of human IgG1 isotype. Graphs show the mean (n=2) ± SD. Example 14 Assessment of TriNKET binding to human NKG2D expressed by cells

[0241] 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. mAb or TriNKET dilutions were used to stain cells, and a fluorophore-conjugated anti-human IgG secondary antibody was used to detect binding of TriNKET or mAb. Cells were analyzed by flow cytometry, and binding MFI was normalized to secondary antibody control to obtain fold over background values. Assessment of TriNKET binding to human cancer antigens expressed by cells

[0242] Human cancer cell lines expressing either CD33 or HER2 were used to evaluate tumor antigen binding of TriNKET derived from different NKG2D-targeting clones. The human AML cell line MV4-11 was used to evaluate TriNKET binding to cell-expressed CD33. 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 the respective cells. TriNKET binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and binding MFI to cell-expressed CD33 and HER2 was normalized to secondary antibody control to obtain fold over background values. Determination of antibody binding capacity of human HER2-positive cancer cell lines

[0243] Antibody binding capacity (ABC) of HER2 positive human cancer cell lines was measured. Antibody-labeled beads were prepared using the Quantum Simply Cellular kit from Bangs Lab (#815) according to the manufacturer's instructions. Briefly, each of the four populations of beads were stained with saturating amounts of anti-HER2 antibody and the cell population was stained with saturating amounts of the same antibody. Sample data were acquired for each bead population, and for the cell population. The QuickCal worksheet provided with the kit was used to generate standard curves and extrapolate the ABC values ​​for each of the cell lines. Activation of primary NK cells by TriNKET

[0244] 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 for activation or rested overnight without cytokines. IL-2-activated NK cells were used after 24–48 h. Resting NK cells were always used the day after purification.

[0245] Human cancer cell lines expressing the cancer target of interest were harvested from culture and cells were cultured at 2 × 10 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 / mL. IL-2 and fluorophore-conjugated anti-CD107a were added to the NK cells for activation cultures. Brefeldin-A and monensin were diluted in culture medium to block protein export from the cells for intracellular cytokine staining. To a 96-well plate, 50 μl of tumor targets, mAb / TriNKET, BFA / monensin, and NK cells were added in a total culture volume of 200 μl. Plates were cultured for 4 hours before samples were prepared for FACS analysis.

[0246] After 4 hours of 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. Cytotoxicity assay of primary human NK cells

[0247] 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. The following day, IL-2-activated or resting NK cells were used for cytotoxicity assays. Cyto Tox96 LHD Release Assay:

[0248] 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, the cells were 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 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 diluted mAb or TriNKET was added to each well. Resting and / or activated NK cells were harvested from the cultures, the cells were washed and resuspended in culture medium at 10 / mL 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. Plates were incubated at 37° C. with 5% CO2 for 3 hours and 15 minutes. After incubation, 10× lysis buffer was added to the target cell only wells and to wells containing media only for maximum lysis and volume adjustment. Plates were then returned to the incubator for an additional 45 minutes for a total of 4 hours of incubation before color development.

[0249] After incubation, the plates were removed from the incubator and the cells were pelleted by centrifugation at 200g for 5 min. 50 μl of culture supernatant was transferred to a clean microplate and 50 μl of substrate solution was added to each well. The plates were protected from light and incubated at room temperature for 30 min. 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:

[0250] Human cancer cell lines expressing the target of interest were harvested from culture, the cells were washed with PBS, and incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with PBS and resuspended in growth medium 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 the wells in triplicate to avoid disturbing the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Wells were reserved for spontaneous release from the target cells and wells were prepared for maximum lysis of the target cells by the addition of 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target 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 cultures, the cells were washed and 10 μl of culture medium was added 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 with 5% CO2 for 2-3 hours before the assay was developed.

[0251] After 2-3 hours of incubation, plates were removed from the incubator and cells were pelleted by centrifugation at 200g for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate provided by the manufacturer and 200 μl of room temperature europium solution was added to each well. Plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. Plates were read using either a 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:

[0252] SkBr-3 target cells were labeled with BacMam 3.0 NucLight Green (#4622) to allow tracking of target cells. 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 already labeled SkBr-3 cells. 50 ul of complete culture medium was added for a total culture volume of 200 μl.

