RU2809125C2 - Polyvalent binding proteins for activation of natural killer cells and their therapeutic application for treatment of malignant neoplasm - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: polyvalent binding protein that binds to NKG2D, CD16 and tumor-specific antigen. Also a pharmaceutical composition containing this protein is presented. A method for enhancing the death of tumor cells; method for treating malignant neoplasms are disclosed.

EFFECT: effective treatment of malignant neoplasms.

16 cl, 9 tbl, 70 dwg, 17 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62/456535, filed February 8, 2017, the entire contents of which are incorporated herein by reference for all purposes.

LIST OF SEQUENCES

[0002] This application contains a sequence listing that is provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on February 6, 2018, is called DFY-001PC_SL.txt and is 71,169 bytes in size.

TECHNICAL FIELD

[0003] The invention relates to multispecific binding proteins that bind to tumor-specific antigen, NKG2D receptor and CD16.

State of the art

[0004] Malignancy continues to be a serious health problem despite significant research efforts and scientific advances for the treatment of this disease reported in the literature. Some of the most commonly diagnosed types of cancer include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of malignancy in men. Breast cancer remains the leading cause of death in women. Current treatment options for these types of malignancies are not effective for all patients and/or may have significant side effects. Other types of malignancies also remain difficult to treat with current treatment options.

[0005] Antitumor immunotherapy is desirable because it is highly specific and can promote the destruction of tumor cells using the patient's own immune system. Fusion proteins, such as bispecific T cell recruiter proteins, are antitumor immunotherapies described in the literature that bind to tumor cells and T cells to facilitate tumor cell destruction. Antibodies that bind to certain tumor-specific antigens and to certain immune cells have been described in the literature. See, for example, WO 2016/134371 and WO 2015/095412.

[0006] Natural killer (NK) cells are a component 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, that is, through cytolytic granules that contain perforin and granzymes, and through death receptor pathways. Activated NK cells also secrete inflammatory cytokines, such as IFN-gamma and chemokines, which promote the recruitment of other leukocytes to target tissues.

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

The essence of the invention

[0008] The invention relates to multispecific binding proteins that bind to a tumor-specific antigen on a tumor cell and the NKG2D receptor and CD16 receptor on natural killer cells to activate natural killer cells, to pharmaceutical compositions containing such multispecific binding proteins, and to therapeutic methods , using such polyspecific proteins, and to pharmaceutical compositions, including for the treatment of malignant neoplasms. Such proteins may interact with more than one type of NK-activating receptor and may block the binding of natural ligands to NKG2D. In some embodiments, the protein may be an NK cell agonist in humans and in other species, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in more detail below.

[0009] In some embodiments, the polyspecific binding protein may include a first antigen binding site that binds NKG2D; a second antigen-binding site that binds to a tumor-specific antigen; and the Fc domain of the antibody, a portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16.

[0010] In some embodiments, the polyspecific binding protein is trivalent, which includes a first and a second antigen binding site that both bind to the same tumor-specific antigen; a third antigen binding site that binds NKG2D; and the Fc domain of the antibody, the part that is sufficient to bind to CD16.

[0011] In some embodiments, the polyspecific binding protein is tetravalent, which includes first and second antigen binding sites that both bind to the same tumor-specific antigen; third and fourth antigen binding sites, which both bind NKG2D; and the Fc domain of the antibody, the part that is sufficient to bind to CD16.

[0012] Each antigen binding site may include an antibody heavy chain variable domain and an antibody light chain variable domain (eg, located as in an antibody, or fused together with a scFv), or one or more antigen binding sites may be a single domain antibody, such as a _VH antibody H, such as camel antibody, or V _NAR antibody, such as those found in cartilaginous fish. In some cases, the tumor-specific 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 invention relates to a method of treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein for the treatment of cancer. Exemplary cancers for treatment using multispecific binding proteins include, for example, carcinoma whose cells express HER2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a multispecific binding protein that contains an NKG2D binding domain (right arm), a tumor-specific antigen binding domain (left arm), and an Fc domain or portion thereof that binds CD16.

[0015] FIG. 2 is a multispecific binding protein that contains an NKG2D binding domain in scFv format (right arm), a tumor-specific antigen binding domain (left arm), and an Fc domain or part thereof that binds CD16.

[0016] FIG. 3 is TriNKET in the form of Triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half antibodies, each with one light and one heavy chain, which are derived from two parent antibodies. The Triomab form may be a heterodimeric construct containing 1/2 rat antibody and 1/2 mouse antibody.

[0017] FIG. 4 is TriNKET in KiH common light chain (LC) form using knob-in-hollow (KIH) technology. KiH is a heterodimer containing 2 target-binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations. TriNKET in KiH format can be a heterodimeric construct with 2 fabs binding to target 1 and target 2, containing 2 different heavy chains and a common light chain that pairs with both HCs.

[0018] FIG. 5 is TriNKET in the form of Dual Variable Domain Immunoglobulin (DVD-Ig™), which combines the target-binding domains of two monoclonal antibodies through flexible, naturally occurring linkers to produce a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the antigen 2-targeting variable domain is fused to the N-terminus of the antigen 1-targeting Fab variable domain. The construct contains normal Fc.

[0019] FIG. 6 is a TriNKET in the form of an orthogonal Fab interface (Ortho-Fab), which is a heterodimeric construct that contains 2 Fabs binding to target 1 and target 2 fused to an Fc. LC-HC pairing is provided by an orthogonal interface. Heterodimerization is mediated by mutations in Fc.

[0020] FIG. 7 is TrinKET in 2 in 1 Ig format.

[0021] FIG. 8 is TriNKET in ES form, which is a heterodimeric construct containing 2 different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is mediated by mutations in the electrostatic interaction in Fc.

[0022] FIG. 9 is TriNKET in Fab-exchange form: antibodies that exchange Fab fragments by exchanging a heavy chain (HC) and attached light chain (LC) (half-molecule) with a heavy-light chain pair from another molecule, producing bispecific antibodies. The Fab-exchanged form (cFae) is a heterodimer containing 2 target-binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0023] FIG. 10 is TriNKET in SEED-body form, which is a heterodimer containing 2 target-binding Fabs 1 and 2, and an Fc stabilized by heterodimerization mutations.

[0024] FIG. 11 is a TriNKET in the form of LuZ-Y, in which the leucine zipper domain is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing 2 different target-binding scFabs 1 and 2 fused to an Fc. Heterodimerization is mediated by leucine zipper motifs fused to the C terminus of the Fc.

[0025] FIG. 12 is TriNKET in Cov-X-Body form.

[0026] FIG. 13A-13B are TriNKETs in κλ-Body forms, which are heterodimeric constructs with two different Fabs fused to Fc, stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains kappa LC, and a second Fab targeting antigen 2 contains contains lambda LC. FIG. 13A is an illustrative view of one κλ-Body shape; FIG. 13B is an illustrative view of another κλ-Body.

[0027] 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] FIG. 15 is a graph showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant cynomolgus NKG2D in an ELISA assay.

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

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

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

[0032] FIG. 19 is a graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competition with the natural ligand ULBP-6.

[0033] FIG. 20 is a graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competition with the natural MICA ligand.

[0034] FIG. 21 is a graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant mouse NKG2D-Fc by competition with the natural ligand Rae-1 delta.

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

[0036] FIG. 23 is a graph showing activation of murine NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells that express murine NKG2D-CD3 zeta fusion proteins.

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

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

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

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

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

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

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

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

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

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

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

[0048] FIG. 35 is a graph showing polyspecific binding proteins induced by higher levels of cytotoxicity to target tumor cells by murine NK cells.

[0049] FIG. 36 is a graph showing polyspecific binding proteins induced by higher levels of cytotoxicity to target tumor cells by murine NK cells.

[0050] FIG. 37 shows the binding profile of CD33-targeted TriNKET to NKG2D expressed on EL4 cells. In FIG. 37 shows the binding of two TriNKETs when the CD33 binding domain is used as the second targeting moiety.

[0051] FIG. 38 shows the binding profile of HER2-targeted TriNKET to NKG2D expressed on EL4 cells. In FIG. 38 shows the same two NKG2D binding domains now coupled to a second fragment targeting HER2.

[0052] FIG. 39 is the binding profile of BCMA-targeted TriNKET to NKG2D expressed on EL4 cells.

[0053] FIG. 40 is a histogram of CD20-targeted TriNKETs that bind to NKG2D expressed on EL4 cells. Unstained EL4 cells were used as a negative control for the fluorescence signal. Not painted: painted over; CD20-TriNKET-F04: solid line; CD20-TriNKET-C26: dotted line.

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

[0055] FIG. 42 is a binding profile of HER2-targeted TriNKETs to HER2 expressed on human renal cell carcinoma 786-O cells.

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

[0057] FIG. 44 is a histogram of CD20-targeting TriNKETs that bind to CD20 expressed on human Raji lymphoma cells. Unstained cells were used as a negative control for the fluorescent signal. Not painted: painted over; CD20-TriNKET-F04: solid line; CD20-TriNKET-C26: dotted line.

[0058] FIG. 45A-45C are histograms of synergistic NK cell activation using CD16 and NKG2D. Fig. 45A shows the level of CD107a; FIG. 45B shows IFNγ levels; FIG. 45C demonstrates CD107a and IFNγ levels. Graphs show mean (n=2) ± SD. Data are representative of five independent experiments using five different healthy donors.

[0059] FIG. 46 is a bar graph showing NK cell activation using TriNKET targeting NKG2D and CD16. The antibodies tested are of the human IgG1 isotype. Graphs show mean (n=2) ± SD.

[0060] FIG. 47A-47C are histograms demonstrating 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 production of the cytokine IFNγ. Compared to the monoclonal antibody trastuzumab, both TriNKETs demonstrated superior activation of human NK cells using a variety of human HER2 tumor cells. In FIG. 47A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. In FIG. 47B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. FIG. 47C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells.

[0061] FIG. 48A-48B are line graphs demonstrating TriNKET-mediated activation of quiescent or IL-2-activated human NK cells in coculture with the CD33-expressing human AML cell line MV4-11. In FIG. 48A shows TriNKET-mediated activation of resting human NK cells. In FIG. 48B shows TriNKET-mediated activation of IL-2-activated human NK cells from the same donor.

[0062] FIG. 49A-49B are graphs demonstrating the increase in cytotoxic activity of TriNKET using IL-2-activated and resting human NK cells. In FIG. 49A shows the percentage of specific lysis of SkBr-3 tumor cells by resting human NK cells. In FIG. 49B shows the percentage of specific lysis of SkBr-3 tumor cells by IL-2-activated human NK cells.

[0063] FIG. 50A-50B are graphs demonstrating that TriNKETs provide greater benefit against intermediate and low HER2 cancers compared to trastuzumab. In FIG. 50A shows the killing of activated human NK cells by SkBr-3 tumor cells with high levels of HER2. In FIG. 50B shows human NK cell killing of 786-O tumor cells with low HER2 levels. TriNKETs provide greater benefit over trastuzumab against tumor cells with low HER2 expression.

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

[0065] FIG. 52A-52B are line graphs of monoclonal antibodies or TriNKET-mediated activation of human NK cells in coculture with other Molm-13 (FIG. 52B) or THP-1 (FIG. 52A) cells.

[0066] FIG. 53A-53C are line graphs of human NK cell cytotoxicity assays using three human AML cell lines as targets. In FIG. 53A shows that Mv4-11 cells, which express CD64, but at a lower level than THP-1, showed reduced efficacy with an anti-CD33 monoclonal antibody. FIG. 53B demonstrates that the anti-CD33 monoclonal antibody exhibits good efficacy against Molm-13 cells that do not express CD64. FIG. 53C demonstrates that THP-1 cells have no effect with anti-CD33 monoclonal antibody individually. The identity of the line graphs marked in FIG. 53C also applies to the line graphs in FIG. 53A-53B.

[0067] FIG. 54A and 54B are histograms showing that healthy donor B cells are sensitive to TriNKET-mediated lysis.

[0068] FIG. 54C and 54D are histograms showing that myeloid cells are resistant to TriNKET-mediated lysis.

[0069] FIG. 55 is line plots of TriNKET-mediated hPBMC killing of SkBr-3 tumor cells in long-term co-cultures.

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

[0071] FIG. 57 are line graphs showing that SC2.2 does not affect RMA/S-HER2 subcutaneous tumor growth.

[0072] FIG. 58A-58B are graphs showing in vitro binding by mcFAE-C26.99 TriNKET. 60 μg/ml of the indicated antibodies with fourfold dilutions were added to 2×10 ⁵ B16F10 tumor cells (FIG. 58A) or EL4-mNKG2D cells (FIG. 58B). Binding was assessed using a goat anti-mouse PE secondary antibody followed by flow cytometry analysis.

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

[0074] In FIG. 60A-60B show the antitumor efficacy of mcFAE-C26.99 TriNKET in s.c. models. B16F10. Mice were treated intraperitoneally with (FIG. 60A) mouse IgG2a isotype control mab C1.18.4 and mouse anti-Tyrp-1 monoclonal antibody or (FIG. 60B) mouse IgG2a isotype control mab C1.18.4 and mcFAE-C26.99 TriNKET, which were dosed 150 mcg (days 6, 8, 10, 12, 14, 16 and 21). Tumor growth was assessed over 28 days. Graphs show tumor growth curves of individual mice.

[0075] In FIG. 61A-61B show the antitumor efficacy of mcFAE-C26.99 TriNKE T in i.v. models. B16F10. FIG. 61A represents the tumor load when antibodies were administered at a dose of 150 μg (days 4, 6, 8, 11, 13, 15). FIG. 61B represents the tumor burden when antibodies were administered at a dose of 150 μg (days 7, 9, 11, 13, 15). Eighteen days after tumor infection, mice were sacrificed and superficial lung metastases were assessed.

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

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

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

[0079] FIG. 65 is a graph showing that BCMA-targeting TriNKETs with different NKG2D binding domains enhance human NK cell lysis of KMS12-PE myeloma cells.

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

[0081] FIG. 67 is a heterodimeric Oasc-Fab construct that includes Fab binding to target 1 and scFab binding to target 2 fused to an Fc. Heterodimerization is mediated by mutations in Fc.

[0082] FIG. 68 is a DuetMab, which is a heterodimeric construct containing 2 different Fabs binding to antigen 1 and 2, and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain different SS bridges that ensure proper pairing of LC and HC.

[0083] FIG. 69 is a CrossmAb that is a heterodimeric construct with 2 different target binding Fabs 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are switched, for example, CH1 is fused inline with VL while CL is fused inline with VH.

[0084] FIG. 70 is a Fit-Ig, which is a homodimeric construct in which a Fab that binds to antigen 2 is fused to the N-terminus of an HC Fab that binds to antigen 1. The construct contains a wild-type Fc.

DETAILED DESCRIPTION

[0085] The invention relates to multispecific binding proteins that bind to a tumor-specific antigen on a tumor cell and to the NKG2D receptor and the CD16 receptor on natural killer cells to activate the natural killer cell, to pharmaceutical compositions containing such multispecific binding proteins, and to therapeutic methods using such polyspecific proteins, and pharmaceutical compositions, including for the treatment of malignant neoplasms. Various aspects of the invention are set forth below in the sections; however, aspects of the invention described in one specific section are not intended to be limited to any particular section.

[0086] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0087] Terms in the singular as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0088] As used herein, the term “antigen-binding site” refers to the portion of the 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 divergent regions in the V regions of the heavy and light chains are called "hypervariable regions", which are located between more conserved flanking regions known as "framework regions" or "FR". Thus, the term "FR" refers to the amino acid sequences that are naturally present in immunoglobulins between and adjacent to the hypervariable regions. In a human antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are located relative to each other in three-dimensional space, forming 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 designated “complementarity determining regions,” or “CDRs.” In some animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain to form a "single domain antibody". Antigen binding sites may exist 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 a scFv, using a peptide linker to join a heavy chain variable domain to a light chain variable domain in a single polypeptide.

[0089] As used herein, the term “tumor-specific antigen” means any antigen, including, but not limited to, protein, glycoprotein, ganglioside, carbohydrate, lipid, that is associated with a malignancy. Such an antigen may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma or immune infiltrates.

[0090] As used herein, the terms “subject” and “patient” refer to the organism to be treated with the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (eg, mice, monkeys, horses, cattle, pigs, dogs, cats, and the like) and more preferably include humans.

[0091] As used herein, the term “effective amount” refers to an amount of a compound (eg, a compound of the present invention) sufficient to achieve the beneficial or intended results. 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 “treatment” includes any effect, such as reduction, reduction, modulation, improvement, or elimination, that results in the improvement of a condition, disease, disorder, etc., or the alleviation of a symptom thereof.

