EP4705348A1

EP4705348A1 - Nk cell engagers binding to nkp80 and uses thereof

Info

Publication number: EP4705348A1
Application number: EP24800314.7A
Authority: EP
Inventors: Thai Leong YAP; Samantha Jo-Leen WONG; Shuet Theng LEE; Kah Fei WAN; Manuel Adrian SUTER
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2023-05-03
Filing date: 2024-04-29
Publication date: 2026-03-11
Also published as: WO2024228674A1; CN121057751A

Abstract

There is provided a multi-specific polypeptide construct comprising: (a) one or more antigen targeting domains binding to one or more cancer-associated antigens; and (b) one or more NK cell-targeting domains, wherein binding to NK cells may stimulate and/or suppress innate immune cell functions. Also disclosed are an antigen binding protein, or an antigen-binding fragment thereof; a nucleic acid sequence; a vector; a host cell; a method of producing the multi-specific polypeptide construct, or the antibody; a method of screening and/or identifying the multi-specific polypeptide construct, or the antibody as disclosed herein; a pharmaceutical composition; and a method for treating cancer. In a specific embodiment, the NK cell-targeting domain can bind to Nkp80 and/or CD16 and the cancer-associated antigen can be HER2 and/or EGFR.

Description

NK CELL ENGAGERS BINDING TO NKP80 AND USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to the field of multi-specific polypeptide constructs engineered to engage NK cells and bind a cell surface antigen, inducing a desired immune response in various disease indications.

BACKGROUND

[0002] Natural killer (NK) cells are part of the innate immune system, constituting 5-15% of circulating lymphocytes. NK cells perform natural immunosurveillance against stressed cells such as tumor cells or viral-infected cells, triggering their lysis upon identification. Typically, NK cell activity is mediated by a delicate balance between activating and inhibitory receptors expressed on its cell surface. Normal healthy cells express HLA Class I molecules, which suppress NK cells activity by binding to the killer-cell immunoglobulin-like receptors (KIRs) on NK cell surfaces. In contrast, ligands expressed by stressed cells bind to the activating receptors.

[0003] Both activating and inhibitory receptors are expressed on the surface of NK cells, contributing to the execution of the functions performed by the NK cell. Inhibitory receptors specific for MHC-I (major histocompatibility complex class I) antigens tightly regulate NK cell- mediated cytotoxicity and lymphokine production. The inhibitory signal from the MHC-I specific receptor is essential for hematopoietic target cells to avoid destruction by NK cells. This concept is termed the “missing self" and was originally proposed by Ljunggren and Karre. Such MHC-I- recognizing inhibitory receptors form three families of NK-cell surface receptors, namely KIRs (killer cell immunoglobulin-like receptors), LI Rs (Leukocyte immunoglobin-like receptors), and NKG2A (natural killer group 2 A). KIRs, members of the immunoglobulin superfamily, are type I transmembrane molecules that recognize classical human leukocyte antigens A, B, and C (HLA class la). LIRs, also known as ILTs (immunoglobin-like transcripts), form the second set of receptors and mainly recognize non-classical HLA-G (class lb) molecules, in addition to HLA class la. LIRs belong to the same Ig superfamily as KIRs. NKG2A, a member of the NKG2 group of seven receptors, namely A, B, C, D, E, F, and H, dimerizes with CD94 to form the NKG2A/CD94 receptor. It belongs to the C-type lectin family of receptors that recognizes non-classical HLA-E class I molecule as its ligand. [0004] Destruction by NK cells not only require detection of MHC-I molecules on transforming cells by inhibitory receptors but also activation of the NK cell by activating receptors. Natural cytotoxicity receptors (NCRs) represent the group of natural killer cell surface activating receptors that includes NKp46, NKp30, and NKp44. These receptors, as well as NKG2D, DNAM-1 (DNAX accessory molecule-1) and NKp80, recognize ligands expressed on the surface of virally infected or malignantly transformed cells. CD16 (or FcyRIII), also an activating receptor, is expressed mainly by the CD56dim NK-cell subset and is essential for antibody-dependent cellular cytotoxicity (ADCC) against IgG-coated target cells.