[0253] Image collection was set up in an IncuCyte S3. Images for phase, green, and red channels were collected every hour, with two images per well. Image analysis was performed using the IncuCyte S3 software. Masks for the green and red channels were created 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 overlapping objects) / (number of green objects)). * 100%. Comparison of TriNKET and SC2.2 targeting HER+ cancer cells

[0254] 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. TriNKET of the present disclosure targeting HER2-expressing tumor / cancer cells is more effective than SC2.2, a single-chain bispecific molecule constructed from scFv derived from trastuzumab linked to ULBP-6, a ligand for NKG2D. SC2.2 binds simultaneously 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 showed that SC2.2 was effective in activating and killing NK cells. However, SC2.2 failed to show efficacy in the RMA / S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using a RMA / S-HER2 overexpressing syngeneic mouse model. In this mouse model, SC2.2 failed to show tumor growth control compared to vehicle controls. Thus, although SC2.2 can activate and kill NK cells and bind to HER2+ cancer cells, these properties were insufficient to effectively control HER2+ tumor growth. Assessment of SC2.2 serum half-life in C57Bl / 6 mice

[0255] 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, 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.

[0256] 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 was 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

[0257] To test the efficacy of SC2.2 against subcutaneous RMA / S-HER2 tumors, an in vivo study was designed according to Figure 56. 6 RMA / S cells were injected subcutaneously into the flank of 20 C57Bl / 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 for the duration of the study. Tumor volume was calculated using the following formula: tumor volume = length x width x height. TriNKET binds to cells expressing human NKG2D

[0258] The ability of TriNKET to bind to cells expressing human NKG2D was determined. Figure 37 and Figure 38 show the dose-response binding of two TriNKETs that contain different NKG2D binding domains. Figure 37 shows the binding of two TriNKETs when CD33 binding domain is used as the second targeting arm. Figure 38 shows that the same two NKG2D binding domains are now paired with the second targeting arm of HER2. The six NKG2D binding domains retain the same binding profile as both tumor targeting domains. TriNKET binds to cells expressing human cancer antigens

[0259] The ability of TriNKET to bind to cells expressing human cancer antigens was determined. Figures 41 and 42 show the binding of TriNKET to CD33 (Figure 41) and HER2 (Figure 42) expressed by cells. TriNKET binding to antigens expressed by cells was consistent across the NKG2D binding domain. TriNKET bound at a level comparable to the parent monoclonal antibody. Antibody binding ability of human HER2-positive cancer cell lines

[0260] Table 9 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 (+).

[0261] [Table 9] Primary human NK cells are activated by TriNKET in co-culture with human cancer lines expressing various levels of HER2

[0262] Figures 47A-47C show that TriNKET and trastuzumab were able to activate primary human NK cells in co-culture with HER2-positive human tumor cells, as indicated by increased CD107a degranulation and IFNγ cytokine production. Compared to the monoclonal antibody trastuzumab, both TriNKETs showed superior activation of human NK cells with various human HER2 cancer cells.

[0263] Figure 47A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. Figure 47B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. Figure 47C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells. TriNKET enhances the activity of resting and IL-2-activated human NK cells

[0264] Figures 48A-48B show TriNKET-mediated activation of resting human NK cells or IL-2-activated human NK cells in co-culture with the CD33-expressing human AML cell line MV4-11. Figure 48A shows TriNKET-mediated activation of resting human NK cells. Figure 48B shows TriNKET-mediated activation of IL-2-activated human NK cells from the same donor. Resting NK cells showed less background IFNγ production and CD107a degranulation than IL-2-activated NK cells. Resting NK cells showed greater changes in IFNγ production and CD107a degranulation compared to IL-2-activated NK cells. IL-2-activated NK cells showed a greater percentage of cells becoming IFNγ+;CD107a after stimulation with TriNKET. TriNKET enhances the cytotoxicity of resting and IL-2-activated human NK cells