[0092] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0093] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (eg, such as oil/water or water/oil emulsions), and various types of wetting agents. agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].

[0094] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (eg, acid or base) of a compound of the present invention that, when administered to a patient, is capable of providing the compound of the present invention or an active metabolite or residue thereof. As is known to those skilled in the art, "salts" of the compounds of the present invention can be prepared from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic , benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid and the like. Other acids, such as oxalic acid, which are not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts.

[0095] Typical bases include, but are not limited to, alkali metal hydroxides (eg, sodium), alkaline earth metal hydroxides (eg, magnesium), ammonia, and compounds of the formula NW4 ⁺ where W is C ₁ - ₄ alkyl and the like.

[0096] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate and the like. Other examples of salts include the anions of the compounds of the present invention coupled with a suitable cation such as Na ⁺ , NH4 ⁺ and NW4 ⁺ (where W represents a C ₁ - ₄ alkyl group), and the like.

[0097] For therapeutic use, salts of the compounds of the present invention are contemplated to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0098] Throughout the specification where compositions are described as having, including or containing specific components, or where processes and methods are described as having, including or containing specific steps, it is intended that there are additionally compositions of the present invention that consist essentially or consist of of the listed components, and that there are processes and methods according to the present invention that consist essentially of or consist of the listed processing steps.

[0099] Generally, if a percentage is stated in a composition, it is a percentage by weight unless otherwise stated. Additionally, if a variable is not accompanied by a definition, then the previous definition of the variable takes precedence.

I. Proteins

[00100] The invention relates to multispecific binding proteins that bind to a tumor-specific antigen on a tumor cell and to the NKG2D receptor and CD16 receptor on natural killer cells to activate natural killer cells. Polyspecific binding proteins are useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of the polyspecific binding protein to the NKG2D receptor and CD16 receptor on the natural killer cell enhances the activity of the natural killer cell to destroy the tumor cell. Binding of the multispecific binding protein to a tumor-specific antigen on a tumor cell brings the tumor cell closer to the natural killer cell, facilitating direct and indirect destruction of the tumor cell by the natural killer cell. Further descriptions of examples of multispecific binding proteins are provided below.

[00101] The first component of the multispecific binding proteins binds to cells expressing the NKG2D receptor, which may include, but is not limited to, NK cells, γδ T cells, and CD8 ⁺ αβ T cells. Once NKG2D binds, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D.

[00102] The second component of the multispecific binding proteins binds to one or more tumor-specific 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.

[00103] The third component for multispecific binding proteins 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.

[00104] Polyspecific binding proteins can take several formats, as shown, but are not limited to the examples below. 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 includes a first Fc domain (hinge-CH2-CH3), a first heavy chain variable domain, and optionally a first heavy chain domain CH1. The light chain of an immunoglobulin includes a light chain variable domain and a light chain constant domain; Together with the first immunoglobulin heavy chain, the immunoglobulin light chain forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain contains a second Fc domain (hinge-CH2-CH3), a second heavy chain variable domain, and optionally a second heavy chain domain CH1, which can pair with an immunoglobulin light chain identical to that which pairs with the first immunoglobulin heavy chain, for except when an immunoglobulin light chain is paired with a second immunoglobulin heavy chain, the resulting antigen binding site binding to a tumor antigen. The first Fc domain and the second Fc domain together can bind CD16 (FIG. 1).

[00105] Another exemplary format includes a heterodimeric multispecific antibody that includes a first immunoglobulin heavy chain, an immunoglobulin light chain, and a second immunoglobulin heavy chain. The first immunoglobulin heavy chain includes a first Fc domain (hinge-CH2-CH3) fused through either a linker or an antibody hinge to a single chain Fv (scFv) that binds NKG2D. Various linkers can be used to link the scFv to the first Fc domain or within the scFv itself. In addition, the scFv may include mutations that allow the formation of a disulfide bond to stabilize the overall structure of the scFv. ScFv may also include mutations to modify the isoelectric point of the entire first immunoglobulin heavy chain and/or to allow for easier subsequent purification. The second immunoglobulin heavy chain includes a second Fc domain (hinge-CH2-CH3) and a second heavy chain variable domain and a second optional heavy chain domain CH1. The light chain of an immunoglobulin includes 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 the tumor antigen. The first Fc domain and the second Fc domain together can bind CD16 (FIG. 2).

[00106] An alternative format of heterodimeric multispecific proteins includes 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 includes a first Fc domain (hinge-CH2-CH3), a first heavy chain variable domain, and optionally a first heavy chain CH1 domain. The first light chain of an immunoglobulin includes a light chain variable domain and a light chain constant domain. Together with the first immunoglobulin heavy chain, the first immunoglobulin light chain forms an antigen-binding site that binds to tumor antigen. The second immunoglobulin heavy chain contains a second Fc domain (hinge-CH2-CH3), a second heavy chain variable domain, and optionally a second heavy chain domain CH1. The second immunoglobulin light chain includes a light chain variable domain and a light chain constant domain. Together with the second immunoglobulin heavy chain, the immunoglobulin light chain forms an antigen-binding site that binds to the same tumor antigen. The second immunoglobulin heavy chain may be paired with an immunoglobulin light chain, which may be identical to the immunoglobulin light chain that is paired with the first immunoglobulin heavy chain, except that the immunoglobulin light chain is paired with the second immunoglobulin heavy chain, the resulting antigen binding site being the second antigen-binding site that binds to tumor antigen. In some embodiments, the first Fc domain and the second Fc domain together may bind CD16 (FIG. 1).

[00107] One or more additional binding motifs may be fused to the C-terminus of the CH3 domain of the constant region, optionally through a linker sequence. In some embodiments, the antigen binding site may be a single chain or disulfide stabilized variable region (ScFv) or may form a tetravalent or trivalent molecule.

[00108] In some embodiments, the multispecific binding protein is in the form of Triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half antibodies, each with one light and one heavy chain, which are derived from two parent antibodies.

[00109] In some embodiments, the polyspecific binding protein is a common light chain (LC) form of KiH that utilizes knobs-in-holes (KIH) technology. KIH includes engineered CH3 domains to create either a “bump” or a “cavity” in each heavy chain to promote heterodimerization. The concept of the knob-in-hollow (KiH) Fc technology was to introduce a knob in one CH3 domain (CH3A) by replacing a small residue with a bulky one (i.e., T366WCH3A in EU numbering). To accommodate the overhang, a complementary indentation surface was created on another CH3 domain (CH3B) by replacing the nearest adjacent residues to the overhang with smaller ones (i.e., T366S/L368A/Y407VCH3B). The cavity mutation was optimized by structure-driven 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 structures of KiH Fc variants (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) demonstrated that heterodimerization is thermodynamically supported by hydrophobic interactions driven by steric complementarity at the core interface between CH3 domains, while the knob-knob and valley-gap interfaces do not favor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[00110] In some embodiments, the polyspecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies through flexible natural linkers to produce a tetravalent IgG-like molecule.

[00111] In some embodiments, the polyspecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab). In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. (2014 ) 32 (2): 191-8), structure-based regional design introduces complementary mutations at the LC and HC _VH - _CH1 interface in only one Fab without any changes in the other Fab.

[00112] In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In some embodiments, the polyspecific binding protein is in the form of an ES, which is a heterodimeric construct containing 2 different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is mediated by mutations in the electrostatic interaction in Fc. In some embodiments, the multispecific binding protein is in the form of Bodyλ-Body, which are heterodimeric constructs with 2 different Fc-fused Fabs stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains kappa-LC, while the second Fab targeting for antigen 2, contains lambda LC. FIG. 13A is an exemplary representation of one κλ-Body shape; FIG. 13B is an exemplary representation of another κλ-Body.

[00113] In some embodiments, the multispecific binding protein is in a Fab-exchange form (antibodies that exchange Fab fragments by exchanging the heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, such that leads to the production of bispecific antibodies). In some embodiments, the multispecific binding protein is in the form of a SEED-Body (a strand exchange engineered domain (SEED) platform has been designed to generate asymmetric and bispecific antibody-like molecules, allowing for expanded therapeutic applications of natural antibodies. This protein engineered platform is based on exchange of structurally related immunoglobulin sequences in conserved CH3 domains. The SEED design allows efficient generation of AG/GA heterodimers while preventing homodimerization of the CH3 AG and GA SEED domains. (Muda M. et al., Protein Eng. Des. Sel. (2011 , 24 (5): 447-54. In some embodiments, the polyspecific binding protein is in the form of LuZ-Y, in which a leucine zipper domain is used to induce heterodimerization of two different HCs (Wranik, B. J. Et al., J. Biol Chem (2012), 287: 43331-9).

[00114] In some embodiments, the multispecific binding protein is in the form of a Cov-X-Body (in bispecific CovX-Body, two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. While the pharmacophores responsible for functional activity, the antibody scaffold provides a long half-life and Ig-like distribution. Pharmacophores can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107 (52) ; 22611-22616).

[00115] In some embodiments, the multispecific binding protein is in an Oasc-Fab heterodimeric form that includes a target-binding Fab 1 and a target-binding scFab 2 fused to an Fc. Heterodimerization is ensured by mutations in Fc.

[00116] In some embodiments, the polyspecific binding protein is in the form of a DuetMab, which is a heterodimeric construct containing 2 different Fabs binding to antigen 1 and 2, and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain different SS bridges that ensure correct pairing of LC and HC.

[00117] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a heterodimeric construct with 2 different target binding Fabs 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are switched, for example, CH1 is fused to VL in the same polypeptide, while CL is fused to VH in the same polypeptide.

[00118] In some embodiments, the polyspecific binding protein is in the form of Fit-Ig, which are homodimeric constructs where a Fab that binds antigen 2 is fused to the N-terminus of an HC Fab that binds antigen 1. The construct contains a wild-type Fc .

[00119] Table 1 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to NKG2D.

Table 1 Clones Amino acid sequence of the heavy chain variable region Amino acid sequence of the light chain variable region ADI-27705 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:1) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPITFGGGTKVEIK
(SEQ ID NO:2) ADI-27724 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:3) EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGGGTKVEIK
(SEQ ID NO:4) ADI-27740
(A40) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:5) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFYTFGGGTKVEIK
(SEQ ID NO:6) ADI-27741 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:7) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSNSYYTFGGGTKVEIK
(SEQ ID NO:8) ADI-27743 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:9) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPTFGGGTKVEIK
(SEQ ID NO:10) ADI-28153 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWGFDPWGQGTLVTVSS
(SEQ ID NO:11) ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIK
(SEQ ID NO:12) ADI-28226
(C26) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:13) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPITFGGGTKVEIK
(SEQ ID NO:14) ADI-28154 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:15) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK
(SEQ ID NO:16) ADI-29399 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:17) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPTFGGGTKVEIK
(SEQ ID NO:18) ADI-29401 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:19) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDIYPTFGGGTKVEIK
(SEQ ID NO:20) ADI-29403 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:21) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSYPTFGGGTKVEIK
(SEQ ID NO:22) ADI-29405 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:23) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPTFGGGTKVEIK
(SEQ ID NO:24) ADI-29407 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:25) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSFPTFGGGTKVEIK
(SEQ ID NO:26) ADI-29419 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:27) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSFSTFGGGTKVEIK
(SEQ ID NO:28) ADI-29421 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:29) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESYSTFGGGTKVEIK
(SEQ ID NO:30) ADI-29424 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:31) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSFITFGGGTKVEIK
(SEQ ID NO:32) ADI-29425 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:33) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSYPTFGGGTKVEIK
(SEQ ID NO:34) ADI-29426 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:35) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFPTFGGGTKVEIK
(SEQ ID NO:36) ADI-29429 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:37) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYELYSYTFGGGTKVEIK
(SEQ ID NO:38) ADI-29447
(F47) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:39) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTFITFGGGTKVEIK
(SEQ ID NO:40) ADI-27727 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGDSSIRHAYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:41) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGGGTKVEIK
(SEQ ID NO:42) ADI-29443
(F43) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSDRFHPYFDYWGQGTLVTVSS
(SEQ ID NO:43) EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFDTWPPTFGGGTKVEIK
(SEQ ID NO:44) ADI-27744
(A44) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSS
(SEQ ID NO:45)
CDR1 (SEQ ID NO:51) - FTFSSYAMS
CDR2 (SEQ ID NO:52) - AISGSGGSTYYADSVKG
CDR3 (SEQ ID NO:53) - AKDGGYYDSGAGDY DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK
(SEQ ID NO:46)
CDR1 (SEQ ID NO:54) - RASQGIDSWLA
CDR2 (SEQ ID NO:55) - AASSLQS
CDR3 (SEQ ID NO:56) - QQGVSYPRT ADI-27749
(A49) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSS
(SEQ ID NO:47)
CDR1 (SEQ ID NO:57) - FTFSSYSMN
CDR2 (SEQ ID NO:58) - SISSSSSYIYYADSVKG
CDR3 (SEQ ID NO:59) - ARGAPMGAAAGWFDP DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK
(SEQ ID NO:48)
CDR1 (SEQ ID NO:60) - RASQGISSWLA
CDR2 (SEQ ID NO:61) - AASSLQS
CDR3 (SEQ ID NO:62) - QQGVSFPRT ADI-29463
(F63) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDVWGQGTTVTVSS
(SEQ ID NO:49)
CDR1 (SEQ ID NO:63) - YTFTGYYMH
CDR2 (SEQ ID NO:64) - WINPNSGGTNYAQKFQG
CDR3 (SEQ ID NO:65) - ARDTGEYYDTDDHGMDV EIVLTQSPGTLLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK
(SEQ ID NO:50)
CDR1 (SEQ ID NO:66) - RASQSVSSNLA
CDR2 (SEQ ID NO:67) - GASTRAT
CDR3 (SEQ ID NO:68) - QQDDYWPPT ADI-29404
(F04) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:78) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCEQYDSYPTFGGGTKVEIK
(SEQ ID NO:79) ADI-28200 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGRKASGSFYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:80) DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK
(SEQ ID NO:81)

[00120] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 69 can pair with the light chain variable domain defined by SEQ ID NO: 70 to form an antigen binding site that can bind NKG2D, as illustrated in US Pat. No. 9,273,136.

SEQ ID NO:69

QVQLVESGGGLVKPGGSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS

SEQ ID NO: 70 QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[00121] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 71 can pair with the light chain variable domain defined by SEQ ID NO: 72 to form an antigen binding site that can bind NKG2D, as illustrated in US Pat. No. 7,879,985.

SEQ ID NO:71

QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS

SEQ ID NO: 72 EIVLTQSPGTLSLSPGERAATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[00122] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mainly concentrated on the amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - Ile 332, Leu 234 - Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (See Sondermann et al., Nature, 406 (6793): 267-273). Based on known domains, mutations can be selected to enhance or decrease CD16 binding affinity, such as using phage display libraries or yeast display cDNA libraries, or mutations can be designed based on a known three-dimensional interaction structure.

[00123] Assembly 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 assembly of homodimers of each antibody heavy chain, as well as the assembly of heterodimers. Promotion of preferential heterodimer assembly can be achieved by introducing various mutations into the CH3 domain of each antibody heavy chain constant region, as shown in US13/494870, US16/028850, US11/533709, US12/875015, US13/289934, US14/773418, US12/ 811207, US13/866756, US14/647480, US14/830336. For example, mutations can be made in the CH3 domain of human IgG1 and include different pairs of amino acid substitutions in the first polypeptide and the second polypeptide that allow the two chains to selectively heterodimerize with each other. The amino acid substitution positions shown below are numbered according to the EU index, as in Kabat.

[00124] In one scenario, an amino acid substitution in the first polypeptide replaces the parent amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W) and at least one amino acid substitution in the second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V), so that the larger amino acid replacement (bulge) fits into the surface of the smaller amino acids replacements (cavity). For example, one polypeptide may include the T366W substitution, and another may include three substitutions, including T366S, L368A, and Y407V.

[00125] The heavy chain variable domain of an antibody of the invention may optionally be linked to an amino acid sequence at least 90% identical to an antibody constant region, such as an IgG constant region, including hinge domains, CH2 and CH3 domains with or without a CH1 domain. . In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to the constant region of an antibody from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be included in the constant region compared to the human IgG1 constant region, for example in 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. Typical replacements include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q 362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K3 92E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[00126] In some embodiments, mutations that may be included in the CH1 constant region of human IgG1 may be at amino acid V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In some embodiments, mutations that may be included in the Cκ constant region of human IgG1 may be at amino acid E123, F116, S176, V163, S174, and/or T164.