[0005] As a promising alternative platform for cellular immunotherapy, NK cells have recently gained attention as an important type of innate immune regulatory cell. NK cells can rapidly kill multiple adjacent cancer cells through non-MHC-restrictive effects. Although tumors may develop multiple resistance mechanisms to endogenous NK cell attack, in vitro activation, expansion, and genetic modification of NK cells can greatly enhance their anti-tumor activity and give them the ability to overcome drug resistance. Some of these approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies and solid tumors have thus far yielded many encouraging clinical results. However, there are many challenges to overcome, such as meeting clinical-grade ex vivo expansion, limited in vivo persistence, limited infiltration to solid tumors, and tumor editing to evade NK cell activity. Therefore, there is a need to provide an alternative multi-specific polypeptide construct.

SUMMARY

[0006] In one aspect, there is provided a multi-specific polypeptide construct comprising:

(a) one or more antigen targeting domains binding to one or more cancer-associated antigens; and

(b) one or more NK cell-targeting domains, wherein binding to NK cells may stimulate and/or suppress innate immune cell functions.

[0007] In some examples, one of the NK cell-binding domains is an NKp80-targeting domain.

[0008] In some examples, the NKp80-targeting domain comprises:

(1) a heavy chain variable domain (VH) comprising 1 , 2, or 3 complementarity determining region (CDR) selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(2) a light chain variable domain (VL) comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO:17-31 ; 248, and/or VLCDR3 of SEQ ID NQ:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[0009] In some examples, the NKp80-targeting domain comprises:

(1) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141 -155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(2) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253, a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO:138- 140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[0010] In some examples, the NKp80-targeting domain comprises:

(1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NQ:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO: 17-31 ; 248, and VLCDR3 of SEQ ID NO:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141-155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(4) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253 a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO:138- 140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof. [0011] In some examples, the multi-specific polypeptide construct as disclosed herein further comprises a functional Fc domain.

[0012] In some examples, the Fc domain is

(i) a native / wild type Fc domain (FcWT) or a diminished Fc (FcX) domain of SEQ ID NO:224; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(ii) an enhanced Fc domain (FcE) of SEQ ID NO:226; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(iii) a silent Fc domain / inactivated mutant Fc domain (FcLALA) of SEQ ID NO:225; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[0013] In some examples, the multi-specific polypeptide construct as disclosed herein comprises:

(a) a first domain targeting NKp80;

(b) a second domain targeting CD16;

(c) one or more antigen targeting domains binding to one or more tumor-associated antigen.

[0014] In some examples, the one or more antigen targeting domains bind to a member selected from HER-2, EGFR and CD20.

[0015] In some examples, the one or more antigen targeting domains comprise:

(1) a VH of amino acid sequence SEQ ID NO:231 (VH Cetuximab), a VL of amino acid sequence SEQ ID NO:232 (VL Cetuximab), a CH of amino acid sequence SEQ ID NO:233, and/or a CL of amino acid sequence SEQ ID NO:234; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(2) a VH of amino acid sequence SEQ ID NO:227 (VH Trastuzumab), a VL amino acid sequence SEQ ID NO:228 (VL Trastuzumab), a CH of amino acid sequence SEQ ID NO:229, and/or a CL of amino acid sequence SEQ ID NO:230; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof. [0016] In some examples, the multi-specific polypeptide construct as disclosed herein comprises:

(A) an NKp80-targeting domain comprising:

(2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO: 17-31 ; 248, and VLCDR3 of SEQ ID NQ:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141 -155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(4) 1 , 2, 3, or 4 VL FR selected from a FR1 of SEQ ID NO: 105-118; 253, a of SEQ ID NO: 119- 121 ; 254, a VLFR3 of SEQ ID NO: 122-137; 255, and/or a VLFR4 of SEQ ID NO: 138-140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and

(B) one or more antigen targeting domains comprising:

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;. [0017] In some examples, the multi-specific polypeptide construct of as disclosed herein comprises:

(A) an NKp80 targeting domain comprising:

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141 -155; 257, a VHFR2 of SEQ ID NO: 156-162; 258, a VHFR3 of SEQ ID NO: 163-179; 259 and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(B) one or more antigen targeting domains comprising:

(2) a VH of amino acid sequence SEQ ID NO:227 (VH Trastuzumab), a VL amino acid sequence SEQ ID NO:228 (VL Trastuzumab), a CH of amino acid sequence SEQ ID NO:229, and/or a CL of amino acid sequence SEQ ID NQ:230; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and (C) a Fc domain having an amino acid sequence selected from SEQ ID:224-226; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[0018] In some examples, the polypeptide construct is a tri-specific antigen binding construct comprising:

(a) first targeting domain binding NKp80;

(b) a second targeting domain binding CD16; and

(c) a third targeting domain binding to a target antigen, wherein the targeting domains are selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies.