[0265] Figures 49A-49B show TriNKET enhancement of cytotoxic activity using IL-2-activated and resting human NK cells. Figure 49A shows the percentage of specific lysis of SkBr-3 tumor cells by resting human NK cells. Figure 49B shows the percentage of 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 to trastuzumab, TriNKET more strongly directs the response against SkBr-3 cells by either activated or resting NK cell populations. TriNKET enhances NK cell cytotoxicity against targets with low surface expression

[0266] Figures 50A-50B show that TriNKET provides greater benefit against HER2-moderate and low cancers compared to trastuzumab. Figure 50A shows activated human NK cell killing of HER2-high SkBr-3 tumor cells. Figure 50B shows human NK cell killing of HER2-low 786-O tumor cells. TriNKET provides greater benefit against cancer cells with low HER2 expression compared to trastuzumab. TriNKET provides greatest benefit against targets with low surface expression. The benefits of TriNKET in treating cancers with high FcR expression or in tumor microenvironments with high levels of FcR

[0267] Monoclonal antibody therapy has been approved for the treatment of many cancer types, including both hematological and solid tumors. The use of monoclonal antibodies in cancer treatment has improved patient outcomes but still has limitations. Mechanistic studies have shown that monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blocking, among others.

[0268] Most notably, ADCC is believed to be the primary mechanism by which monoclonal antibodies exert their effects. ADCC depends on antibody-Fc engagement of the low affinity FcγRIII (CD16) on the surface of natural killer cells, which mediates direct lysis of tumor cells. Among the FcγRs, CD16 has the lowest affinity for IgG Fc, while FcγRI (CD64) is a high affinity FcR that binds approximately 1000-fold more tightly to IgG Fc than CD16.

[0269] 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, are also known to express CD64 and 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 effect of CD64 expression on monoclonal antibody therapy. FcRγI (CD64) expression in three AML cell lines

[0270] An in vitro culture system was developed to test the activity of TriNKET and monoclonal antibodies against tumors that surface express high and low levels of CD64. Molm-13 and THP-1 are two human AML cell lines that similarly express surface CD33, but Molm-13 cells do not express CD64, whereas THP-1 cells express CD64 on their surface (Figures 51A-51C). Using monoclonal antibodies or TriNKET directed to target CD33, the effect of tumor CD64 expression on monoclonal antibody or TriNKET therapy was tested. Figures 51A-51C show the expression of high affinity FcRγI (CD64) in three human AML cell lines: Molm-13 cell line (Figure 51A), Mv4-11 cell line (Figure 51B), and THP-1 cell line (Figure 51C). Molm-13 cells do not express CD64, while Mv4-11 cells have low levels of cell surface CD64 and THP-1 have high levels of cell surface CD64. TriNKET is advantageous for targeting tumor cells with high surface expression of FcR

[0271] Figures 52A-52B show monoclonal antibody or TriNKET-mediated activation of human NK cells in co-culture with either Molm-13 cells (Figure 52B) or THP-1 cells (Figure 52A). Monoclonal antibodies against human CD33 showed good activation of human NK cells in the Molm-13 co-culture system as evidenced by increased CD107a degranulation and IFNγ production. Monoclonal antibodies have no effect in the THP-1 co-culture system where high levels of CD64 are present in the tumor. Interestingly, although TriNKET was effective against both Molm-13 cells (Figure 52B) and THP-1 cells (Figure 52A), the monoclonal antibodies were unable to activate NK cells in culture with FcR-Hi THP-1 cells, indicating that TriNKET can overcome binding to CD64 in the tumor and effectively target NK cells for activation. Dual targeting of two activating receptors in NK cells resulted in stronger specific binding to NK cells. Monoclonal antibodies that target only CD16 on NK cells can bind to other high affinity FcRs and block engagement of CD16 on NK cells.