[00127] Alternatively, amino acid substitutions may be selected from the following sets of substitutions presented in Table 2.

table 2 First polypeptide Second polypeptide Set 1 S364E/F405A Y349K/T394F Set 2 S364H/D401K Y349T/T411E Set 3 S364H/T394F Y349T/F405A Set 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6 S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357Q L368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11 K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370S S364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

[00128] Alternatively, amino acid substitutions may be selected from the following sets of substitutions presented in Table 3.

Table 3 First polypeptide Second polypeptide Set 1 K409W D399V/F405T Set 2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set 5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409W E357W/D399V/F405T

[00129] Alternatively, amino acid substitutions may be selected from the following set of substitutions presented in Table 4.

Table 4 First polypeptide Second polypeptide Set 1 T366K/L351K L351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349D Set 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368E Set 6 E356K/D399K K392D/K409D

[00130] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 5.

Table 5 First polypeptide Second polypeptide L351Y, D399R, D399K, S400K, S400R, Y407A, Y407I, Y407V T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

[00131] Alternatively, the at least one amino acid substitution may be selected from the following set of substitutions in Table 6, wherein the position(s) indicated in the "First Polypeptide" column is replaced by any known negatively charged amino acid, and the position(s) listed in the "Second Polypeptide" column is replaced by any known positively charged amino acid.

Table 6 First polypeptide Second polypeptide K392, K370, K409, or K439 D399, E356, or E357

[00132] Alternatively, the at least one amino acid substitution may be selected from the following set in Table 7, wherein the position(s) indicated in the "First Polypeptide" column is replaced by any known positively charged amino acid, and said position(s) listed in the "Second Polypeptide" column is replaced by any known negatively charged amino acid.

Table 7 First polypeptide Second polypeptide D399, E356, or E357 K409, K439, K370, or K392

[00133] Alternatively, amino acid substitutions may be selected from the following set in Table 8.

Table 8 First polypeptide Second polypeptide T350V, L351Y, F405A, and Y407V T350V, T366L, K392L, and T394W

[00134] Alternatively or additionally, the structural stability of the heteromultimeric protein can be increased by introducing S354C on either the first or second polypeptide chain, and Y349C on the opposite polypeptide chain, which forms an artificial disulfide bridge within the interface of the two polypeptides.

[00135] The polyspecific proteins described above can be produced using recombinant DNA technology well known to one skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain may be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain may 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; the first, second and third expression vectors can be stably transfected together into host cells to produce multimeric proteins.

[00136] To achieve the highest yield of polyspecific protein, various ratios of the first, second, and third expression vector may be experimented with to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated to generate a cell bank using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy or Clonepix.

[00137] Clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of polyspecific protein expression. 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. Characteristics of TriNKET

[00138] In certain embodiments, the TriNKETs described herein, which include an NKG2D binding domain and a tumor-specific antigen binding domain, bind to cells expressing human NKG2D. In some embodiments, TriNKETs that include an NKG2D binding domain and a tumor-specific antigen binding domain bind to a tumor-specific antigen at a level comparable to that of a monoclonal antibody having the same tumor-specific antigen binding domain. For example, TriNKETs, which include the NKG2D-binding domain and the HER2-binding domain from trastuzumab, can bind to HER2 expressed on cells at levels comparable to trastuzumab.

[00139] However, the TriNKETs described herein are more effective in reducing tumor growth and killing tumor cells. For example, the TriNKET of the present invention, which targets HER2-expressing tumors/tumor cells, is more effective than SC2.2, a single-chain bispecific molecule constructed from a trastuzumab-derived scFv bound to ULBP-6, a ligand for NKG2D. SC2.2 binds to HER2+ tumor cells and NKG2D+ NK cells simultaneously. Therefore, the effectiveness of SC2.2 in reducing the number of HER2+ tumor cells was investigated. In vitro activation and cytotoxicity studies showed that SC2.2 was effective in activating and killing NK cells. However, SC2.2 failed to demonstrate efficacy in the RMA/S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using a syngeneic mouse model overexpressing RMA/S-HER2. In this mouse model, SC2.2 failed to demonstrate tumor growth control compared to vehicle controls. Thus, although SC2.2 was able to activate and kill NK cells and bind to HER2+ tumor cells, these properties were not sufficient to effectively control HER2+ tumor growth.

[00140] In certain embodiments, the TriNKETs described herein, which include an NKG2D-binding domain and a tumor-specific antigen binding domain, activate primary human NK cells when cultured with tumor cells expressing the antigen. NK cell activation is marked by increased CD107a degranulation and production of IFNγ cytokines. Additionally, compared to a monoclonal antibody that includes a tumor-specific antigen-binding domain, TriNKETs demonstrate superior activation of human NK cells in the presence of antigen-expressing tumor cells. For example, compared to the monoclonal antibody trastuzumab, the TriNKETs of the present invention having a HER2-binding domain have superior activation of human NK cells in the presence of HER2-expressing tumor cells.

[00141] In certain embodiments, the TriNKETs described herein, which include an NKG2D binding domain and a tumor-specific antigen binding domain, enhance the activity of resting and IL-2-activated human NK cells in the presence of tumor cells expressing the antigen. Resting NK cells showed less background IFNγ production and CD107a degranulation than IL-2-activated NK cells. In some embodiments, resting NK cells exhibit a greater change in IFNγ production and CD107a degranulation compared to IL-2-activated NK cells. In some embodiments, IL-2-activated NK cells exhibit a higher percentage of cells becoming IFNγ+; CD107a+ after stimulation with TriNKET.

[00142] In certain embodiments, the TriNKETs described herein, which include an NKG2D-binding domain and a tumor-specific antigen binding domain (non-limiting examples of tumor-specific antigens include CD20, BCMA, and HER2), enhance the cytotoxic activity of quiescent and IL-2-activated human NKs -cells in the presence of tumor cells expressing the antigen. Additionally, TriNKETs (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET), which include a binding domain for a tumor-specific antigen (non-limiting examples tumor-specific antigens include CD20, BCMA and HER2) more effectively enhance the response of directly activated and resting NK cells against tumor cells compared to a monoclonal antibody that includes the same tumor-specific antigen binding site. In some embodiments, TriNKETs are advantageous against tumor cells that express moderate to low levels of tumor antigens over monoclonal antibodies that include the same tumor antigen binding site. Therefore, therapy including TriNKET may be superior to monoclonal antibody therapy.

[00143] In certain embodiments, compared to monoclonal antibodies, TriNKETs described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET and F63-TriNKET), which include a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2), are useful in the treatment of cancers with high expression of the Fc receptor (FcR) or cancers located in the tumor microenvironment with high levels of FcR. Monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blockade, among others. Among the FcγRs, CD16 has the lowest affinity for IgG Fcs; FcγRI (CD64) is a high affinity FcR that binds approximately 1000 times more strongly to IgG Fc than to CD16. CD64 is commonly expressed on many cells of hematopoietic lineages, such as the myeloid lineage, and can be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, also express CD64 and are known to infiltrate into the tumor microenvironment. Expression of CD64 by the tumor or in the tumor microenvironment may have a detrimental effect on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to interact with CD16 on the surface of NK cells, as the antibodies prefer to bind to the high-affinity receptor. TriNKETs, by targeting two activating receptors on the surface of NK cells, may overcome the detrimental effects of CD64 expression (either on the tumor or the tumor microenvironment) on monoclonal antibody therapy. Regardless of CD64 expression on tumor cells, TriNKETs are capable of mediating human NK cell responses against all tumor cells because dual targeting of two activating receptors to NK cells allows for stronger NK cell specific binding.

[00144] In some embodiments, the TriNKETs described herein (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET), which include binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2) provide a better safety profile by reducing side effects associated with off-tumor mistargeting. Natural killer cells and CD8 T cells are able to directly lyse tumor cells, although the mechanisms by which NK cells and CD8 T cells distinguish their own healthy cells from tumor cells differ. NK cell activity is regulated by a balance of signals from activating (NCR, NKG2D, CD16, etc.) and inhibitory (KIR, NKG2A, etc.) receptors. The balance of these activating and inhibitory signals allows NK cells to identify healthy self cells from stressed, virus-infected, or transformed self cells. This “built-in” self-tolerance mechanism will help protect normal healthy tissue from NK cell responses. To extend this principle, NK cell self-tolerance would allow TriNKET to target antigens expressed on both self and tumor cells without the side effects associated with off-tumor mistargeting or an extended therapeutic window. Unlike natural killer cells, T cells require recognition of a specific peptide presented by MHC molecules for activation and effector functions. T cells have been a major target of immunotherapy, and many strategies have been developed to redirect T cell responses against the tumor. Bispecific T cells, checkpoint inhibitors, and CAR T cells are FDA approved but often suffer from dose-limiting toxicity. Bispecific T cells and CAR-T cells function around the TCR-MHC recognition system, using binding domains to target antigens on the surface of tumor cells and using engineered signaling domains to transmit activation signals to the effector cell. Although these treatments are effective in eliciting an antitumor immune response, they are often associated with cytokine release syndrome (CRS) and side effects associated with off-tumor mistargeting. In this context, TriNKETs are unique because they do not “override” the natural NK cell activation and inhibition systems. Instead, TriNKETs are designed to alter the balance and provide additional activation signals to NK cells while maintaining NK tolerance to self cells.

[00145] In some embodiments, TriNKETs described herein comprising an NKG2D binding domain (e.g., A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63 -TriNKETs) that include a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2) are more effective at delaying tumor progression than monoclonal antibodies that include the same tumor antigen-binding domain. In some embodiments, TriNKETs comprising an NKG2D binding domain and a tumor antigen binding domain are more effective against cancer metastases than monoclonal antibodies that include the same tumor antigen binding domain.

[00146] The above description describes many aspects and embodiments of the invention. The patent application specifically addresses all combinations and permutations of aspects and embodiments.

III. Therapeutic Applications

[00147] The invention relates to methods of treating cancer using a multispecific binding protein described herein (for example, A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET and F63-TriNKET) that include a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2), and/or to a pharmaceutical composition described herein. The methods can be used to treat various types of malignancies, including solid tumor, lymphoma and leukemia. The type of malignancy to be treated is preferably matched to the type of tumor cell to which the multispecific binding protein binds. For example, treatment of a cancer whose cells express epithelial cell adhesion molecule (EpCAM), such as colon cancer expressing EpCAM, is desirably treated using a multispecific binding protein described herein that binds thereto. Additional aspects and embodiments of the therapeutic methods are described below.

[00148] Accordingly, one aspect of the invention relates to a method of treating cancer in a patient, wherein the method includes 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), which includes a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2) for the treatment of cancer. Examples of treatable malignancies include solid tumor, leukemia and lymphoma.

[00149] The therapeutic method can be characterized depending on the malignancy to be treated. For example, in certain embodiments, the malignancy is a solid tumor. In some 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, colorectal cancer, kidney cancer, stomach cancer, testicular cancer or uterine cancer. In yet other embodiments, the malignancy is a vascularized tumor, 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, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, glandular squamous cell carcinoma, anal cancer, anal cancer, rectal cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, liver cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choriodpapilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell cancer, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer , endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cancer, ependymal cancer, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, bile duct cancer bladder, gastric cavity cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangiblastoma, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepis ithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, small bowel cancer, connective tissue cancer, Kaposi's sarcoma, pelvic cancer, large cell cancer, colon cancer, leiomyosarcoma, lentigo melanoma, lymphoma, cancer of the male genital organs, malignant melanoma, malignant mesothelial tumors, 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, nonepithelial skin cancer, non-Hodgkin's lymphoma , oat cell carcinoma, oligodendroglial cancer, oral cancer, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma , sinus cancer, skin cancer, small cell cancer, small intestinal cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, urethral cancer, bladder cancer, genitourinary cancer, cervical cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous cancer, VIPoma, vulvar cancer, well differentiated carcinoma or Wilms tumor.

[00150] In some other embodiments, the cancer is non-Hodgkin's lymphoma, such as B-cell lymphoma or T-cell lymphoma. In some 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, B-cell extranodal marginal zone 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 some other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as lymphoblastic T-cell precursor lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer/T-cell lymphoma cells, nodular T-cell lymphoma, cutaneous T-cell lymphoma, enteropathic-type T-cell lymphoma, subcutaneous panniculitis-type T-cell lymphoma, anaplastic large cell lymphoma, or nodular T-cell lymphoma.

[00151] A cancer to be treated can be characterized according to the presence of a specific antigen expressed on the surface of the tumor cell. In some embodiments, the tumor cell expresses 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.

[00152] In certain embodiments, the protein of the present invention is used in a method of enhancing the death of tumor cells (synonymous with lysis, killing, ablation, reducing cell survival or proliferation, etc.) directly or indirectly, or for the production of a drug for use in a method of enhancing the death of tumor cells (synonymous with lysis, destruction, ablation, decreasing cell survival or proliferation, etc.) directly or indirectly, by affecting tumor cells and natural killer cells with the protein of the present invention (for example, A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET, and F63-TriNKET), which includes a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA, and HER2). A tumor cell that responds to a protein as described above expresses a tumor-specific antigen to which a second antigen binding site of the protein binds. For example, in an exemplary embodiment, C26-TriNKET-CD20 is used to target a CD20-expressing tumor cell (either quiescent or activated); in another exemplary embodiment, C26-TriNKET-BMCA is used to target a BMCA-expressing tumor cell (either quiescent or activated).

[00153] In some embodiments, the protein of the present invention is used in a method of treating a cancer in a patient in need thereof, or in the manufacture of a medicament for use in a method of treating a cancer in a patient in need thereof, which includes administering the protein or composition to the patient comprising a protein of the present invention (for example, A40-TriNKET, A44-TriNKET, A49-TriNKET, C26-TriNKET, F04-TriNKET, F43-TriNKET, F47-TriNKET and F63-TriNKET), which includes a binding domain for a tumor-specific antigen ( non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2). A tumor cell that responds to a protein as described above expresses a tumor-specific antigen to which a second antigen-binding site of the protein binds. For example, in an exemplary embodiment, C26-TriNKET-CD20 is used to target a CD20-expressing tumor cell (either quiescent or activated); in another exemplary embodiment, C26-TriNKET-BMCA is used to target a BMCA-expressing tumor or tumor cell (either quiescent or activated).

IV. Combination therapy

[00154] Another aspect of the invention provides combination therapy. The polyspecific binding proteins described herein are used in combination with additional therapeutic agents for the treatment of cancer.

[00155] Typical therapeutic agents that may be used as part of combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin , nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxan, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxiflu ridin, etretinate, isotretinoin, streptozocin, nimustine, vindesines, flutamide, drogenil, butocin, carmofur, razoxane, sisofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocytabine, lisuride, oxymetetholone, tamoxifen, progesterone, mepithiostane, epithiostanol, formestane , interferon alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diphytox, interleukin-2, luteinizing hormone releasing factor and variations of the above agents that may exhibit differential binding to their related receptor and increase or decrease serum half-life.

[00156] An additional class of agents that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Typical immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T lymphocyte associated antigen 4 (CTLA-4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, ( iv) LAG-3, (v) B7-H3, (vi) B7-H4 and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for the treatment of melanoma.

[00157] Other agents that may be used as part of combination therapy in the treatment of cancer are monoclonal antibody agents that target targets other than checkpoints (eg, Herceptin) and noncytotoxic agents (eg, tyrosine kinase inhibitors) .

[00158] Other categories of antitumor agents include, for example: (i) an inhibitor selected from ALK inhibitor, ATR inhibitor, A2A antagonist, base exclusion repair inhibitor, Bcr-Abl tyrosine kinase inhibitor, Bruton's tyrosine kinase inhibitor, CDC7 inhibitor, CHK1 inhibitor, cyclin inhibitor -dependent kinase, DNA-PK inhibitor, both DNA-PK and mTOR inhibitor, DNMT1 inhibitor, DNMT1 inhibitor plus 2-chloro-deoxyadenosine, HDAC inhibitor, Hedgehog signaling pathway inhibitor, IDO inhibitor, JAK inhibitor, mTOR inhibitor, MEK inhibitor , MELK inhibitor, MTH1 inhibitor, PARP inhibitor, phosphoinositide 3-kinase inhibitor, inhibitor of both PARP1 and DHODH, proteasome inhibitor, topoisomerase-II inhibitor, tyrosine kinase inhibitor, VEGFR inhibitor and WEE1 inhibitor; (ii) an agonist for OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF and G-CSF.

[00159] The proteins of the invention can also be used as an adjunct to surgical removal of a primary lesion.