[0019] In some examples, the polypeptide construct is a tri-specific antigen binding construct comprising:

(a) a first targeting domain binding to NKp80, wherein the targeting domain is selected from a Fab fragment, a Fv fragment; a sdAb fragment, an isolated CDR, a scFv, a dsFv, a scAb, a STAb, a sdAb, a single domain CH antibody, a single domain CL antibody, a VHH, a VNAR, and a sdAb based on the VNAR structure from shark;

(b) a first targeting domain binding to CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO:224), FcX (SEQ ID NO:224), silent Fc I Fc inactivated mutant (FcLALA) (SEQ ID NO:225), or FcE (SEQ ID NO:226); and

(c) a third targeting domain binding to a tumor-associated antigen, optionally HER2, EGFR or CD20, wherein the targeting domain is selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies.

[0020] In some examples, the NKp80-targeting domain comprises:

(1) a VH comprising an amino acid sequence selected from SEQ ID NO:203-222; 236; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(2) a VL comprising an amino acid sequence selected from SEQ ID NO: 183-202; 235; wherein the VH and the VL are paired to produce clone 13, clone 28, clone 36, clone 37, clone 45, clone 50, clone 51 , clone 63, clone 71 , clone 74, clone 78, clone 79, clone 81 , clone 82, clone 83, clone 87, clone 94, clone 101 , clone 102, clone 106, or humanized clone 87-2; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[0021] In some examples, the multi-specific polypeptide construct as disclosed herein comprises:

(i) an antigen targeting domain consisting of a Fd fragment or a Fab fragment; a first NK cell targeting domain consisting of a Fc domain; a first [(G4S)n] linker; and a second NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(ii) a first NK cell targeting domain consisting of a Fd fragment or a Fab fragment; a second NK targeting domain consisting of a Fc domain; a first [(G4S)n] linker; and an antigen targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(iii) a first NK cell targeting domain consisting of a Fd fragment or a Fab fragment; a first [(G4s)n] linker; an antigen targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL; and a second NK cell targeting domain consisting of a Fc domain comprising a CH2 and a CH3; or

(iv) an antigen targeting domain consisting of a Fd fragment (comprising of a VH and a CH1) or a Fab fragment; a first [(G4S)n] linker; a first NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL; and a second NK cell targeting domain consisting of a Fc domain comprising a CH2 and a CH3.

[0022] In some examples, the NKp80-targeting domain comprises a member selected from:

(1) VLFR1 (SEQ ID NO:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO:180) (clone 13);

(2) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NO:180) (clone 28);

(3) VLFR1 (SEQ ID NQ:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NQ:70), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 36);

(4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NQ:180) (clone 37);

(5) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NQ:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NO:180) (clone 45);

(6) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:36), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:71), VHFR3 (SEQ ID NO:168), VHCDR3 (SEQ ID NO:90), and VHFR4 (SEQ ID NO:180) (clone 50);

(7) VLFR1 (SEQ ID NO:109), VLCDR1 (SEQ ID NO:5), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:22), VLFR3 (SEQ ID NO:126), VLCDR3 (SEQ ID NO:37), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:56), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:72), VHFR3 (SEQ ID NO:169), VHCDR3 (SEQ ID NO:91), and VHFR4 (SEQ ID NO:180) (clone 51);

(8) VLFR1 (SEQ ID NO:110), VLCDR1 (SEQ ID NO:6), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:127), VLCDR3 (SEQ ID NO:38), VLFR4 (SEQ ID NO:139), (VHFR1 (SEQ ID NO:145), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:73), VHFR3 (SEQ ID NQ:170), VHCDR3 (SEQ ID NO:92), and VHFR4 (SEQ ID NO:181) (clone 71);

(9) VLFR1 (SEQ ID NQ:110), VLCDR1 (SEQ ID NO:7), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:23), VLFR3 (SEQ ID NO:128), VLCDR3 (SEQ ID NO:39), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:146), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:159), VHCDR2 (SEQ ID NO:74), VHFR3 (SEQ ID NO:171), VHCDR3 (SEQ ID NO:93), and VHFR4 (SEQ ID NO:181) (clone 74);