[0272] Cytotoxicity assays of human NK cells using Molm-13 and THP-1 co-culture systems provide further evidence supporting the efficacy of TriNKET in the presence of high levels of CD64. In these cytotoxicity assays, a third human AML cell line, Mv4-11, was used. Mv4-11 cells (Figure 51B) express low levels of CD64 and are between THP-1 cells (Figure 51C) and Molm-13 cells (Figure 51A) in terms of the levels of CD64 on their surface. TriNKET demonstrates efficacy against AML cell lines regardless of FcγRI expression

[0273] Figures 53A-53C show human NK cytotoxicity assays using three human AML cell lines as targets. Monoclonal antibodies against CD33 show good efficacy against Molm-13 cells, which do not express CD64 (Figure 53B). Mv4-11 cells (Figure 53A), which express CD64 but at lower levels than THP-1, showed reduced efficacy with monoclonal anti-CD33. THP-1 cells (Figure 53C) showed no effect with monoclonal anti-CD33 alone. Regardless of CD64 expression in the tumor cells, TriNKET was able to mediate human NK cell responses against all tumor cells tested here.

[0274] Figures 53A-53C show that THP-1 cells were protected against monoclonal antibody therapy due to high levels of high affinity FcR expression on their surface. TriNKET circumvented this protection by targeting two activating receptors on the surface of NK cells. The cytotoxicity data directly correlated with those seen in the co-culture activation experiments. TriNKET was able to circumvent the protection from mAb therapy seen with THP-1 cells and was able to induce NK cell-mediated lysis despite high levels of FcR. 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

[0275] 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 different. NK cell activity is regulated by the balance of signals from activating receptors (NCR, NKG2D, CD16, etc.) and inhibitory receptors (KIR, NKG2A, etc.). The balance of these activating and inhibitory signals allows NK cells to discriminate healthy self cells from stressed, virus-infected, or transformed self cells. This "built-in" self-tolerance mechanism helps protect normal, healthy tissues from NK cell responses. Extending this principle, NK cell self-tolerance allows TriNKET to target antigens expressed in both self and tumor without extratumoral side effects or with an increased therapeutic window.

[0276] Unlike natural killer cells, T cells require recognition of specific peptides presented by MHC molecules for activation and effector function. T cells are a major 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 deliver activation signals to effector cells. These therapies are effective in eliciting antitumor immune responses but are often associated with cytokine release syndrome (CRS) and on-target and off-tumor side effects. In this context, TriNKETs are unique because they do not “disable” the natural system of NK cell activation and inhibition. Rather, TriNKETs are designed to tip this balance and provide additional activation signals to NK cells while maintaining NK tolerance to healthy self.

[0277] 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 10 6 The PBMCs were adjusted to 10 μg / 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 ug / mL. Cells were cultured overnight at 37C with 5% CO2. The next day (24 hours later), PBMCs were harvested from the cultures 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.

[0278] Figures 54B and 54D show that autologous bone marrow cells are protected from TriNKET-mediated NK cell responses. Figures 54A and 54B show that B cells from healthy donors are sensitive to TriNKET-mediated lysis, while bone marrow cells are resistant to TriNKET lysis. PBMCs treated with TriNKET targeting CD20 showed a reduced frequency of CD19+ B cells with CD45+ lymphocyte populations (Figure 54A), but had no effect on CD45+, CDD3-, CD56- lymphocyte populations (Figure 54C). These cultures did not change the frequency of CD45+, CD19+ bone marrow cells (Figure 54B), or the frequency of CD33+, CD33+, CD11b+ bone marrow cells (Figure 54D). TriNKET mediates hPBMC killing of SkBr-3 tumor cells in long-term co-culture Primary human PBMC cytotoxicity assay

[0279] Figure 55 shows the long-term killing of SkBr-3 cells in culture with human PBMC. When cultured alone, SkBr-3 cells proliferate and nearly double in 60 hours. Addition of human PBMC to SkBr-3 cells in culture slows the proliferation rate, and to a lesser extent addition of the isotype control TriNKET targeting CD33. When cultures are treated with trastuzumab SkBr-3 no longer proliferates, and after 60 hours only 80% of the cells from time zero remain. Since SkBr-3 cells are sensitive to HER2 signal blockade, the effect on SkBr-3 cell growth may be mediated by HER2 signal blockade or via Fc effector functions such as ADCC. Example 15 Antitumor effect of mcFAE-C26.99 TriNKET in vitro

[0280] To validate the binding activity of murine cFAE-C26.99 TriNKET, direct binding was measured in comparison to its monoclonal antibody by flow cytometry assays on Tyrp-1 positive B16F10 melanoma cells (Figure 58A) and the EL4 line overexpressing murine NKG2D (EL4-mNKG2D, Figure 58B).