[00160] The amount of polyspecific 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 combination therapy to a patient in need of such administration, the therapeutic agents in combination or the pharmaceutical composition or compositions containing the therapeutic agents may be administered in any order, such as, for example, sequentially, simultaneously, together, simultaneously, and the like. In addition, for example, the multispecific binding protein may be administered during the time that the additional therapeutic agent(s) are exerting their prophylactic or therapeutic effects, or vice versa.

V. Pharmaceutical compositions

[00161] The present disclosure also relates to pharmaceutical compositions that contain 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), which includes a binding domain for a tumor-specific antigen (non-limiting examples of tumor-specific antigens include CD20, BCMA and HER2). The composition can be prepared for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for suitable formulation. Suitable formulations for use in the present disclosure are available from Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief overview of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990) .

[00162] The intravenous drug delivery composition of the present invention may be contained in a pouch, pen, or syringe. In some embodiments, the package 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 frozen-dried (lyophilized) and may be contained in approximately 12-60 vials. In some embodiments, the composition may be lyophilized, and 45 mg of the lyophilized composition may be contained in one vial. In some embodiments, about 40-100 mg of the lyophilized composition may be contained in one vial. In some embodiments, a lyophilized formulation of 12, 27, or 45 vials is combined to produce a therapeutic dose of protein in an intravenous drug formulation. In some embodiments, the composition may be a liquid composition and stored at a concentration of from about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the composition may be a liquid composition and stored at a concentration of about 600 mg/vial. In some embodiments, the composition may be a liquid composition and stored at a concentration of about 250 mg/vial.

[00163] The present disclosure may exist in liquid form as an aqueous solution of a pharmaceutical composition comprising a therapeutically effective amount of protein in a buffer solution to form the composition.

[00164] These compositions can be sterilized by conventional sterilization methods or can be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the formulations is typically 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single-dose units, each containing a fixed amount of the abovementioned agent or agents. The composition in solid form may also be packaged in a variable quantity container.

[00165] In certain embodiments, the present disclosure relates to an extended shelf life formulation comprising the protein of the present invention in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water and hydroxide sodium

[00166] In certain embodiments, an aqueous composition is prepared comprising the protein of the present invention in a pH buffer solution. The buffer of the present invention may have a pH in the range of about 4 to about 8, such as about 4.5 to about 6 or about 4.8 to about 5.5, or may have a pH from about 5 to about 5.2. Ranges intermediate to the above pH values are also intended to be part of this disclosure. For example, value ranges that use a combination of any of the above values as upper and/or lower limits are intended to be included. Examples of buffers that will control pH in this range include acetate (such as sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

[00167] In certain embodiments, the composition includes a buffer system that contains citrate and phosphate to maintain a pH in the range of about 4 to about 8. In some embodiments, the pH range can be from about 4.5 to about 6, or about pH 4.8 to about 5.5, or the pH range may be from about 5 to about 5.2. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), about 1.5 mg /ml disodium phosphate. dihydrate (eg, 1.53 mg/ml), about 0.9 mg/ml sodium dihydrogen phosphate dihydrate (eg, 0.86), and about 6.2 mg/ml sodium chloride (eg, 6.165 mg/ml). In some embodiments, the buffer system includes 1 to 1.5 mg/mL citric acid, 0.25 to 0.5 mg/mL sodium citrate, 1.25 to 1.75 mg/mL disodium phosphate dihydrate, 0. 7 to 1.1 mg/ml sodium dihydrogen phosphate dihydrate and 6 to 6.4 mg/ml sodium chloride. In some embodiments, the pH of the composition is adjusted using sodium hydroxide.

[00168] A polyol that acts as a tonic and can stabilize the antibody may also be included in the formulation. The polyol is added to the formulation in an amount that may vary depending on the target isotonicity of the formulation. In some embodiments, the aqueous composition may be isotonic. The amount of polyol added can also be varied relative to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (such as mannitol) may be added compared to a disaccharide (such as trehalose). In some embodiments, the polyol that can be used in the composition as a tonicity agent is mannitol. In some embodiments, the concentration of mannitol may be from about 5 to about 20 mg/ml. In some embodiments, the concentration of mannitol may be about 7.5-15 mg/ml. In some embodiments, the concentration of mannitol may be about 10-14 mg/ml. In some embodiments, the concentration of mannitol may be about 12 mg/ml. In some embodiments, a sorbitol polyol may be included in the composition.

[00169] A detergent or surfactant may also be added to the composition. Typical detergents include nonionic detergents such as polysorbates (eg, polysorbates 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of detergent added is such that it reduces aggregation of the antibody in the formulation and/or minimizes particle formation in the formulation and/or reduces adsorption. In certain embodiments, the composition may include a surfactant that is a polysorbate. In some embodiments, the composition may contain the detergent polysorbate 80 or Tween 80. Tween 80 is the term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In some embodiments, the composition may contain from about 0.1 to about 10 mg/ml polysorbate 80 or from about 0.5 to about 5 mg/ml. In some embodiments, approximately 0.1% polysorbate 80 may be added to the composition.

[00170] In some embodiments, the protein product of the present invention is formulated as a liquid composition. The liquid formulation may be presented at a concentration of 10 mg/ml in any USP/Ph Eur Type I 50R vial, capped with a rubber stopper and sealed using an aluminum crimp seal. The stopper can be made of elastomer conforming to USP and Ph Eur. In some embodiments, the vials may be filled with 61.2 mL of protein product solution to provide an extractable volume of 60 mL. In some embodiments, the liquid composition may be diluted with a 0.9% saline solution.

[00171] In some embodiments, the liquid composition of the invention may be formulated as a solution at a concentration of 10 mg/ml in combination with sugar at a stabilizing level. In some embodiments, the liquid composition may be formulated in an aqueous carrier. In some embodiments, the stabilizer may be added in an amount no greater than that which would result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugar may be a disaccharide, such as sucrose. In some embodiments, the liquid composition may also include one or more of a buffering agent, a surfactant, and a preservative.

[00172] In some embodiments, the pH of the liquid formulation may be adjusted by adding a pharmaceutically acceptable acid and/or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[00173] In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, cell collection/clarification, purification, drug substance/drug product storage, and during sample analysis. Deamidation is the loss of NH ₃ from a protein to form a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate reduces the mass of the parent peptide by 17 daltons. Subsequent hydrolysis results in an increase in mass of 18 daltons. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. Essentially, deamidation is usually defined as an increase in mass of 1 Dalton. Deamidation of asparagine produces aspartic or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain influence deamidation rates. Gly and Ser following Asn in protein sequences result in higher sensitivity to deamidation.

[00174] In some embodiments, the liquid composition of the present invention may be maintained under pH and humidity conditions to prevent deamination of the protein product.

[00175] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and suitable for the preparation of a liquid composition. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00176] A preservative may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a reusable (multi-dose) formulation.

[00177] Intravenous (IV) formulations may be the preferred route of administration in specific cases, for example, when a patient is in the hospital after a transplant receiving all medications via the IV route. In some embodiments, the liquid composition is diluted with a 0.9% sodium chloride solution prior to administration. In some embodiments, the diluted injectable drug product is isotonic and suitable for administration by intravenous infusion.

[00178] In some embodiments, salts or buffer components may be added in an amount of 10-200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with metals or amines forming the base. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[00179] A preservative may optionally be added to the formulations to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a reusable (multi-dose) formulation.

[00180] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of a liquid composition. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00181] The present disclosure may exist in the form of a lyophilized composition comprising proteins and a lyoprotectant. The lyoprotector can be sugar, for example disaccharides. In some embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, an excipient and/or a preservative.

[00182] The amount of sucrose or maltose useful for stabilizing the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In some embodiments, the protein/sucrose or maltose weight ratio may be from 1:2 to 1:5.

[00183] In some embodiments, the pH of the composition may be adjusted prior to lyophilization by adding a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[00184] Prior to lyophilization, the pH of the solution containing the protein of the present invention may be adjusted to a range of 6 to 8. In some embodiments, the pH range for the lyophilized drug product may be 7 to 8.

[00185] In some embodiments, salts or buffer components may be added in an amount of 10-200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with metals or amines forming the base. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[00186] In some embodiments, "filler" may be added. A "filler" is a compound that adds bulk to the lyophilized mixture and contributes to the physical structure of the lyophilized tablet (eg, facilitates the production of a substantially uniform lyophilized tablet that maintains an open-cell structure). Exemplary excipients include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized compositions of the present invention may contain such excipients.

[00187] A preservative may optionally be added to the formulations to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a reusable (multi-dose) formulation.

[00188] In some embodiments, the lyophilized drug product may consist of an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (eg, safe and non-toxic for administration to humans) and suitable for the preparation of a liquid composition after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00189] In some embodiments, the lyophilized drug product of the present invention is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into the solution.

[00190] In some embodiments, the lyophilized protein product of the present invention consists of about 4.5 ml of water for injection and is diluted with 0.9% saline (sodium chloride solution).

[00191] Actual dosage levels of active ingredients in pharmaceutical compositions may be varied to provide an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient.

[00192] The specific dose may be a unit dose for each patient, for example, 50-5000 mg of protein. Alternatively, the patient dose may be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Those skilled in the art will routinely further refine the calculations necessary to determine the appropriate dosage for treatment, especially in light of the dosage information and assays disclosed herein. Dosage can also be determined by using known dose determination assays used in conjunction with appropriate dose-response data. An individual patient's dosage may be adjusted as disease progression is monitored. The patient's blood levels of the targeting construct or complex may be measured to see if dosage adjustments need to be made to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targeting constructs and/or complexes and their doses are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al. Clinica Chimica Acta 308 : 33-41, 2001).

[00193] Typically, dosages based on body weight are from about 0.01 μg to about 100 mg per kg body weight, for example, from about 0.01 μg to about 100 mg/kg body weight, from about 0.01 µg to approximately 50 mg/kg body weight, approximately 0.01 µg to approximately 10 mg/kg body weight, approximately 0.01 µg to approximately 1 mg/kg body weight, approximately 0.01 µg to approximately 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/kg body weight, from about 0.1 µg to about 100 mg/kg body weight, from about 0.1 µg to about 50 mg/kg body weight, from about 0 .1 μg to approximately 10 mg/kg body weight, approximately 0.1 μg to approximately 1 mg/kg body weight, approximately 0.1 μg to approximately 100 μg/kg body weight, approximately 0.1 μg to approximately 50 µg/kg body weight, from about 0.1 µg to about 10 µg/kg body weight, from about 0.1 µg to about 1 µg/kg body weight, from about 1 µg to about 100 mg/kg body weight, from about 1 µg to about 50 mg/kg body weight, from about 1 µg to about 10 mg/kg body weight, from about 1 µg to about 1 mg/kg body weight, from about 1 µg to about 100 µg/kg body weight body, from about 1 μg to about 50 μg/kg body weight, from about 1 μg to about 10 μg/kg body weight, from about 10 μg to about 100 mg/kg body weight, from about 10 μg to about 50 mg/ kg body weight, from about 10 μg to about 10 mg/kg body weight, from about 10 μg to about 1 mg/kg body weight, from about 10 μg to about 100 μg/kg body weight, from about 10 μg to about 50 µg/kg body weight, from about 50 µg to about 100 mg/kg body weight, from about 50 µg to about 50 mg/kg body weight, from about 50 µg to about 10 mg/kg body weight, from about 50 µg to about up to 1 mg/kg body weight, from approximately 50 μg to approximately 100 μg/kg body weight, from approximately 100 μg to approximately 100 mg/kg body weight, from approximately 100 μg to approximately 50 mg/kg body weight, from approximately 100 µg to approximately 10 mg/kg body weight, approximately 100 μg to approximately 1 mg/kg body weight, approximately 1 mg to approximately 100 mg/kg body weight, approximately 1 mg to approximately 50 mg/kg body weight, approximately from 1 mg to about 10 mg/kg body weight, from about 10 mg to about 100 mg/kg body weight, from about 10 mg to about 50 mg/kg body weight, from about 50 mg to about 100 mg/kg body weight .

[00194] Doses may be administered one or more times daily, weekly, monthly or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the repetition rate for dosing based on the measured retention time and concentrations of the targeting construct or complex in body fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter, or by direct intracellular injection. This may occur one or more times per day, one or more times per week, one or more times per month, and one or more times per year.

[00195] The above description describes many aspects and embodiments of the invention. The patent application specifically addresses all combinations and permutations of aspects and embodiments.

EXAMPLES

[00196] The present invention has now been described generally and will be better understood by reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention in any way.

Example 1 - NKG2D-binding domains bind to NKG2D

NKG2D-binding domains bind to purified recombinant NKG2D

[00197] Human, mouse, or cynomolgus NKG2D ectodomain nucleic acid sequences were fused to nucleic acid sequences encoding human IgG1 Fc domains and introduced into mammalian cells for expression. After purification, NKG2D-Fc fusion proteins were adsorbed into the wells of microplates. After blocking the wells with bovine serum albumin to prevent nonspecific binding, the NKG2D-binding domains were titrated and added to wells that had previously been adsorbed with NKG2D-Fc fusion proteins. Primary antibody binding was determined using a secondary antibody that is conjugated to horseradish peroxidase and specifically recognizes human kappa light chain to avoid Fc cross-reactivity. 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, was added to the wells to visualize the binding signal, the absorbance of which was measured at 450 nM and corrected at 540 nM. An NKG2D binding domain clone, isotype control or positive control (selected from SEQ ID NOs: 45-48 or anti-mouse clones NKG2D, MI-6 and CX-5 available from eBioscience) was added to each well.

[00198] The isotype control showed minimal binding to the recombinant NKG2D-Fc proteins, while the positive control bound most strongly to the recombinant antigens. The NKG2D binding domains produced by all clones exhibited binding to recombinant human (FIG. 14), mouse (FIG. 16), and cynomolgus (FIG. 15) NKG2D-Fc proteins, although with varying affinities from clone to clone. Typically, each anti-NKG2D clone binds to recombinant human (FIG. 14) and cynomolgus (FIG. 15) NKG2D-Fc with similar affinity, but lower affinity to recombinant mouse NKG2D-Fc (FIG. 16).

NKG2D-binding domains bind to cells expressing NKG2D

[00199] EL4 murine lymphoma cell lines have been engineered to express human or mouse NKG2D-CD3-zeta signaling domain chimeric antigen. NKG2D-binding clone, isotype control, or positive control was used at a concentration of 100 nM to stain extracellular NKG2D expressed on EL4 cells. Antibody binding was determined using fluorophore-conjugated anti-human IgG secondary antibodies. 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.

[00200] NKG2D-binding domains produced by all clones associated with human and mouse NKG2D-expressing EL4 cells. Positive control antibodies (selected from SEQ ID NO: 45-48 or anti-mouse clones NKG2D, MI-6 and CX-5 available from eBioscience) gave the best FOB binding signal. The NKG2D binding affinity for each clone was similar between cells expressing human (FIG. 17) and mouse (FIG. 18) NKG2D (FIG. 17-18, respectively).

Example 2 - NKG2D-binding domains block natural ligand binding to NKG2D

Competition with ULBP-6

[00201] Recombinant human NKG2D-Fc proteins were adsorbed to microplate wells, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. A saturated concentration of ULBP-6-His-biotin was added to the wells, followed by the addition of NKG2D-binding domain clones. After a 2-hour incubation, the wells were washed and ULBP-6-His-biotin, which remained bound to the NKG2D-Fc-coated wells, was detected with horseradish peroxidase-conjugated streptavidin and the substrate TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated from the percentage of ULBP-6-His-biotin that was blocked from binding to NKG2D-Fc proteins in the wells. A positive control antibody (selected from SEQ ID NOs: 45-48) and various NKG2D binding domains blocked ULBP-6 binding to NKG2D, while the isotype control showed little competition with ULBP-6 (FIG. 19).

Competition with MICA

[00202] Recombinant human MICA-Fc proteins were adsorbed into microplate wells, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. NKG2D-Fc-biotin was added to the wells, followed by the addition of NKG2D-binding domains. After incubation and washing, NKG2D-Fc-biotin that remained bound to MICA-Fc-coated wells was detected using streptavidin-HRP substrate and TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated from the percentage of NKG2D-Fc-biotin that was blocked from binding to MICA-Fc in the wells. The positive control antibody (selected from SEQ ID NOs: 45-48) and various NKG2D binding domains blocked MICA binding to NKG2D, while the isotype control showed little competition with MICA (FIG. 20).