(10) VLFR1 (SEQ ID NO:111), VLCDR1 (SEQ ID NO:8), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:24), VLFR3 (SEQ ID NO:129), VLCDR3 (SEQ ID NQ:40), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:147), VHCDR1 (SEQ ID NO:58), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:75), VHFR3 (SEQ ID NO:172), VHCDR3 (SEQ ID NO:94), and VHFR4 (SEQ ID NO:181) (clone 78);

(11) VLFR1 (SEQ ID NO:112), VLCDR1 (SEQ ID NO:9), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:25), VLFR3 (SEQ ID NQ:130), VLCDR3 (SEQ ID NO:41), VLFR4 (SEQ ID NQ:140), (VHFR1 (SEQ ID NO:148), VHCDR1 (SEQ ID NO:59), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:76), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:95), and VHFR4 (SEQ ID NO:181) (clone 79);

(12) VLFR1 (SEQ ID NO:113), VLCDR1 (SEQ ID NQ:10), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:26), VLFR3 (SEQ ID NO:131), VLCDR3 (SEQ ID NO:42), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:149), VHCDR1 (SEQ ID NQ:60), VHFR2 (SEQ ID NQ:160), VHCDR2 (SEQ ID NO:77), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:96), and VHFR4 (SEQ ID NO:181) (clone 81);

(13) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:43), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:78), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:97), and VHFR4 (SEQ ID NO:181) (clone 82); (14) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:12), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:27), VLFR3 (SEQ ID NO:133), VLCDR3 (SEQ ID NO:44), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:151), VHCDR1 (SEQ ID NO:62), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:79), VHFR3 (SEQ ID NO:175), VHCDR3 (SEQ ID NO:98), and VHFR4 (SEQ ID NO:180) (clone 87);

(15) VLFR1 (SEQ ID NO:115), VLCDR1 (SEQ ID NO:13), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:28), VLFR3 (SEQ ID NO:127), VLCDR3 (SEQ ID NO:45), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:152), VHCDR1 (SEQ ID NO:63), VHFR2 (SEQ ID NO:160), VHCDR2 (SEQ ID NO:80), VHFR3 (SEQ ID NO:176), VHCDR3 (SEQ ID NO:99), and VHFR4 (SEQ ID NO:181) (clone 94);

(16) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:29), VLFR3 (SEQ ID NO:134), VLCDR3 (SEQ ID NO:46), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO: 153), VHCDR1 (SEQ ID NO:64), VHFR2 (SEQ ID NO: 158), VHCDR2 (SEQ ID NO:81), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:100), and VHFR4 (SEQ ID NO:182) (clone 101);

(17) VLFR1 (SEQ ID NO:116), VLCDR1 (SEQ ID NO:14), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:25). VLFR3 (SEQ ID NO:135), VLCDR3 (SEQ ID NO:47), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:153), VHCDR1 (SEQ ID NO:65), VHFR2 (SEQ ID NO:162), VHCDR2 (SEQ ID NO:82), VHFR3 (SEQ ID NO:177), VHCDR3 (SEQ ID NO:101), and VHFR4 (SEQ ID NO:181) (clone 102);

(18) VLFR1 (SEQ ID NO:117), VLCDR1 (SEQ ID NO:15), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:30), VLFR3 (SEQ ID NO:136), VLCDR3 (SEQ ID NO:48), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:154), VHCDR1 (SEQ ID NO:66), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:83, VHFR3 (SEQ ID NO:178), VHCDR3 (SEQ ID NO:102), and VHFR4 (SEQ ID NO:181) (clone 106);

(19) VLFR1 (SEQ ID NO:118), VLCDR1 (SEQ ID NO:16), VLFR2 (SEQ ID NO:121), VLCDR2 (SEQ ID NO:31), VLFR3 (SEQ ID NO:137), VLCDR3 (SEQ ID NO:49), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:155), VHCDR1 (SEQ ID NO:67), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:84), VHFR3 (SEQ ID NO:179), VHCDR3 (SEQ ID NO:103), and VHFR4 (SEQ ID NO:181) (clone 63);

(20) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:50), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:85), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:104), and VHFR4 (SEQ ID NO:181) (clone 83); or

(21) VLFR1 (SEQ ID NO:253), VLCDR1 (SEQ ID NO:247), VLFR2 (SEQ ID NO:254), VLCDR2 (SEQ ID NO:248), VLFR3 (SEQ ID NO:255), VLCDR3 (SEQ ID NO:249), VLFR4 (SEQ ID NO:256), (VHFR1 (SEQ ID NO:257), VHCDR1 (SEQ ID NO:250), VHFR2 (SEQ ID NO:258), VHCDR2 (SEQ ID NO:251), VHFR3 (SEQ ID NO:259), VHCDR3 (SEQ ID NO:252), and VHFR4 (SEQ ID NO:260) (humanized clone 87-2).