[0281] To test whether mcFAE-C26.99 TriNKET retained the ability to mediate cytotoxicity, we measured the killing of Tyrp-1 positive B16F10 tumor targets by murine IL-2-activated NK cells. As shown in Figure 59, murine NK cells increased their cytotoxic activity in the presence of mcFAE-C26.99. Importantly, the anti-Tyrp-1 monoclonal antibody TA99 had only a marginal effect. Increased NK cytotoxicity mediated by mcFAE-C26.99 TriNKET

[0282] Approximately 5 x 10 per well 3 B16F10 melanoma cells were seeded 2 days before the assay. On the day of the experiment, 5 × 10 4Murine IL-2-activated NK cells were added in the presence of TA99 mab or mcFAE-C26.99 TriNKET (mcFAE-C26.99 is a heterodimer of mC26 and TA99 with mouse IgG2c as Fc. Gm mutation refers to the heterodimerization mutation used to generate the heterodimer). 20 μg / mL antibody was used in 4-fold dilutions. After 4 hours of co-culture, the percentage of cytotoxicity was assessed using the CytoTox96 kit for LDH release. The dotted line represents baseline cytotoxicity in the absence of antibody. [ka] [ka] Example 16 Antitumor effect of mcFAE-C26.99 TriNKET in vivo

[0283] To test whether mcFAE-C26.99 exerts an antitumor function in vivo, 2 × 10 5 B16F10 tumor cells were subcutaneously injected. Mice were treated with either the isotype control monoclonal TA99 antibody or mcFAE-C26.99 TriNKET. Treatment with monoclonal TA99 antibody showed tumor progression similar to that in the isotype-treated control group. However, administration of mcFAE-C26.99 TriNKET resulted in delayed tumor progression compared to the isotype-treated group. Approximately 2×10 5B16F10 melanoma cells were injected subcutaneously into the flank of C57BL / 6 mice. Six days after tumor inoculation, mice were randomized (n=10 / group). Mice were treated intraperitoneally with (Figure 60A) isotype control mouse IgG2a mab C1.18.4 and mouse anti-Tyrp-1 monoclonal antibody or (Figure 60B) isotype control mouse IgG2a mab C1.18.4 and mcFAE-C26.99 TriNKET injected at a dose of 150 μg (days 6, 8, 10, 12, 14, 16, and 21). Tumor growth was evaluated for 28 days. Graphs show tumor growth curves of individual mice.

[0284] In addition to the subcutaneous B16F10 tumor model, mcFAE-C26.99 TriNKET was also tested for its antitumor efficacy in a disseminated tumor environment. 5 Mice were injected intravenously with B16F10 cells. Treatment was initiated on either day 4 or day 7 with low (300 μg / injection) and high (600 μg / injection) antibody doses. Lung metastases were counted on day 18 after tumor inoculation. Treatment initiated on days 4 and 7 after tumor inoculation resulted in a reduced number of lung metastases when TA99 monoclonal antibody or mcFAE-C26.99 TriNKET was used at higher concentrations compared to isotype-treated controls. At lower concentrations, only mcFAE-C26.99 TriNKET reduced tumor burden (Figure 61A). A similar effect was seen when the antibody was administered starting on day 7 after tumor inoculation. Overall, mcFAE-C26.99 TriNKET therapy resulted in a lower number of lung metastases compared to monoclonal TA99 antibody in all tested conditions. Approximately 1×10 5B16F10 melanoma cells were injected intravenously into the tail vein of C57BL / 6 mice (n=8 / group). Mice were left untreated or treated intraperitoneally with control mab (isotype, clone C1.18.4), monoclonal TA99 antibody or TA99 TriNKET (mcFAE-C26.99). Figure 61A represents the tumor burden when antibody was administered at a 150 μg dose (days 4, 6, 8, 11, 13, 15). Figure 61B represents the tumor burden when antibody was administered at a 150 μg dose (days 7, 9, 11, 13, 15). 18 days after tumor challenge, mice were euthanized and surface lung metastases were scored (Figure 61B). (Example 17) Cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies, or bispecific antibodies against HER2-positive cells