Competition with Rae-1 delta

[00203] Recombinant mouse Rae-1delta-Fc (purchased from R&D Systems) was adsorbed to microplate wells, and the wells were blocked with bovine serum albumin to reduce nonspecific binding. Mouse NKG2D-Fc-biotin was added to the wells, followed by the addition of NKG2D-binding domains. After incubation and washing, NKG2D-Fc-biotin that remained bound to Rae-1delta-Fc-coated wells was detected using streptavidin-HRP substrate and TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D-binding domains to NKG2D-Fc proteins was calculated from the percentage of NKG2D-Fc-biotin that was blocked from binding to Rae-1delta-Fc-coated wells. Positive control (selected from SEQ ID NO: 45-48 or anti-mouse clones NKG2D, MI-6 and CX-5 available from eBioscience) and various NKG2D-binding domain clones blocked Rae-1-delta binding to mouse NKG2D, in while the isotype control antibody showed little competition with Rae-1delta (FIG. 21).

Example 3 - NKG2D-binding domain clones activate NKG2D

[00204] Human and mouse NKG2D nucleic acid sequences were fused to nucleic acid sequences encoding the CD3 zeta signaling domain to produce chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells to generate the retrovirus. EL4 cells were infected with viruses containing NKG2D-CAR along with 8 μg/ml polybrene. At 24 hours postinfection, NKG2D-CAR expression levels in EL4 cells were analyzed by flow cytometry, and clones that expressed high levels of NKG2D-CAR on the cell surface were selected.

[00205] To determine whether NKG2D binding domains activate NKG2D, they were adsorbed to microplate wells and NKG2D-CAR EL4 cells were cultured in antibody fragment-coated wells for 4 hours in the presence of brefeldin-A and monensin. Intracellular production of TNF-alpha, an indicator of NKG2D activation, was analyzed by flow cytometry. The percentage of TNF-alpha-positive cells was normalized to that of cells treated with the positive control. All NKG2D binding domains activated both human (FIG. 22) and mouse (FIG. 23) NKG2D.

Example 4 - NKG2D-binding domains activate NK cells

Primary human NK cells

[00206] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMC, and the purity of the isolated NK cells was typically >95%. The isolated NK cells were then cultured in a medium containing 100 ng/ml IL-2 for 24-48 hours, after which they were transferred into the wells of a microplate on which NKG2D-binding domains were adsorbed and cultured in a medium containing conjugated fluorophore antibody against CD107a, brefeldin-A and monensin. After culture, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD107a and IFN-gamma staining were analyzed in CD3-CD56 ⁺ cells to assess NK cell activation. The increase in the number of CD107a/IFN-gamma double positive cells indicates better activation of NK cells due to the recruitment of two activating receptors rather than a single receptor. NKG2D binding domains and positive control (selected from SEQ ID NOs: 45-48) showed a higher percentage of NK cells becoming CD107a ⁺ and IFN-gamma ⁺ than isotype control (FIG. 24 and FIG. 25 represent two independent experiments , each using a different PBMC donor to prepare NK cells).

Primary mouse NK cells

[00207] Spleens were obtained from C57Bl/6 mice and ground 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 then isolated from spleen cells using a negative depletion method using magnetic beads with a purity of >90%. Purified NK cells were cultured in medium containing 100 ng/ml mIL-15 for 48 hours, after which they were transferred to the wells of a microplate on which NKG2D-binding domains were adsorbed and cultured in medium containing a fluorophore-conjugated antibody against CD107a, brefeldin-A and monensin. After culture in NKG2D-binding domain-coated wells, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1, and IFN-gamma. CD107a and IFN-gamma staining were analyzed in CD3-NK1.1 ⁺ cells to assess NK cell activation. The increase in the number of CD107a/IFN-gamma double positive cells indicates better activation of NK cells due to the recruitment of two activating receptors rather than a single receptor. The NKG2D-binding domains and the positive control (selected from the anti-mouse NKG2D clones, MI-6 and CX-5, available from eBioscience) showed a higher percentage of NK cells becoming CD107a ⁺ and IFN-gamma ⁺ than the isotype control ( FIGURE 26 and FIGURE 27 represent two independent experiments, each using different mice to prepare NK cells).

Example 5 - NKG2D-binding domains provide cytotoxicity to target tumor cells

[00208] Primary human and mouse NK cell activation assays demonstrate increased levels of NK cell cytotoxicity markers following incubation with NKG2D binding domains. To determine whether this results in increased tumor cell lysis, a cell-based assay was used in which each NKG2D-binding domain was converted into a monospecific antibody. The Fc region was used as one targeting moiety, while the Fab region (NKG2D binding domain) acted as the other targeting moiety to activate NK cells. THP-1 cells, which are of human origin and express Fc receptors at high levels, were used as a tumor target and a Perkin Elmer DELFIA cytotoxicity kit was used. THP-1 cells were labeled with BATDA reagent and resuspended at 10 ⁵ /ml in culture medium. Labeled THP-1 cells were then combined with NKG2D antibodies and isolated murine NK cells in microplate wells at 37°C for 3 hours. After incubation, 20 μl of the culture supernatant was removed, mixed with 200 μl of europium solution and incubated with shaking for 15 minutes in the dark. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (337 nm excitation, 620 nm emission), and specific lysis was calculated according to the kit instructions.

[00209] The positive control, ULBP-6, a natural ligand for NKG2D, showed increased specific lysis of THP-1 target cells by murine NK cells. NKG2D antibodies also increased specific lysis of THP-1 target cells, whereas the isotype control antibody showed decreased specific lysis. The dotted line indicates specific lysis of THP-1 cells by murine NK cells without the addition of antibodies (FIG. 28).

Example 6 - NKG2D antibodies show high thermal stability

[00210] The melting temperature of the NKG2D binding domains was analyzed using differential scanning fluorimetry. The extrapolated apparent melting points are high compared to typical IgG1 antibodies (FIG. 29).

Example 7 Polyspecific binding proteins exhibit increased ability to activate NK cells.

[00211] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMC, and the purity of the isolated NK cells was typically >95%. The isolated NK cells were then cultured in a medium containing 100 ng/ml IL-2 for 24-48 hours, after which they were transferred to the wells of a microplate on which polyspecific and bispecific binding proteins were adsorbed, respectively, and cultured in a medium containing containing a fluorophore-conjugated antibody against CD107a, brefeldin-A and monensin. After culture, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56, and IFN-gamma. CD107a and IFN-gamma staining were analyzed in CD3-CD56 ⁺ cells to assess NK cell activation. An increase in the number of 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. This was done using a controlled Fab fragment exchange (cFAE) reaction starting with a trastuzumab homodimer and a ULBP-6-Fc homodimer (see Labrijn et al. Nature Protocols 9, 2450-2463). SC2.2 is a single chain protein comprising a trastuzumab-derived scFv and ULBP-6 (SEQ ID NO: 73).

SEQ ID NO: 73

MAAAAIPALLLCLPLLFLLFGWSRARRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDST LEPSAGAPLAMSSGTTQLRATATTLILCCLLIILPCFILPGI

[00212] CD107a and IFN-gamma staining analysis revealed that the isotype control IgG showed no NK cell activation, while a higher percentage of NK cells became CD107a ⁺ and IFN-gamma ⁺ after stimulation with polyspecific binding protein compared to bispecific protein, demonstrating stronger activation of NK cells due to the recruitment of two activating receptors (NKG2D and CD16), rather than just one (NKG2D) (FIG. 30). This increase in NK cell activation is expected to result in greater tumor cell killing.

Example 8. Polyspecific binding proteins exhibit increased cytotoxicity towards target tumor cells.

Cytotoxicity assay of primary human NK cells

[00213] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMC, and the purity of the isolated NK cells was typically >95%. NK cells were then cultured overnight in medium containing 100 ng/ml IL-2 before use in cytotoxicity assays. The next day, NK cells were resuspended in an amount of 5×10 ⁵ /ml in fresh culture medium. Human breast cancer cell line SkBr-3 cells were labeled with BATDA reagent in accordance with the Perkin Elmer DELFIA cytotoxicity kit and resuspended at 5×10 ⁴ /ml in culture medium. Various dilutions of polyspecific binding proteins were prepared in culture medium. NK cells, SkBr-3 labeled cells, and polyspecific binding proteins were then pooled into microplate wells and incubated at 37°C for 3 hours. After incubation, 20 μl of the culture supernatant was removed, mixed with 200 μl of europium solution and incubated with shaking for 15 minutes in the dark. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (337 nm excitation, 620 nm emission), and specific lysis was calculated according to the kit instructions. AL0.2 is a multispecific binding protein containing a HER2 binding domain (trastuzumab), an NKG2D binding domain (selected from SEQ ID NO: 1-44)) and a human IgG1 Fc domain. This was done using a controlled Fab fragment exchange (cFAE) reaction, starting with a trastuzumab homodimer and an anti-NKG2D homodimer. AL0.2si is based on AL0.2 and contains an additional mutation D265A in the Fc domain, which abolishes CD16 binding. Trastuzumab-si is based on trastuzumab and contains an additional mutation D265A in the Fc domain, which abolishes CD16 binding. AL2.2 is a multispecific binding protein containing a HER2-binding domain (trastuzumab), an NKG2D-binding domain (ULBP-6), and a human IgG1 Fc domain. SC2.2 is a single-chain protein comprising a trastuzumab-derived scFv and ULBP-6.

[00214] AL0.2 showed enhanced lysis of SkBr-3 target cells by human NK cells compared to trastuzumab in a dose-dependent manner, with a p-value of 0.0311 at EC50 (FIG. 31). AL0.2si (FIG. 32) and trastuzumab-si (FIG. 33) showed a decrease in both activity and maximum specific cell lysis of SkBr-3 compared to AL0.2, with a p-value of 0.0002 and 0.0001 at EC50, respectively (FIG. 32-33). In addition, AL0.2 showed enhanced dose-dependent lysis of SkBr-3 cells than AL2.2 (FIG. 34). The IgG isotype control showed no increase in specific lysis at any of the concentrations tested. Together, the data demonstrated that multispecific binding proteins recruiting 2 activating receptors on NK cells and one tumor antigen induced greater tumor cell killing by human NK cells compared with bispecific proteins recruiting one activating receptor on NK cells and one tumor antigen .

Primary Mouse NK Cell Cytotoxicity Assay

[00215] Spleens were obtained from C57Bl/6 mice and ground 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 then isolated from spleen cells using a negative depletion method using magnetic beads with a purity of >90%. Purified NK cells were cultured in medium containing 100 ng/ml mIL-15 for 48 hours, then resuspended in culture medium at 10 ⁶ /ml for cytotoxicity assays. RMA-HER2-dTomato, a mouse tumor cell line engineered to express HER2 and dTomato, and its control counterpart, RMA cells expressing zsGreen, were used as targets. They were resuspended at a concentration of 2×10 ⁵ /ml in a culture medium and seeded into the wells of a microplate in a 1:1 ratio. Dilutions of the polyspecific protein were added to the culture medium and added to RMA cells along with NK cells. After incubation overnight at 37°C with 5% CO2. The percentage of RMA-HER2-dTomato and RMA-zsGreen cells was determined by flow cytometry using a fluorescent reporter to identify the two cell types. Specific target cell death = (1- ((% RMA-Ca2T-dTomato cells in treatment group * % RMA-zsGreen cells in control group)/(% RMA-Ca2T-dTomato cells in control group * % RMA-zsGreen cells in treatment group))) * 100%.

[00216] AL2.2 is more effective in redirecting NK cell responses to tumor targets than SC2.2 (FIG. 36) and trastuzumab (FIG. 35). The control protein showed little effect on target specific killing. These data demonstrate that multispecific binding proteins recruiting 2 activating receptors on NK cells and one tumor antigen induce greater tumor cell killing by murine NK cells compared with bispecific proteins recruiting one activating receptor on NK cells and one tumor antigen .

Example 9 - Polyspecific binding proteins bind to NKG2D

[00217] Murine EL4 lymphoma cell lines engineered to express human NKG2D trispecific binding proteins (TriNKET), each of which contains an NKG2D binding domain, a tumor-specific antigen binding domain (such as a CD33-, HER2-, CD20-, or BCMA-binding domain) and an Fc domain that binds CD16, as shown in FIG. 1 were tested for their affinity for extracellular NKG2D expressed on EL4 cells. Binding of polyspecific binding proteins to NKG2D was determined using fluorophore-conjugated anti-human IgG secondary antibodies. 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.

[00218] TriNKETs tested include 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), CD20-TriNKET-C26 (ADI-28226 and CD20-binding domain), CD20-TriNKET-F04 (ADI-29404 and CD20-binding domain ), BCMA-TriNKET-C26 (ADI-28226 and BCMA-binding domain), BCMA-TriNKET-F04 (ADI-29404 and BCMA-binding domain), BCMA-TriNKET-F43 (ADI-29443 and BCMA-binding domain) and BCMA-TriNKET-F47 (ADI-29447 and BCMA-binding domain). The HER2-binding domain used in the tested molecules consisted of a heavy chain variable domain and a trastuzumab light chain variable domain. The CD33 binding domain consisted of a heavy chain variable domain and a light chain variable domain, listed below.

SEQ ID NO:74:

SEQ ID NO:75:

The CD20 binding domain used in the tested molecules consisted of a heavy chain variable domain and a light chain variable domain. The BCMA binding domain used in the test molecules consisted of a heavy chain variable domain and a light chain variable domain, as follows.

EM-801 Heavy Chain Variable Domain (SEQ ID NO: 82):

EM-801 Light Chain Variable Domain (SEQ ID NO: 83):

EM-901 Heavy Chain Variable Domain (SEQ ID NO: 76)

EM-901 Light Chain Variable Domain (SEQ ID NO: 77)

[00219] Data show that the TriNKET of the present invention binds to NKG2D when the protein includes a tumor antigen binding domain such as CD33, HER2, CD20 and BCMA.

Example 10 - Polyspecific binding proteins bind to human tumor antigens

Trispecific binding proteins bind to CD33, HER2, CD20 and BCMA

[00220] The human AML cell line MV4-11 expressing CD33 was used to assay TriNKET binding to tumor-specific antigen. TriNKET and the parental anti-CD33 monoclonal antibody were incubated with cells, and binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and excess over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and the parental anti-CD33 monoclonal antibody normalized to secondary antibody controls. CD33-TriNKET-C26 and CD33-TriNKET-F04 showed comparable levels of binding to CD33 compared to the parental anti-CD33 antibody (FIG. 41).

[00221] Human tumor cell lines expressing HER2 were used to assay TriNKET binding to tumor-specific antigen. The renal cell carcinoma cell line 786-O expresses HER2 at low levels. TriNKET and optionally the parental anti-HER2 monoclonal antibody (trastuzumab) were incubated with cells and binding was determined using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and excess over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and trastuzumab normalized to the secondary antibody control. HER2-TriNKET-C26 and HER2-TriNKET-F04 showed comparable levels of binding to HER2 expressed on 786-O cells compared to trastuzumab (FIG. 42).

[00222] Human myeloma cells MM.1S expressing BCMA were used to assay the binding of TriNKET to the tumor-specific antigen BCMA. TriNKET and optionally the parental anti-BCMA monoclonal antibody (EM-801) were incubated with cells, and binding was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and excess over background (FOB) was calculated using 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 showed comparable levels of binding to BCMA expressed on MM.1S cells compared to EM-801 (FIG. 43).

[00223] Human Raji lymphoma cells expressing CD20 were used to assay the binding of TriNKET to the tumor-specific antigen CD20. TriNKET was incubated with cells, and binding was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and a histogram was generated. As shown in FIG. 44, CD20-TriNKET-C26 and CD20-TriNKET-F04 bind equally well to CD20.

Example 11 - Polyspecific binding proteins activate NK cells

[00224] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMC, and the purity of the isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated NK cells were used within 24-48 hours of activation.

[00225] Human tumor cells expressing tumor antigen were collected and resuspended in culture medium at a concentration of 2×10 ⁶ /ml. Monoclonal antibodies or TriNKETs targeting tumor antigen were diluted in culture medium. Activated NK cells were collected, washed and resuspended at a concentration of 2×10 ⁶ /ml in culture medium. Tumor cells were then mixed with monoclonal antibodies/TriNKET and NK cells were activated in the presence of IL-2. Brefeldin-A and monensin were also added to the mixed culture to block protein transport out of the cell for intracellular cytokine staining. Fluorophore-conjugated anti-CD107a antibody was added to the mixed culture and the culture was incubated for 4 hours before samples were prepared for FACS analysis using fluorophore-conjugated anti-CD3, CD56, and IFN-gamma antibodies. CD107a and IFN-gamma staining were analyzed in CD3-CD56 ⁺ cells to assess NK cell activation. The increase in the number of CD107a/IFN-gamma double positive cells indicates better activation of NK cells due to the involvement of two activating receptors rather than a single receptor.