[0023] In another aspect, there is provided an antigen-binding protein, or an antigen-binding fragment thereof, comprising the CDR sequences selected from:

(1) VLCDR1 (SEQ ID NO:1), VLCDR2 (SEQ ID NO:17), VLCDR3 (SEQ ID NO:32), HCDR1 (SEQ ID NO:51), VHCDR2 (SEQ ID NO:68), and VHCDR3 (SEQ ID NO:86) (clone 13);

(2) VLCDR1 (SEQ ID NO:2), VLCDR2 (SEQ ID NO:18), VLCDR3 (SEQ ID NO:33), VHCDR1 (SEQ ID NO:52), VHCDR2 (SEQ ID NO:69), and VHCDR3 (SEQ ID NO:87) (clone 28);

(3) VLCDR1 (SEQ ID NO:3), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NO:34), VHCDR1 (SEQ ID NO:53), VHCDR2 (SEQ ID NO:70), and VHCDR3 (SEQ ID NO:88) (clone 36);

(4) VLCDR1 (SEQ ID NO:3), VLCDR2 (SEQ ID NO:20), VLCDR3 (SEQ ID NO:35), VHCDR1 (SEQ ID NO:54), VHCDR2 (SEQ ID NO:70), and VHCDR3 (SEQ ID NO:88) (clone 37);

(5) VLCDR1 (SEQ ID NO:4), VLCDR2 (SEQ ID NO:21), VLCDR3 (SEQ ID NO:35), VHCDR1 (SEQ ID NO:55), VHCDR2 (SEQ ID NO:70), and VHCDR3 (SEQ ID NO:89) (clone 45);

(6) VLCDR1 (SEQ ID NO:4), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NO:36), VHCDR1 (SEQ ID NO:55), VHCDR2 (SEQ ID NO:71), and VHCDR3 (SEQ ID NO:90) (clone 50);

(7) VLCDR1 (SEQ ID NO:5), VLCDR2 (SEQ ID NO:22), VLCDR3 (SEQ ID NO:37), VHCDR1 (SEQ ID NO:56), VHCDR2 (SEQ ID NO:72), and VHCDR3 (SEQ ID NO:91) (clone 51);

(8) VLCDR1 (SEQ ID NO:6), VLCDR2 (SEQ ID NO:18), VLCDR3 (SEQ ID NO:38), VHCDR1 (SEQ ID NO:57), VHCDR2 (SEQ ID NO:73), and VHCDR3 (SEQ ID NO:92) (clone 71);

(9) VLCDR1 (SEQ ID NO:7), VLCDR2 (SEQ ID NO:23), VLCDR3 (SEQ ID NO:39), VHCDR1 (SEQ ID NO:57), VHCDR2 (SEQ ID NO:74), and VHCDR3 (SEQ ID NO:93) (clone 74);

(10) VLCDR1 (SEQ ID NO:8), VLCDR2 (SEQ ID NO:24), VLCDR3 (SEQ ID NO:40), VHCDR1 (SEQ ID NO:58), VHCDR2 (SEQ ID NO:75), and VHCDR3 (SEQ ID NO:94) (clone 78); (11) VLCDR1 (SEQ ID NO:9), VLCDR2 (SEQ ID NO:25), VLCDR3 (SEQ ID NO:41), VHCDR1 (SEQ ID NO:59), VHCDR2 (SEQ ID NO:76), and VHCDR3 (SEQ ID NO:95) (clone 79);

(12) VLCDR1 (SEQ ID NQ:10), VLCDR2 (SEQ ID NO:26), VLCDR3 (SEQ ID NO:42), VHCDR1 (SEQ ID NO:60), VHCDR2 (SEQ ID NO:77), and VHCDR3 (SEQ ID NO:96) (clone 81);