[0285] 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 the next day for cytotoxicity assays.

[0286] DELFIA Cytotoxicity Assay:

[0287] Human cancer cell lines expressing the target of interest were harvested from culture, the cells were washed with HBS, and incubated for 10 min for labeling with BATDA reagent (Perkin Elmer AD0116). 6 The target cells were labeled according to the manufacturer's instructions. After labeling, the cells were washed three times with HBS and resuspended in growth medium at 0.5–1.0 × 10 5The 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 the wells in triplicate to avoid disturbing the pelleted cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Wells were reserved for spontaneous release from the target cells and wells were prepared for maximum lysis of the target cells by the addition of 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target 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 cultures, the cells were washed and 10 μl of culture medium was added 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 with 5% CO2 for 2-3 hours before the assay was developed.

[0288] After 2-3 hours of incubation, plates were removed from the incubator and cells were pelleted by centrifugation at 200g for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate provided by the manufacturer and 200 μl of room temperature europium solution was added to each well. Plates were protected from light and incubated for 15 minutes at 250 rpm on a plate shaker. Plates were read using either a 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 recapitulate TriNKET activity:

[0289] Figure 66 shows the cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies, or bispecific antibodies against HER2-positive Colo-201 cell line. TriNKET (ADI-29404 (F04)) targeting HER2 induced maximum lysis of Colo-201 cells by resting human NK cells. 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, indicating the importance of dual targeting of CD16 and NKG2D to NK cells. To further demonstrate the importance of dual targeting to NK cells, monoclonal antibody trastuzumab was used to target HER2 and mediate ADCC by NK cells. Trastuzumab alone was able to increase NK cell lysis of Colo-201 cells, but the maximum lysis achieved by trastuzumab alone was about four-fold lower compared to TriNKET. To understand the importance of CD16 and NKG2D targeting the same molecule, TriNKET (ADI-29404 (F04)) activity was compared to the activity of a bispecific antibody targeting HER2 and NKG2D in combination with trastuzumab. When used at equimolar concentrations, the combination of bispecific and trastuzumab failed to mediate maximum lysis of Colo-201 cells by resting human NK cells. The failure of the trastuzumab + bispecific combination indicates the importance of containing the trispecific binding of TriNKET in one molecule. Incorporation by Reference

[0290] The entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes. Equivalent