[00226] TriNKETs mediate activation of human NK cells co-cultured with HER2-expressing SkBr-3 cells (FIG. 47A), Colo201 cells (FIG. 47B), and HCC1954 cells (FIG. 47C), respectively, as shown by increased CD107a degranulation and production of IFN-gamma. SkBr-3 cells and HCC1954 cells have high levels of HER2 surface expression, while Colo201 has moderate HER2 expression. Compared to the monoclonal antibody trastuzumab, TriNKETs demonstrate superior activation of human NK cells in the presence of human tumor cells. NK cells individually, NK cells plus SkBr-3 cells were used as negative controls.

[00227] TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) mediate activation of human NK cells co-cultured with CD33-expressing human AML Mv4-11 cells, as shown by increased CD107a degranulation and IFN-gamma production. Compared with anti-CD33 monoclonal antibody, TriNKET (C26-TriNKET-HER2 and F04-TriNKET-HER2) showed superior activation of human NK cells in the presence of human tumor cells expressing HER2 (FIGS. 47A-47C).

Primary human NK cells are activated by TriNKET in coculture with target-expressing human tumor cell lines.

[00228] Co-culture of primary human NK cells with CD20-positive human tumor cells resulted in TriNKET-mediated activation of primary human NK cells (FIG. 62). TriNKETs targeting CD20 (e.g., C26-TriNKET-CD20 and F04-TriNKET-CD20) mediated the activation of human NK cells co-cultured with CD20-positive Raji cells, as evidenced by increased CD107a degranulation and IFNγ cytokine production (FIG. 62). Compared to the monoclonal antibody rituximab, both TriNKETs (eg, C26-TriNKET-CD20 and F04-TriNKET-CD20) showed superior activation of human NK cells (FIG. 62).

Rituximab_vH

Rituximab_vL

[00229] Co-culture of primary human NK cells with BCMA-positive MM.1S myeloma cells resulted in TriNKET-mediated activation of primary human NK cells. TriNKETs targeting BCMA (e.g., C26-TriNKET-BMCA and F04-TriNKET-BMCA) mediated the activation of human NK cells co-cultured with MM.1S myeloma cells, as evidenced by increased CD107a degranulation and IFNγ cytokine production (FIG. 63). Compared to the TriNKET isotype, TriNKETs targeting BCMA (e.g., A44-TriNKET-BMCA, A49-TriNKET-BMCA, C26-TriNKET-BMCA, F04-TriNKET-BMCA, F43-TriNKET-BMCA, F43-TriNKET-BMCA, F47-TriNK-BMCA and F63-TriNKET-BMCA) showed increased NK cell activity (FIG. 63).

Example 12 Trispecific binding proteins provide cytotoxicity to target tumor cells.

[00230] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56 ⁺ ) were isolated using negative selection using magnetic beads from PBMC, and the purity of the isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

[00231] To test the ability of human NK cells to lyse tumor 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 tumor cells expressing tumor antigen were collected, washed and resuspended in culture medium at a concentration of 1-2×10 ⁵ /ml. Resting and/or activated NK cells were collected, washed and resuspended at 10 ⁵ -2×10 ⁶ /ml in the same culture medium as the tumor cells. In each well of a 96-well plate, 50 μl of tumor cell suspension was mixed with 50 μl of NK cell suspension with or without TriNKET targeting a tumor antigen expressed on tumor cells. After incubation at 37°C with 5% CO2 for 3 hours and 15 minutes, 10× lysis buffer was added to wells containing only tumor cells and to wells containing only maximum lysis and negative control reagent media, respectively. The plate was then placed back into the incubator for an additional 45 minutes to achieve a total of 4 hours of incubation. The cells were then pelleted and the culture supernatant was transferred to a new 96-well plate and mixed with the development substrate. The new plate was incubated for 30 minutes at room temperature and the absorbance was read at 492 nm on a SpectraMax i3x. The percentage of specific lysis of tumor cells was calculated as follows: % Specific lysis = ((experimental lysis - spontaneous lysis from NK cells alone - spontaneous lysis from tumor cells alone)/(Maximum lysis - negative control reagents)) x 100%.

[00232] TriNKETs mediate the cytotoxicity of human NK cells against the CD33-positive Molm-13 human AML cell line. As shown in FIG. 53B, resting human NK cells were mixed with Molm-13 tumor cells, and TriNKET (eg, C26-TriNKET-CD33 and F04-TriNKET-CD33) were able to enhance the cytotoxic activity of resting human NK cells in a dose-dependent manner against tumor cells. The dotted line indicates the cytotoxic activity of resting NK cells without TriNKET. Activated human NK cells were mixed with Molm-13 tumor cells, and TriNKET further enhanced the cytotoxic activity of activated human NK cells, compared to anti-CD33 antibody, in a dose-dependent manner against tumor cells (FIG. 53B).

[00233] TriNKETs enhance the cytotoxicity of NK cells against low surface expression targets compared to the cytotoxic activity of trastuzumab, an anti-HER2 monoclonal antibody. Resting human NK cells were mixed with SkBr tumor cells expressing high levels of HER2 and tumor cells 786-O expressing low levels of HER2, and the ability of TriNKET to enhance the cytotoxic activity of resting human NK cells against tumor cells expressing high levels of HER2 was analyzed. and at low levels in a dose-dependent manner. The dotted lines in FIG. 50A and FIG. 50B indicate the cytotoxic activity of resting NK cells against tumor cells in the absence of TriNKET. As shown in FIG. 50B, after mixing activated human NK cells with 786-O cells expressing low levels of HER2 and TriNKET (e.g. CD26-TriNKET-HER2 and F04-TriNKET-HER2), dose-dependent cytotoxic activity of activated human NK cells against tumors was observed cells.

[00234] TriNKET-mediated lysis of BCMA-positive myeloma cells was analyzed. FIG. 64 shows TriNKET-mediated lysis of BCMA-positive KMS12-PE myeloma cells by resting human effector NK 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 the same extent, but TriNKETs using the EM-901-targeting domain provided increased activity.

[00235] FIG. 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. Altering the NKG2D-binding domain in BCMA-targeting TriMAKET resulted in changes in maximum killing as well as TriNKET efficiency. All TriNKETs demonstrated increased killing of KMS12-PE target cells compared to monoclonal antibody EM-901 (FIG. 65).

Example 13

[00236] Synergistic activation of human NK cells by cross-linking NKG2D and CD 16 was studied.

Primary Human NK Cell Activation Assay

[00237] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy platelet layer of peripheral blood using density gradient centrifugation. NK cells were purified from PBMCs using negative magnetic beads (StemCell #17955). NK cells were >90% CD3-CD56 ⁺ as determined by flow cytometry. Cells were then expanded for 48 hours in medium containing 100 ng/ml hIL-2 (Peprotech #200-02) before use in activation assays. Antibodies were applied to a 96-well flat bottom plate at a concentration of 2 μg/ml (anti-CD16, Biolegend #302013) and 5 μg/ml (anti-NKG2D, R&D #MAB139) in 100 μl sterile PBS overnight at 4°C followed by thorough washing of the wells to remove excess antibodies. To assess degranulation, IL-2-activated NK cells were resuspended at a concentration of 5x10 ⁵ cells/ml in culture medium supplemented with 100 ng/ml hIL2 and 1 μg/ml APC-conjugated anti-CD107a mAb (Biolegend #328619). Then 1×10 ⁵ cells/well were added to the antibody-coated plates. The protein transport inhibitors Brefeldin A (BFA, Biolegend #420601) and Monensin (Biolegend #420701) were added at final dilutions of 1:1000 and 1:270, respectively. The seeded cells were incubated for 4 hours at 37°C in 5% CO2. For intracellular IFN-γ staining, NK cells were labeled with anti-CD3 (Biolegend #300452) and anti-CD56 mAb (Biolegend #318328) and then fixed and permeabilized and labeled with anti-IFN-γ mAb (Biolegend #506507), NK- cells were analyzed for CD107a and IFN-γ expression by flow cytometry after gating of live CD56 ⁺ CD3 − cells.

[00238] To investigate the relative effectiveness of the receptor combination, cross-linking of NKG2D or CD16 and co-cross-linking of both receptors through stimulation in a plate-bound state were performed. As shown in FIG. 45 (FIG. 45A-45C), combined stimulation of CD16 and NKG2D resulted in highly elevated CD107a levels (degranulation) (FIG. 45A) and/or IFN-γ production (FIG. 45B). The dotted lines represent the additive effect of individual stimulations of each receptor.

[00239] CD107a levels and intracellular IFN-γ production of IL-2-activated NK cells were analyzed after 4 hours of stimulation in the plate-bound state with anti-CD16, anti-NKG2D, or a combination of both monoclonal antibodies. Graphs show mean (n=2) ± SD. FIG. 45A shows CD107a levels; FIG. 45B shows IFNγ levels; FIG. 45C demonstrates CD107a and IFN-γ production levels. The data shown in FIG. 45A-45C represent five independent experiments using five different healthy donors.

[00240] CD107a degranulation and intracellular IFN-γ production of IL-2-activated NK cells were analyzed after 4 hours of stimulation in the plate-bound state with trastuzumab, anti-NKG2D, or TriNKET derived from the binding domains of trastuzumab and anti-NKG2D antibody (FIG. 46). In all cases, the antibodies tested were of the human IgG1 isotype. Graphs show mean (n=2) ± SD.

Example 14

Assessment of TriNKET binding to cell-expressed human NKG2D

[00241] EL4 cells transduced with human NKG2D were used to test binding to cell-expressed human NKG2D. TriNKET was diluted to 20 μg/ml and then serially diluted. Dilutions of mAb or TriNKET were used to stain cells, and TriNKET or mAb binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and MFI binding was normalized to secondary control antibodies to obtain excess relative to background values.

Evaluation of TriNKET binding to human tumor antigens expressed by cells

[00242] Human tumor cell lines expressing CD33 or HER2 were used to assess tumor antigen binding to TriNKETs derived from various NKG2D-targeting clones. The human AML cell line MV4-11 was used to assess the binding of TriNKET to cell-expressed CD33. The human renal cell carcinoma cell line 786-O expresses low levels of HER2 and was used to assess the binding of TriNKET to cell-expressed HER2. TriNKET was diluted to 20 μg/ml and incubated with the appropriate cells. TriNKET binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, MFI binding to cell-expressed CD33, and HER2 was normalized to secondary antibody controls to obtain elevations over background values.

Determination of the ability of antibodies to bind to HER2-positive cells of human tumor cell lines

[00243] The antibody binding capacity (ABC) of HER2-positive human tumor cell lines was measured. The Quantum Simply Cellular kit from Bangs Lab (#815) was used and the manufacturer's instructions were followed to make antibody-labeled beads. Briefly, each of the four bead populations was stained with a saturating amount of anti-HER2 antibody, and the cell populations were also stained with a saturating amount of the same antibody. Sampling data were obtained for each granule population as well as for the cell populations. The QuickCal protocol provided with the kit was used to generate a standard curve and extrapolate ABC values for each of the cell lines.

Activation of Primary NK Cells by TriNKET

[00244] PBMC were isolated from the buffy coat of peripheral blood using density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection method with 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 left overnight without the cytokine. IL-2-activated NK cells were used after 24-48 hours; resting NK cells were always used the day after purification.

[00245] Human tumor cell lines expressing the target tumor antigen of interest were harvested from culture and the cells were adjusted to 2×10 ⁶ /ml. Monoclonal antibodies or TriNKETs targeting the target tumor of interest were diluted in culture medium. Resting and/or activated NK cells were collected from the culture, the cells were washed and resuspended at a concentration of 2×10 ⁶ /ml in the culture medium. IL-2 and fluorophore-conjugated anti-CD107a antibody were added to NK cells to activate the culture. Brefeldin-A and monensin were diluted in culture medium to block protein transport out of the cell for intracellular cytokine staining. 50 μL of tumor targets, mAb/TriNKET, BFA/monensin, and NK cells were added to a 96-well plate to a total culture volume of 200 μL. The plate was cultured for 4 hours before samples were prepared for FACS analysis.

[00246] After 4 hours of culture activation, cells were prepared for flow cytometry analysis using fluorophore-conjugated antibodies against CD3, CD56, and IFNγ. CD107a and IFNγ staining were analyzed in CD3 − CD56 ⁺ populations to assess NK cell activation.

Cytotoxicity Analysis of Primary Human NK Cells

[00247] PBMC were isolated from the buffy coat of peripheral blood using density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection method with 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 left overnight without cytokines. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

Cyto Tox 96 LHD Release Assay:

[00248] The ability of human NK cells to lyse tumor cells was measured with or without the addition of TriNKET using the cyto Tox 96 non-radioactive cytotoxicity assay from Promega (G1780). Human cell lines expressing the tumor target of interest were collected from culture, the cells were washed with PBS and resuspended in culture medium at a concentration of 1-2×10 ⁵ /ml for use as target cells. 50 μl of target cell suspension was added to each well. Monoclonal antibodies or TriNKETs targeting the tumor antigen of interest were diluted in culture medium, and 50 μl of the diluted mAb or TriNKET was added to each well. Resting and/or activated NK cells were collected from the culture, the cells were washed and resuspended at a concentration of 10 ⁵ -2×10 ⁶ /ml in culture medium depending on the desired E:T ratio. 50 μl of NK cells was added to each well of the plate to obtain a total of 150 μl of culture volume. The plate was incubated at 37°C with 5% CO2 for 3 hours and 15 minutes. After incubation, 10× lysis buffer was added to target cell-only wells and media-only wells to maximize lysis and volume control. The plate was then placed back into the incubator for an additional 45 minutes to bring the total development incubation time to 4 hours.

[00249] After incubation, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 50 μl of culture supernatant was transferred to a clean microplate, and 50 μl of substrate solution was added to each well. The plate was protected from light and incubated for 30 minutes at room temperature. 50 μL of stop solution was added to each well and absorbance was measured 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:

[00250] Human tumor cell lines expressing the target of interest were harvested from culture, the cells were washed with PBS and resuspended in growth medium at a concentration of 10 ⁶ /ml for labeling with BATDA reagent (Perkin Elmer AD0116). The manufacturer's instructions for labeling target cells were followed. After labeling, cells were washed 3 times with PBS and resuspended at 0.5-1×10 ⁵ /ml in culture medium. To prepare background wells, an aliquot of labeled cells was removed and the cells were removed from the medium. 100 μl of medium was carefully added to the wells in triplicate to avoid damaging the pelleted cells. 100 μl of BATDA-labeled cells was added to each well of a 96-well plate. Wells were reserved for spontaneous release from target cells, and wells were prepared to maximize target cell lysis by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the target tumor 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 collected from the culture, the cells were washed and resuspended at a concentration of 10 ⁵ -2×10 ⁶ /ml in culture medium depending on the desired E:T ratio. 50 μl of NK cells was added to each well of the plate to obtain a total of 200 μl of culture volume. The plate was incubated at 37°C with 5% CO2 for 2-3 hours before analysis.

[00251] After culturing for 2-3 hours, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μl of the culture supernatant was transferred into a clean microplate provided by the manufacturer, and 200 μl of europium solution at room temperature was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plate was read using Victor 3 or SpectraMax i3X instruments. % Specific Lysis was calculated as follows: % Specific Lysis = ((Experimental release - spontaneous release)/(Maximum release - spontaneous release)) * 100%.

Long-term cytotoxicity assay for human PBMCs:

[00252] SkBr-3 target cells were labeled using BacMam 3.0 NucLight Green (#4622) to track target cells. The manufacturer's protocol was followed to label target cells with SkBr-3. 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 the wells of a 96-well plate already containing labeled SkBr-3 cells; 50 μl of complete culture medium was added to a total culture volume of 200 μl.

[00253] Image acquisition has been configured on the IncuCyte S3. Images for the phase, green, and red channels were collected every hour, 2 images per well. Image analysis was performed using IncuCyte S3 software. Screens 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 % Annexin V-positive Mv4-11 target cells. % Annexin V-positive SkBr-3 cells = ((number of overlapping objects)/(number of green objects)) * 100%.

Comparison of TriNKET, which targets HER ⁺ tumor cells, with SC2.2

[00254] TriNKET targeting HER2 is more effective than trastuzumab in reducing SkBr-3 cells, with only 60% of cells from time zero remaining at 60 hours. The TriNKETs of the present invention, which target HER2-expressing tumors/tumor cells, are more effective than SC2.2, a single-chain bispecific molecule constructed from a trastuzumab-derived scFv bound to ULBP-6, a ligand for NKG2D. SC2.2 binds HER2 ⁺ tumor cells and NKG2D ⁺ NK cells simultaneously. Therefore, the effectiveness of SC2.2 in reducing the number of HER2+ tumor cells was investigated. In vitro activation and cytotoxicity studies showed that SC2.2 was effective in activating and killing NK cells. However, SC2.2 failed to demonstrate efficacy in the RMA/S-HER2 subcutaneous tumor model. The efficacy of SC2.2 was also tested in vivo using a syngeneic mouse model overexpressing RMA/S-HER2. In this mouse model, SC2.2 failed to demonstrate tumor growth control compared to vehicle controls. Thus, although SC2.2 was able to activate and kill NK cells and bind to HER2 ⁺ tumor cells, these properties were insufficient to effectively control HER2 ⁺ tumor growth.