(13) VLCDR1 (SEQ ID NO:11), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NO:43), VHCDR1 (SEQ ID NO:61), VHCDR2 (SEQ ID NO:78), and VHCDR3 (SEQ ID NO:97) (clone 82);

(14) VLCDR1 (SEQ ID NO:12), VLCDR2 (SEQ ID NO:27), VLCDR3 (SEQ ID NO:44), VHCDR1 (SEQ ID NO:62), VHCDR2 (SEQ ID NO:79), and VHCDR3 (SEQ ID NO:98) (clone 87);

(15) VLCDR1 (SEQ ID NO:13), VLCDR2 (SEQ ID NO:28), VLCDR3 (SEQ ID NO:45), VHCDR1 (SEQ ID NO:63), VHCDR2 (SEQ ID NO:80), and VHCDR3 (SEQ ID NO:99) (clone 94);

(16) VLCDR1 (SEQ ID NO:1), VLCDR2 (SEQ ID NO:29), VLCDR3 (SEQ ID NO:46), VHCDR1 (SEQ ID NO:64), HCDR2 (SEQ ID NO:81), and VHCDR3 (SEQ ID NQ:100) (clone 101);

(17) VLCDR1 (SEQ ID NO:14), VLCDR2 (SEQ ID NO:25), VLCDR3 (SEQ ID NO:47), VHCDR1 (SEQ ID NO:65), VHCDR2 (SEQ ID NO:82), and VHCDR3 (SEQ ID NO:101) (clone 102);

(18) VLCDR1 (SEQ ID NO:15), VLCDR2 (SEQ ID NO:30), VLCDR3 (SEQ ID NO:48), VHCDR1 (SEQ ID NO:66), VHCDR2 (SEQ ID NO:83, and VHCDR3 (SEQ ID NO:102) (clone 106);

(19) VLCDR1 (SEQ ID NO:16), VLCDR2 (SEQ ID NO:31), VLCDR3 (SEQ ID NO:49), VHCDR1 (SEQ ID NO:67), VHCDR2 (SEQ ID NO:84), and VHCDR3 (SEQ ID NO:103) (clone 63);

(20) VLCDR1 (SEQ ID NO:11), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NO:50), VHCDR1 (SEQ ID NO:61), VHCDR2 (SEQ ID NO:85), and VHCDR3 (SEQ ID NO:104) (clone 83); or

(21) VLCDR1 (SEQ ID NO:247), VLCDR2 (SEQ ID NO:248), VLCDR3 (SEQ ID NO:249), VHCDR1 (SEQ ID NO:250), VHCDR2 (SEQ ID NO:251), and VHCDR3 (SEQ ID NO:252) (humanized clone 87-2), wherein the CDR sequences share at least about 90% homology to an amino acid sequence selected from SEQ ID NO: 1-104; 247-252, and/or wherein the CDR sequences selected from SEQ ID NO:1-104; 247-252 comprise 2, or 3 amino acid substitutions.

[0024] In yet another aspect, there is provided an antigen-binding protein, or an antigen-binding fragment thereof, comprising the CDR and FR sequences selected from: (1) VLFR1 (SEQ ID NO:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO:180) (clone 13);

(2) VLFR1 (SEQ ID NQ:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NO:180) (clone 28);

(3) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 36);

(4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NQ:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 37);

(5) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NQ:180) (clone 45);

(8) VLFR1 (SEQ ID NO:110), VLCDR1 (SEQ ID NO:6), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:127), VLCDR3 (SEQ ID NO:38), VLFR4 (SEQ ID NO:139), (VHFR1 (SEQ ID NO:145), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:73), VHFR3 (SEQ ID NO:170), VHCDR3 (SEQ ID NO:92), and VHFR4 (SEQ ID NO:181) (clone 71);

(9) VLFR1 (SEQ ID NQ:110), VLCDR1 (SEQ ID NO:7), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:23), VLFR3 (SEQ ID NO:128), VLCDR3 (SEQ ID NO:39), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:146), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:159), VHCDR2 (SEQ ID NO:74), VHFR3 (SEQ ID NO:171), VHCDR3 (SEQ ID NO:93), and VHFR4 (SEQ ID NO:181) (clone 74);

(10) VLFR1 (SEQ ID NO:111), VLCDR1 (SEQ ID NO:8), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:24), VLFR3 (SEQ ID NO:129), VLCDR3 (SEQ ID NQ:40), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:147), VHCDR1 (SEQ ID NO:58), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:75), VHFR3 (SEQ ID NO:172), VHCDR3 (SEQ ID NO:94), and VHFR4 (SEQ ID NO:181) (clone 78);