[0291] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. 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 a tumor-associated antigen; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; Proteins including: (Item 2) The protein of item 1, wherein the first antigen-binding site binds to human, non-human primate, and rodent NKG2D. (Item 3) 3. The protein according to item 1 or 2, wherein the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 4) 4. The protein according to item 3, wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide. (Item 5) 5. The protein according to item 3 or 4, wherein the second antigen-binding site also comprises a heavy chain variable domain and a light chain variable domain. (Item 6) 6. The protein according to item 5, wherein the light chain variable domain of the first antigen-binding site has an amino acid sequence identical to an amino acid sequence of the light chain variable domain of the second antigen-binding site. (Item 7) 3. The protein according to item 1 or 2, wherein the first antigen-binding site is a single domain antibody. (Item 8) The single domain antibody is HH fragment or V NAR 8. The protein according to item 7, which is a fragment. (Item 9) 9. The protein of any one of items 1 to 4 or 7 to 8, wherein the second antigen-binding site is a single domain antibody. (Item 10) The second antigen-binding site is H H fragment or V NAR 10. The protein according to item 9, which is a fragment. (Item 11) 9. The protein according to item 1, 2, 7 or 8, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain. (Item 12) 13. 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 13) 12. The protein according to any of items 1 to 11, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 41 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 42. (Item 14) 12. The protein according to any of items 1 to 11, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 43 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 44. (Item 15) 12. The protein according to any of items 1 to 11, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 69 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 70. (Item 16) 12. The protein according to any of items 1 to 11, wherein the first antigen-binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO: 71 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 72. (Item 17) 10. The protein of any one of the preceding items, wherein the tumor associated antigen is selected from the group consisting of HER2, CD20, CD33, BCMA, EpCAM, CD2, CD19, 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. (Item 18) 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 19) 18. The protein according to any one of items 1 to 17, comprising an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody. (Item 20) A formulation comprising a protein according to any one of the preceding items and a pharma- ceutically acceptable carrier. (Item 21) 20. A cell comprising one or more nucleic acids encoding the protein of any one of items 1 to 19. (Item 22) 20. A method for directly or indirectly enhancing tumor cell death comprising exposing tumor and natural killer cells to a protein according to any one of items 1 to 19. (Item 23) A method for treating cancer, comprising administering to a patient a protein according to any one of items 1 to 19 or a formulation according to item 20. (Item 24) 24. The method of claim 23, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumor, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic carcinoma, thyroid cancer, urothelial cancer, cancer infiltrated with myeloid-derived suppressor cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with angiogenesis.

Claims

1. A multispecific binding protein comprising: (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to a tumor-associated antigen; and (c) an antibody Fc domain sufficient to bind CD16, or a CD16-binding portion thereof comprising the hinge and CH2 domain; wherein the multispecific binding protein is capable of binding to the tumor-associated antigen on a cancer cell and to NKG2D and CD16 on a natural killer (NK) cell to activate the NK cell.

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

3. A multispecific binding protein as described in claim 1 or 2, wherein the first antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

4. The multispecific binding protein of claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:1 and the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 79.

5. The multispecific binding protein of claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:41 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

42.

6. The multispecific binding protein of claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:43 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

44.

7. The multispecific binding protein of claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:69 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

70.

8. The multispecific binding protein of claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:71 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

72.

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

10. The multispecific binding protein of claim 1, wherein the second antigen-binding site comprises a heavy chain variable domain and a light chain variable domain.

11. The multispecific binding protein of any one of claims 1 to 9, wherein the second antigen-binding site is a single domain antibody.

12. The multispecific binding protein of any one of claims 1 to 11, wherein the antibody Fc domain sufficient to bind to CD16, or the CD16 binding portion thereof, comprises an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody, numbering according to the EU index of Kabat.

13. The multispecific binding protein of any one of claims 1 to 12, wherein the antibody Fc domain comprises substitutions of K360E and K409W in one polypeptide and substitutions of Q347R, D399V, and F405T in the other polypeptide, numbering according to the EU index of Kabat.

14. The multispecific binding protein of any one of claims 1 to 13, wherein the antibody Fc domain comprises a S354C substitution in one polypeptide and a Y349C substitution in the other polypeptide, numbering according to the EU index of Kabat.

15. A pharmaceutical composition comprising the multispecific binding protein of any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

16. A cell comprising one or more nucleic acids encoding a multispecific binding protein according to any one of claims 1 to 14.

17. A composition comprising a multispecific binding protein according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 for use in therapy.

18. A composition comprising a multispecific binding protein according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 for use in a method of treating cancer, the method comprising administering the composition to a patient having a cancer which expresses the tumor associated antigen.

19. The composition for use of claim 18, wherein the cancer is selected from the group consisting of acute myeloid leukemia, acute myelomonocytic leukemia, B-cell lymphoma, bladder cancer, breast cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumors, ovarian cancer, and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic carcinoma, thyroid cancer, urothelial cancer, cancer infiltrated with myeloid-derived suppressor cells, cancer with extracellular matrix deposition, and cancer with angiogenesis.

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