Estimation of SC2.2 half-life from serum in C57Bl/6 mice

[00255] To determine the serum half-life of SC2.2 in C57Bl/6 mice, SC2.2 was fluorescently labeled to monitor its concentration in vivo. SC2.2 was labeled with IRDye 800CW (Licor #929-70020). The labeled protein was injected intravenously into 3 C57Bl/6 mice, and blood was collected from each mouse at the indicated time points. After collection, blood was centrifuged at 1000 g for 15 minutes and serum was collected from each sample and stored at 4°C until all time points were collected.

[00256] Serum was imaged using an Odyssey CLx infrared imaging system, and fluorescence signal from channel 800 was quantified using Image J software. Image intensities were normalized to the first time point, and the data were fit to the biphasic decay equation. In this experimental system, the half-life of beta SC2.2 was calculated to be approximately 7 hours.

In vivo testing of SC2.2 against RMA/S-HER2 subcutaneous tumors

[00257] The in vivo study was designed in accordance with FIG. 56 to test the efficacy of SC2.2 against subcutaneous RMA/S‐HER2 tumors. 10 ⁶ RMA/S cells transduced with human HER2 were injected subcutaneously into the flank of 20 C57Bl/6 mice. Starting on day 2 after tumor inoculation, SC2.2 was administered daily intraperitoneally. SC2.2 was dosed at high and low concentrations along with a vehicle control. Beginning on day 4 after tumor inoculation, tumors were measured on Monday, Wednesday, and Friday throughout the study. Tumor volume was calculated using the following formula: tumor volume=length x width x height

TriNKET binds to cells expressing human NKG2D

[00258] The ability of TriNKET to bind to cells expressing human NKG2D was determined. In FIG. 37 and FIG. 38 shows dose-dependent binding of two TriNKETs containing different NKG2D-binding domains. In FIG. 37 shows the binding of two TriNKETs when the CD33 binding domain is used as the second targeting moiety. In FIG. 38 shows the same two NKG2D binding domains now coupled to a second HER2 targeting moiety. The six NKG2D-binding domains retain the same binding profile with both tumor-targeting domains.

TriNKETs bind to cells expressing human tumor antigens

[00259] The ability of TriNKET to bind to cells expressing human tumor antigens was determined. In FIG. 41 and FIG. 42 shows the binding of TriNKET to CD33 expressed by cells (FIG. 41) and HER2 (FIG. 42). Binding of TriNKET to cell-expressed antigen was coordinated between NKG2D-binding domains. TriNKET bound at comparable levels as the parental monoclonal antibody.

Ability of antibodies to bind to human HER2-positive cells of human tumor cell lines

[00260] Table 9 shows the results of quantitation of surface HER2. SkBr-3 and HCC1954 cells were found to have high (+++) levels of surface HER2. ZR-75-1 and Colo201 showed moderate levels (++) of surface HER2, and 786-O showed the lowest level of HER2 (+).

[00261]

Table 9: ABC of HER2-positive tumor cell lines

Table 9: ABC of HER2-positive tumor cell lines Cell line HER2 expression level ABC 786-0 Short 28162 Colo201 Average 273568 ZR-75-1 Average 281026 SkBr-3 High 6820532 HCC1954 High 10569869

Primary human NK cells are activated by TriNKET in co-culture with human tumor cell lines expressing HER2 at various levels

[00262] In FIG. 47A-47C showed 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 production of IFNγ cytokines. Compared with the monoclonal antibody trastuzumab, both TriNKETs demonstrated superior activation of human NK cells by various human HER2 tumor cells.

[00263] In FIG. 47A shows that human NK cells are activated by TriNKET when cultured with SkBr-3 cells. In FIG. 47B shows that human NK cells are activated by TriNKET when cultured with Colo201 cells. In FIG. 47C shows that human NK cells are activated by TriNKET when cultured with HCC1954 cells.

TriNKETs enhance the activity of resting and IL-2-activated human NK cells

[00264] In FIG. 48A-48B show TriNKET-mediated activation of quiescent or IL-2-activated human NK cells in coculture with the CD33-expressing human AML cell line MV4-11. In FIG. 48A shows TriNKET-mediated activation of resting human NK cells. In FIG. 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 a greater change in IFNγ production and CD107a degranulation compared to IL-2-activated NK cells. IL-2-activated NK cells showed a higher percentage of cells becoming IFNγ ⁺ ; CD107a ⁺ after stimulation with TriNKET.

TriNKETs enhance the cytotoxicity of resting and IL-2-activated human NK cells

[00265] In FIG. 49A - 49B show TriNKET enhancement of cytotoxic activity using IL-2-activated and quiescent human NK cells. In FIG. 49A shows the percentage of specific lysis of SkBr-3 tumor cells by resting human NK cells. In FIG. 49B shows the percentage of specific lysis of SkBr-3 tumor cells by IL-2-activated human NK cells. IL-2-activated and resting NK cell populations originate from the same donor. Compared with trastuzumab, TriNKET is more effective at directing responses against SkBr-3 cells by either activated or quiescent NK cell populations.

TriNKETs enhance NK cell cytotoxicity towards low surface expression targets

[00266] In FIG. 50A-50B show that TriNKET provides greater benefit against intermediate and low HER2 cancers compared with trastuzumab. In FIG. 50A shows the destruction of SkBr-3 tumor cells with high levels of HER2 by activated human NK cells. In FIG. 50B shows human NK cell killing of 786-O tumor cells with low HER2 levels. TriNKETs provide greater benefit over trastuzumab against tumor cells with low HER2 expression. TriNKETs provide the greatest benefit against targets with low surface expression.

Advantage of TriNKET in the treatment of malignancy with high levels of FcR expression or in the tumor microenvironment with high levels of FcR

[00267] Monoclonal antibody therapy has been approved for the treatment of many types of malignancies, including both hematologic and solid tumors. Although the use of monoclonal antibodies in the treatment of malignancy has improved patient outcomes, limitations still exist. Mechanistic studies have demonstrated that monoclonal antibodies exert their effects on tumor growth through multiple mechanisms, including ADCC, CDC, phagocytosis, and signal blockade, among others.

[00268] In particular, ADCC is believed to be the primary mechanism by which monoclonal antibodies exert their effects. ADCC is based on the interaction of low-affinity Fc antibodies Fc γRIII (CD16) on the surface of natural killer cells, which provide direct lysis of the tumor cell. Among FcγRs, CD16 has the lowest affinity for IgG Fcs, FcγRI (CD64) is a high affinity FcR and binds approximately 1000 times more strongly to IgG Fcs than CD16.

[00269] CD64 is typically expressed on cells of many hematopoietic lineages, such as the myeloid lineage, and can be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor-infiltrating immune cells, such as MDSCs and monocytes, also express CD64 and are known to infiltrate into the tumor microenvironment. Expression of CD64 by the tumor or in the tumor microenvironment may have a detrimental effect on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to interact with CD16 on the surface of NK cells, as the antibodies prefer to bind to the high-affinity receptor. By targeting two activating receptors on the surface of NK cells, TriNKETs may overcome the detrimental effects of CD64 expression on monoclonal antibody therapy.

Expression of FcRγI (CD64) in three AML cell lines

[00270] An in vitro culture system was developed to test the activity of TriNKET and monoclonal antibodies against tumors with high and low levels of CD64 surface expression. Molm-13 and THP-1 are two human AML cell lines that have similar expression of surface CD33, but Molm-13 cells do not express CD64, while THP-1 cells express CD64 on their surface (FIGS. 51A - 51C ). Using monoclonal antibodies or TriNKET targeting CD33, the effect of tumor CD64 expression on monoclonal antibodies or TriNKET therapy was tested. In FIG. 51A-51C show expression of high affinity FcRγI (CD64) on three human AML cell lines, the Molm-13 cell line (FIG. 51A), the Mv4-11 cell line (FIG. 51B) and the THP-1 cell line. (FIG. 51C). Molm-13 cells do not express CD64, while Mv4-11 cells have low levels and THP-1 cells have high levels of CD64 on the cell surface.

TriNKETs have an advantage in targeting tumor cells with high surface FcR expression

[00271] In FIG. 52A-52B show monoclonal antibody or TriNKET mediated activation of human NK cells in coculture with either Molm-13 (FIG. 52B) or THP-1 (FIG. 52A) cells. Anti-human CD33 monoclonal antibody demonstrated good activation of human NK cells in the Molm-13 co-culture system, as evidenced by increased CD107a degranulation and IFNγ production. The monoclonal antibody has no effect in a THP-1 coculture system where the tumor has high levels of CD64. Interestingly, TriNKETs were effective against both Molm-13 (FIG. 52B) and THP-1 (FIG. 52A) cells, while monoclonal antibodies failed to activate NK cells in culture with FcR-Hi THP- cells. 1, indicating the ability of TriNKET to overcome binding to CD64 on tumors and effectively target NK cells for activation. Dual targeting of two activating receptors to NK cells provided stronger specific binding to NK cells. Monoclonal antibodies that target only CD16 on NK cells can bind to other high-affinity FcRs and prevent CD16 recruitment on NK cells.

[00272] Human NK cell cytotoxicity assays using Molm-13 and THP-1 coculture systems provide further evidence supporting the efficacy of TriNKET in the presence of high levels of CD64. A third human AML cell line, Mv4-11, was used in these cytotoxicity assays. Mv4-11 cells (FIG. 51B) express CD64 at low levels and fall between THP-1 (FIG. 51C) and Molm-13 (FIG. 51A) cells in terms of the level of CD64 on their surface.

TriNKETs demonstrate efficacy in AML cell lines independent of FcγRI expression

[00273] In FIG. 53A-53C show human NK cell cytotoxicity assays using three human AML cell lines as targets. Anti-CD33 monoclonal antibody shows good activity against Molm-13 cells (FIG. 53B), which do not express CD64. Mv4-11 cells (FIG. 53A), which express CD64, but at a lower level than THP-1, showed reduced efficacy with the anti-CD33 monoclonal antibody. THP-1 cells (FIG. 53C) had no effect with anti-CD33 monoclonal antibody alone. Regardless of CD64 expression on tumor cells, TriNKETs were able to mediate human NK cell responses against all tumor cells tested herein.

[00274] In FIG. 53A-53C showed that THP-1 cells were protected from monoclonal antibody therapy due to high levels of high-affinity FcR expression on their surface. TriNKETs bypassed this defense by targeting two activating receptors on the surface of NK cells. The cytotoxicity data directly correlated with what was seen in the coculture activation experiments. TriNKETs were able to bypass the protection from mAb therapy observed with THP-1 cells and induce NK cell-mediated lysis despite high FcR levels.

Depletion of normal myeloid and normal B cells in cultured PBMCs: TriNKETs provide a better safety profile due to less off-tumor mistargeting

[00275] Natural killer cells and CD8 T cells are able to directly lyse tumor cells, although the mechanisms by which NK cells and CD8 T cells recognize their own healthy cells from tumor cells are different. The activity of NK cells is regulated by the balance of signals from activating (NCR, NKG2D, CD16, etc.) and inhibitory (KIR, NKG2A, etc.) receptors. The balance of these activating and inhibitory signals allows NK cells to identify healthy self cells from stressed, virus-infected, or transformed self cells. This “built-in” self-tolerance mechanism will help protect normal healthy tissue from NK cell responses. To extend this principle, NK cell self-tolerance will allow TriNKET to target both self-expressed and tumor cell-expressed antigens without the side effects associated with off-tumor mistargeting or an extended therapeutic window.

[00276] Unlike natural killer cells, T cells require recognition of a specific peptide presented by MHC molecules for activation and effector functions. T cells have been a major target of immunotherapy, and many strategies have been developed to redirect T cell responses against the tumor. Bispecific T cells, checkpoint inhibitors, and CAR T cells are FDA approved but often suffer from dose-limiting toxicity. Bispecific T cells and CAR-T cells function around the TCR-MHC recognition system, using binding domains to target antigens on the surface of tumor cells and using engineered signaling domains to transmit activation signals to the effector cell. Although these treatments are effective in eliciting an antitumor immune response, they are often associated with cytokine release syndrome (CRS) and side effects associated with off-tumor mistargeting. In this context, TriNKETs are unique because they do not “override” the natural NK cell activation and inhibition systems. Instead, TriNKETs are designed to alter the balance and provide additional activation signals to NK cells while maintaining NK tolerance to self cells.

[00277] PBMCs were isolated from whole blood by density gradient centrifugation. Any contaminating erythrocytes were lysed by incubation in ACK lysis buffer. PBMCs were washed 3 times in PBS, and the total number of PBMCs was counted. PBMC were adjusted to 10 ⁶ /ml in primary cell culture medium. 1 mL of PBMC was seeded into wells of a 24-well plate, and the indicated TriNKET or mAb was added to the PBMC cultures at a concentration of 10 μg/mL. Cells were cultured overnight at 37°C with 5% CO2. The next day (24 hours later), PBMCs were harvested from culture and prepared for FACS analysis. CD45 ⁺ percentage; CD19 ⁺ B cells and CD45 ⁺ ; CD33 ⁺ ; CD11b ⁺ myeloid cells were analyzed in different treatment groups.

[00278] In FIG. 54B and 54D show that autologous myeloid cells are protected from TriNKET-mediated NK cell responses. In FIG. 54A and 54B show that healthy donor B cells are sensitive to TriNKET-mediated lysis, while myeloid cells are resistant to TriNKET lysis. PBMCs treated with TriNKET targeting CD20 showed a reduced frequency of CD19 ⁺ B cells with a population of CD45 ⁺ lymphocytes (FIG. 54A), but had no effect on the population of CD45 ⁺ , CDD3-, CD56 lymphocytes (FIG. 54C). In these cultures, the frequency of CD45 ⁺ , CD19 ⁺ myeloid cells (FIG. 54B) or the frequency of CD33 ⁺ , CD33 ⁺ , CD11b ⁺ myeloid cells (FIG. 54D) did not change.

TriNKETs mediate the killing of hPBMC SkBr-3 tumor cells in long-term co-cultures

Cytotoxicity assay of primary human PBMCs

[00279] In FIG. 55 shows long-term killing of SkBr-3 cells in culture with human PBMC. When cultured alone, SkBr-3 cells proliferate and nearly double in size within 60 hours. When human PBMCs are added to SkBr-3 cells in culture, the rate of proliferation is slowed, and when the isotype control TriNKET targeting CD33 is added, proliferation is also slowed, but to a lesser extent. When cultures are treated with trastuzumab, SkBr-3 no longer proliferates, and after 60 hours only 80% of cells remain relative to time zero. Because SkBr-3 cells are sensitive to HER2 signal blockade, the effect on SkBr-3 cell growth may be mediated by HER2 signal blockade or through Fc effector functions such as ADCC.

Example 15

Antitumor efficacy of mcFAE-C26.99 TriNKET in vitro

[00280] To test the binding activity of murine cFAE-C26.99 TriNKET, direct binding was measured in comparison to its monoclonal antibodies using flow cytometry assays against Tyrp-1-positive B16F10 melanoma cells (Figure 58A) and an EL4 cell line overexpressing murine NKG2D (EL4-mNKG2D, FIG. 58B).

[00281] To test whether mcFAE-C26.99 TriNKETs retain the ability to mediate cytotoxicity, killing of Tyrp-1-positive B16F10 tumor targets by murine IL-2-activated NK cells was measured. As shown in FIG. 59, murine NK cells increased their cytotoxic activity in the presence of mcFAE-C26.99. It is important to note that the anti-Tyrp-1 monoclonal antibody TA99 showed only minor effects.