(12) VLFR1 (SEQ ID NO:113), VLCDR1 (SEQ ID NO:10), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:26), VLFR3 (SEQ ID NO:131), VLCDR3 (SEQ ID NO:42), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:149), VHCDR1 (SEQ ID NQ:60), VHFR2 (SEQ ID NQ:160), VHCDR2 (SEQ ID NO:77), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:96), and VHFR4 (SEQ ID NO:181) (clone 81);

(13) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:43), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NQ:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:78), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:97), and VHFR4 (SEQ ID NO:181) (clone 82);

(14) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:12), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:27), VLFR3 (SEQ ID NO:133), VLCDR3 (SEQ ID NO:44), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:151), VHCDR1 (SEQ ID NO:62), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:79), VHFR3 (SEQ ID NO:175), VHCDR3 (SEQ ID NO:98), and VHFR4 (SEQ ID NO:180) (clone 87);

(15) VLFR1 (SEQ ID NO:115), VLCDR1 (SEQ ID NO:13), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:28), VLFR3 (SEQ ID NO:127), VLCDR3 (SEQ ID NO:45), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:152), VHCDR1 (SEQ ID NO:63), VHFR2 (SEQ ID NQ:160), VHCDR2 (SEQ ID NO:80), VHFR3 (SEQ ID NO:176), VHCDR3 (SEQ ID NO:99), and VHFR4 (SEQ ID NO:181) (clone 94);

(16) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:29), VLFR3 (SEQ ID NO:134), VLCDR3 (SEQ ID NO:46), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO: 153), VHCDR1 (SEQ ID NO:64), VHFR2 (SEQ ID NO: 158), VHCDR2 (SEQ ID NO:81), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID N0:100), and VHFR4 (SEQ ID NO:182) (clone 101);

(19) VLFR1 (SEQ ID NO:118), VLCDR1 (SEQ ID NO:16), VLFR2 (SEQ ID NO:121), VLCDR2 (SEQ ID NO:31), VLFR3 (SEQ ID NO:137), VLCDR3 (SEQ ID NO:49), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:155), VHCDR1 (SEQ ID NO:67), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:84), VHFR3 (SEQ ID NO:179), VHCDR3 (SEQ ID NO:103), and VHFR4 (SEQ ID NO:181) (clone 63); (20) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:50), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:85), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:104), and VHFR4 (SEQ ID NO:181) (clone 83); or

(21) VLFR1 (SEQ ID NO:253), VLCDR1 (SEQ ID NO:247), VLFR2 (SEQ ID NO:254), VLCDR2 (SEQ ID NO:248), VLFR3 (SEQ ID NO:255), VLCDR3 (SEQ ID NO:249, VLFR4 (SEQ ID NO:256), (VHFR1 (SEQ ID NO:257), VHCDR1 (SEQ ID NO:250), VHFR2 (SEQ ID NO:258), VHCDR2 (SEQ ID NO:251), VHFR3 (SEQ ID NO:259), VHCDR3 (SEQ ID NO:252), and VHFR4 (SEQ ID NO:260) (humanized clone 87-2), wherein the FR and CDR sequences share at least about 90% homology to an amino acid sequence selected from SEQ ID NQ:1-104; 247-260, and/or wherein the FR and CDR sequences selected from SEQ ID NO:1-104; 247-260 comprise 2, or 3 amino acid substitutions.

[0025] In yet another aspect, there is provided a nucleic acid sequence encoding the multispecific polypeptide construct, or the antibody as disclosed herein.

[0026] In yet another aspect, there is provided a vector comprising sequences for the multispecific polypeptide construct, or the antibody as disclosed herein.

[0027] In yet another aspect, there is provided a host cell comprising the vector as disclosed herein.

[0028] In yet another aspect, there is provided a method of producing the multi-specific polypeptide construct, or the antibody as disclosed herein comprising culturing the host cell, and optionally isolating the multi-specific polypeptide construct from said host cell and/or the culture media.

[0029] In yet another aspect, there is provided a method of screening and/or identifying the multispecific polypeptide construct, or the antibody as disclosed herein, wherein the NK cell-targeting domain is antiNKp80.