Increased NK cytotoxicity mediated by mcFAE-C26.99 TriNKET

[00282] Approximately 5 x 103 B16F10 melanoma cells per well were seeded two days prior to analysis. On the day of the experiment, 5x10 ⁴ mouse 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 mutations include to heterodimerization mutations used to produce a heterodimer). 20 μg/ml antibodies with fourfold dilutions were used. After 4 hours of co-culture, the percentage of cytotoxicity was assessed using the CytoTox96 LDH release kit. The dotted line represents baseline cytotoxicity in the absence of antibodies.

mC26_hvL_mCL (bold) (italicized and underlined amino acids are heterodimerization mutations used to produce the heterodimer):

DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY KASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYGSFPIT FGGGTKVEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSSTSPIVKSFNRNEC (SEQ ID NO:86)

mC26_hvH_ IgG2C GmB

QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYYWS WIRQPPGKGLEWIG EIDHSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR ARGPWSFDP WGQGTLVTVSS AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQ TITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMI K GFLPAEIAVDWT SNGRTEQNYKNTATVLDSDGSYFMYS R LRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK (SEQ ID NO:87)

TA99_mvL_ mCL

DIQMSQSPASLSSASVGETVTITC RASGNIYNYLA WYQQKQGKSPHLLVY DAKTLAD GVPSRFSGSGSGTQYSLKISSLQTEDSGNYYC QHFWSLPFT FGSGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS YTCEATHKTSSTSPIVKSFNRNEC (SEQ ID NO:88)

TA99_mvH_ IgG2C GmA

EVQLQQSGAELVRPGALVKLSCKTS GFNIKDYFLH WVRQRPDQGLEWIG WINPDNGNTVYDPKFQG TASLTADTSSNTVYLQLSGLTSEDTAVYFCTR RDYTYEKAALDY WGQGASVIVSS AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTW PSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVD WTSNGRTEQNYKNTATVLDSDGSY L MYSKL T VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK (SEQ ID NO:89)

Example 16

Antitumor efficacy of mcFAE-C26.99 TriNKET in vivo

[00283] To test whether mcFAE-C26.99 induces antitumor functions in vivo, C57BL/6 mice were injected subcutaneously with 2×10 ⁵ B16F10 tumor cells. Mice were treated with either the isotype control monoclonal antibody TA99 or TriNKET mcFAE-C26.99. Treatment with the monoclonal antibody TA99 showed similar tumor progression as the isotype-treated control group. However, administration of mNFAE-C26.99 TriNKET resulted in a delay in tumor progression compared with the isotype group. Approximately 2 x 10 ⁵ B16F10 melanoma cells were injected subcutaneously into the flank of C57BL/6 mice. On day 6 after tumor inoculation, mice were randomized (n=10 per group). Mice were treated intraperitoneally (FIG. 60A) with isotype control mouse IgG2a mab C1.18.4 and mouse anti-Tyrp-1 monoclonal antibody or (FIG. 60B) with isotype control mouse IgG2a ma C1.18.4 and mcFAE-C26.99 TriNKET, which were injected at 150 mcg (days 6, 8, 10, 12, 14, 16 and 21). Tumor growth was assessed over 28 days. Graphs show tumor growth curves of individual mice.

[00284] In addition to the B16F10 subcutaneous tumor model, mcFAE-C26.99 TriNKET was also tested for its antitumor efficacy in the disseminated tumor setting. 1×10 ⁵ B16F10 cells were injected into mice intravenously. Treatment began on either day 4 or 7 with low (300 μg/injection) and high (600 μg/injection) doses of antibody. On day 18 after inoculation, lung metastases were counted. Treatments initiated on days 4 and 7 after tumor inoculation resulted in a reduction in lung metastases when monoclonal antibody TA99 or mcFAE-C26.99 TriNKET was used at high concentrations compared with the isotype control group. At low concentrations, only mcFAE-C26.99 TriNKET reduced tumor burden (FIG. 61A). Similar effects were observed when antibodies were administered starting at day 7 after tumor inoculation. Overall, mcFAE-C26.99 TriNKET therapy resulted in a reduction in lung metastases compared with monoclonal antibody TA99 in all conditions tested. Approximately 1x10 ⁵ B16F10 melanoma cells were injected intravenously into the tail vein of C57BL/6 mice (n=8 per group). Mice were either left untreated or treated intraperitoneally with control mab (isotype, clone C1.18.4), TA99 monoclonal antibody, or Tri99KET TA99 (mcFAE-C26.99). FIG. 61A represents the tumor load when antibodies were administered at a dose of 150 μg (days 4, 6, 8, 11, 13, 15). FIG. 61B represents the tumor load when antibodies were administered at a dose of 150 μg (days 7, 9, 11, 13, 15). 18 days after tumor challenge, mice were sacrificed and superficial lung metastases were assessed (FIG. 61B).

Example 17

Cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies or bispecific antibodies against HER2-positive cells

[00285] PBMC were isolated from the buffy coat of peripheral blood using density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a negative selection method with 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 left overnight without cytokines. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

[00286] DELFIA Cytotoxicity Assay:

[00287] Human tumor cell lines expressing the target of interest were harvested from culture, the cells were washed with HBS and resuspended in growth medium at a concentration of 10 ⁶ ml/ml for labeling with BATDA reagent (Perkin Elmer AD0116). The manufacturer's instructions for labeling target cells were followed. After labeling, the cells were washed 3 times with HBS and resuspended at a concentration of 0.5-1×10 ⁵ /ml in culture medium. To prepare background wells, an aliquot of labeled cells was removed and the cells were removed from the medium. 100 μl of medium was carefully added to the wells in triplicate to avoid damaging the pelleted cells. 100 μl of BATDA-labeled cells was added to each well of a 96-well plate. Wells were maintained for spontaneous release from target cells, and wells were prepared to maximize target cell lysis by adding 1% Triton-X. Monoclonal antibodies or TriNKET against the target tumor 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 collected from the culture, the cells were washed and resuspended at a concentration of 10 ⁵ -2×10 ⁶ /ml in culture medium depending on the desired E:T ratio. 50 μl of NK cells was added to each well of the plate to obtain a total of 200 μl of culture volume. The plate was incubated at 37°C with 5% CO2 for 2-3 hours before analysis.

[00288] After culturing for 2-3 hours, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 minutes. 20 μL of culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 μL of europium solution at room temperature was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plate was read using Victor 3 or SpectraMax i3X instruments. % Specific Lysis was calculated as follows: % Specific Lysis = ((Experimental release - spontaneous release)/(Maximum release - spontaneous release)) * 100%.

The combination of a monoclonal antibody and a bispecific NK cell recruiter does not recapitulate the activity of TriNKET:

[00289] In FIG. 66 shows the cytotoxic activity of resting human NK cells mediated by TriNKET, monoclonal antibodies, or bispecific antibodies against the HER2-positive cell line Colo-201. TriNKET (ADI-29404 (F04)), targeting HER2, induced maximal lysis of Colo-201 cells by resting human NK cells. The D265A mutation was introduced into the CH2 domain of TriNKET to abolish FcR binding. HER2-TriNKET (ADI-29404 (F04)) - D265A fails to mediate lysis of Colo-201 cells, demonstrating the importance of dual targeting of CD16 and NKG2D on NK cells. To further demonstrate the importance of dual NK cell targeting, the 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 approximately 4 times lower compared to TriNKET. To understand the importance of targeting CD16 and NKG2D to the same molecule, the activity of TriNKET (ADI-29404 (F04)) was compared with that of a bispecific antibody targeting HER2 and NKG2D in combination with trastuzumab. When used in equimolar concentrations, the combination of a bispecific antibody and trastuzumab is not able to provide maximum lysis of Colo-201 cells by resting human NK cells. The disadvantage of the trastuzumab+bispecific antibody combination demonstrates the importance of containing the trispecific binding TriNKET in a single molecule.

INCLUSION VIA LINK

[00290] The entire disclosure of each of the patent documents and scientific articles mentioned herein is incorporated by reference for all purposes.

Equivalents

[00291] The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Thus, the above embodiments should be considered in all respects as illustrative and not limiting of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims and not by the preceding description, and all modifications that come within the meaning and scope of the claims are intended to be included therein.

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LIST OF SEQUENCES

<110> ADIMAB, LLC.

<120> POLYSPECIFIC BINDING PROTEINS FOR CELL ACTIVATION -

NATURAL KILLERS AND THEIR THERAPEUTIC USES FOR TREATMENT

MALIGNANT NEOPLASURE

<130>DFY-001PC

<140>

<141>

<150> 62/456.535

<151> 2017-02-08

<160> 89

<170> Patent In version 3.5

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Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 6

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 7

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 7

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Tyr Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 9

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 11

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 12

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 12

Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 13

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 15

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 17

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 18

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 19

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ile Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 21

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 23

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 25

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 26

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 27

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 29

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Ser Tyr Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 31

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 32

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 33

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 34

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 35

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 37

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Leu Tyr Ser Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 39

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 40

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 42

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 43

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 43

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 44

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Thr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 45

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 46

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 47

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 50

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 50

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Asp Tyr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 51

Phe Thr Phe Ser Ser Tyr Ala Met Ser

15

<210> 52

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 52

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 53

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 53

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr

1 5 10

<210> 54

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 54

Arg Ala Ser Gln Gly Ile Asp Ser Trp Leu Ala

1 5 10

<210> 55

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 55

Ala Ala Ser Ser Leu Gln Ser

15

<210> 56

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 56

Gln Gln Gly Val Ser Tyr Pro Arg Thr

15

<210> 57

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 57

Phe Thr Phe Ser Ser Tyr Ser Met Asn

15

<210> 58

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 58

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 59

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 59

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro

1 5 10 15

<210> 60

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 60

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 61

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 61

Ala Ala Ser Ser Leu Gln Ser

15

<210> 62

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 62

Gln Gln Gly Val Ser Phe Pro Arg Thr

15

<210> 63

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 63

Tyr Thr Phe Thr Gly Tyr Tyr Met His

15

<210> 64

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 64

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 65

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 65

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

1 5 10 15

Val

<210> 66

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 66

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 67

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 67

Gly Ala Ser Thr Arg Ala Thr

15

<210> 68

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

peptide

<400> 68

Gln Gln Asp Asp Tyr Trp Pro Pro Thr

15

<210> 69

<211> 121

<212> Protein

<213> Homo sapiens

<400> 69

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 70

<211> 110

<212> Protein

<213> Homo sapiens

<400> 70

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 71

<211> 115

<212> Protein

<213> Homo sapiens

<400> 71

Gln Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Asp Asp Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Ser Tyr Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Asn Trp Asp Asp Ala Phe Asn Ile Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 72

<211> 108

<212> Protein

<213> Homo sapiens

<400> 72

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 73

<211> 246

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 73

Met Ala Ala Ala Ala Ile Pro Ala Leu Leu Leu Cys Leu Pro Leu Leu

1 5 10 15

Phe Leu Leu Phe Gly Trp Ser Arg Ala Arg Arg Asp Asp Pro His Ser

20 25 30

Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg

35 40 45

Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr

50 55 60

Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys

65 70 75 80

Leu Asn Val Thr Met Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu

85 90 95

Val Val Asp Ile Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu Glu Asn

100 105 110

Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

115 120 125

Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Ile Asp

130 135 140

Gly Gln Thr Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr

145 150 155 160

Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys

165 170 175

Asp Val Ala Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys Ile Gly

180 185 190

Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

195 200 205

Ala Gly Ala Pro Leu Ala Met Ser Ser Gly Thr Thr Gln Leu Arg Ala

210 215 220

Thr Ala Thr Thr Leu Ile Leu Cys Cys Leu Leu Ile Ile Leu Pro Cys

225 230 235 240

Phe Ile Leu Pro Gly Ile

245

<210> 74

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 75

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 75

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asn

65 70 75 80

Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Trp Arg Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 76

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 76

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn

20 25 30

Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Pro Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 77

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

polypeptide

<400> 77

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Glu

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile His Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro

85 90 95

Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic polypeptide

<400> 78

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 79

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic polypeptide

<400> 79

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Glu Gln Tyr Asp Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 126

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 81

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 81

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 82

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 82

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 83

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 83

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro

85 90 95

Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 84

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 84

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 85

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 85

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 86

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 87

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 87

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu

115 120 125

Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr Leu Gly Cys

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser

145 150 155 160

Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Leu Leu Gln Ser

165 170 175

Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Asn Thr Trp

180 185 190

Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr

195 200 205

Lys Val Asp Lys Lys Ile Glu Pro Arg Val Pro Ile Thr Gln Asn Pro

210 215 220

Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu

245 250 255

Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

275 280 285

Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

290 295 300

Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

305 310 315 320

Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln

340 345 350

Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe

355 360 365

Ser Leu Thr Cys Met Ile Lys Gly Phe Leu Pro Ala Glu Ile Ala Val

370 375 380

Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala

385 390 395 400

Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Arg Leu Arg

405 410 415

Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val

420 425 430

Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg

435 440 445

Ser Leu Gly Lys

450

<210> 88

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 88

Asp Ile Gln Met Ser Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val

35 40 45

Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Ser Leu Gln Thr

65 70 75 80

Glu Asp Ser Gly Asn Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 89

<211> 456

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 89

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Phe Leu His Trp Val Arg Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Thr Ala Ser Leu Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr

65 70 75 80

Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Ala Ser Val Ile Val Ser Ser Ala Lys Thr Thr Ala Pro Ser

115 120 125

Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val

130 135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu

145 150 155 160

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

165 170 175

Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr

180 185 190

Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro

195 200 205

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Val Pro Ile

210 215 220

Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala

225 230 235 240

Pro Asp Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

245 250 255

Lys Asp Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val

260 265 270

Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

275 280 285

Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

290 295 300

Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

305 310 315 320

Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala

325 330 335

Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val

340 345 350

Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr

355 360 365

Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala

370 375 380

Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr

385 390 395 400

Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Leu Met Tyr

405 410 415

Ser Lys Leu Thr Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe

420 425 430

Ala Cys Ser Val Val His Glu Gly Leu His Asn His Leu Thr Thr Lys

435 440 445

Thr Ile Ser Arg Ser Leu Gly Lys

450 455

<---

Claims (20)

1. Polyspecific binding protein that binds to NKG2D, CD16 and tumor-specific antigen, containing: (a) a first antigen binding site comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of an anti-NKG2D antibody; (b) a second antigen-binding site comprising VH and VL antibodies that binds to said tumor-specific antigen; And (c) a first Fc domain of an antibody and a second Fc domain of an antibody that together bind CD16, where VH or VL of the first antigen-binding site is fused to the N-terminus of the first Fc domain of the antibody, and VH or VL of the second antigen-binding site is fused to the N-terminus of the second Fc domain of the antibody. 2. The polyspecific binding protein of claim 1, wherein the first antigen binding site binds to NKG2D in humans, non-human primates and rodents. 3. The polyspecific binding protein according to claim 1 or 2, wherein the VH and VL of the first antigen binding site are present in the same polypeptide. 4. The polyspecific binding protein according to any one of claims 1 to 3, wherein the VL of the first antigen binding site has an amino acid sequence identical to the amino acid sequence of the VL of the second antigen binding site. 5. The polyspecific binding protein according to any one of claims 1 to 4, wherein the VH of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 1. 6. The polyspecific binding protein according to any one of claims 1 to 4, wherein the VH of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 41, and the VL of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 42. 7. The polyspecific binding protein according to any one of claims 1 to 4, wherein the VH of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 43, and the VL of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 44. 8. The polyspecific binding protein according to any one of claims 1 to 4, wherein the VH of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 69, and the VL of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 70. 9. The polyspecific binding protein according to any one of claims 1 to 4, wherein the VH of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 71, and the VL of the first antigen binding site contains the amino acid sequence of SEQ ID NO: 72. 10. The polyspecific binding protein according to any one of claims 1 to 9, wherein the tumor-specific antigen is HER2 or CD20. 11. Polyspecific binding protein according to any one of claims 1-10, where both the first and second Fc domains of the antibody contain a hinge and CH2 domains. 12. The polyspecific binding protein according to any one of claims 1 to 10, wherein the protein contains an amino acid sequence that is at least 90% identical to amino acids 234-332 of the human IgG1 antibody. 13. A pharmaceutical composition for the treatment of a malignant neoplasm, containing an effective amount of a polyspecific binding protein according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier, wherein the tumor-specific antigen is expressed on a malignant cell or in the tumor microenvironment of a malignant neoplasm. 14. A method for enhancing the death of tumor cells, the method comprising exposing the tumor cell and the natural killer cell to a polyspecific binding protein according to any one of claims 1 to 12, where the tumor-specific antigen is expressed on the tumor cell. 15. A method of treating a malignant neoplasm, where the method includes administering to the patient a polyspecific binding protein according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13, where the tumor-specific antigen is expressed on a malignant cell or in the tumor microenvironment of a malignant neoplasm. 16. The method according to claim 15, where the malignancy 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, stomach cancer, gastrointestinal cancer, gastrointestinal stromal tumors, glioblastoma, head and neck cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumors, ovarian and pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, T-cell lymphoma, testicular cancer, thymic carcinoma, thyroid cancer, urothelial cancer, malignant neoplasms infiltrated by suppressor cells of myeloid origin, malignant neoplasms with the accumulation of extracellular matrix, malignant neoplasms with a high level of reactive stroma and malignant neoplasms with neoangiogenesis.

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