[0030] In yet another aspect, there is provided a pharmaceutical composition comprising the multi-specific polypeptide construct, or the antibody as disclosed herein.

[0031] In yet another aspect, there is provided a method for treating cancer comprising administering to a subject in need thereof the pharmaceutical composition as disclosed herein, wherein the multi-specific polypeptide construct, or the antibody, is administered in an effective amount to treat the cancer in the subject.

[0032] In some examples, the subject has cancer cells that express HER2, CD20, and/or EGFR.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] For a better understanding of the various described implementations, reference should be made to the detailed description below, in conjunction with the following drawings in which like reference numerals refer to corresponding parts throughout the figures.

[0034] FIG. 1 shows the summary of NKp80 activating binders identified from antibody discovery to functional characterization. FcX denotes as an Fc region with diminished ADCC function.

[0035] FIG. 2 shows representative plots of cytotoxicity (% of killing relative to untreated controls) for NKp80 binder clones against a HER2-positive tumor cell line, N87, at the appropriate effectontarget ratio (ET ratio). Screening was performed in 2 non-overlapping batches. NKp80 binders used in this experiment are in tri-specific format containing an Fc region with diminished ADCC capacity (antiHER2-antiNKp80-FcX). An activating clone is defined as a clone that consistently shows greater cytotoxicity than the median cutoff in at least 2 replicates. 20 activating binders in this representative assay are highlighted using triangle symbols. FcX: diminished ADCC function. Triangle: activating binder; Circle: non-activating binder.

[0036] FIG. 3A shows representative cytotoxicity dose-response curves (% killing relative to untreated controls) of four selected NKp80 activating engagers against HER2-positive tumor cells, OVCAR3. These four NKp80 engagers are humanized and expressed in tri-specific format containing a fully functional Fc region (antiHER2-antiNKp80-Fc vs antiHER2-Fc), while the Trastuzumab control does not contain antiNKp80 (hence, antiHER2-Fc). FIG. 3B shows cytotoxicity (% killing relative to untreated controls) of the four selected NKp80 activating engagers against a HER2-positive cell, N87. NKp80 engagers used in this experiment are in tri- specific format containing an Fc region with diminished ADCC capacity (antiHER2-antiNKp80- FcX). The plot shows the average cytotoxicity value calculated from 3 independent assays (n=3). These clones show greater cytotoxicity than Trastuzumab containing a diminished Fc region (antiHER2-FcX), but weaker cytotoxicity than Trastuzumab containing a fully functional Fc (antiHER2-Fc), demonstrating that a functional Fc is needed to boost performance as evidenced in FIG. 3A. This may be due to the lower abundance of NKp80 versus CD16 on NK cells (70,000 copy/cell of CD16 vs 4000 copy/cell of NKp80) as reported in a previous study. FcX: Fc with diminished ADCC function.

[0037] FIG. 4 shows representative cytotoxicity dose-response curves (% killing relative to untreated controls) for an engager containing NKp80 clone 87-2 (tri-specific, antiHER2- antiNKp80-Fc) relative to Trastuzumab. Experiments for each cell line was performed using PBMCs from healthy donors, using a 9-dose series and at identical effector:target ratios. Data were normalized to untreated controls and plotted in Prism.

[0038] FIG. 5 shows representative flow cytometric analysis of CD25- and CD137- expressing population on NK cells and T cells in the presence of indicated antibodies (0.08nM). Secreted IFN-y levels were also measured in the presence of indicated antibodies (0.1 nM). An appropriate ET ratio is used for the experiment, using a HER2-positive tumor cell line, HCT116, as target cells (PBMC: HCT116 cell).

[0039] FIG. 6 shows representative cytotoxicity dose-response curves (% killing relative to untreated controls) for an engager containing NKp80 clone 87-2 (tri-specific, antiEGFR- antiNKp80-Fc) relative to Cetuximab, tested in 2 cell lines (HCT116 and MDA-MB-231 , top and middle). Experiments involving antiEGFR were performed using PBMCs from healthy donors, using a 9-dose series and at identical effectontarget ratios. Data were normalized to untreated controls and plotted in Prism. The bottom panel shows a representative cytotoxicity doseresponse curves (% killing relative to untreated controls) for an engager containing NKp80 clone 87-2 (tri-specific, antiCD20-antiNKp80-Fc) relative to Rituximab, tested in RAJI cells. This experiment involving antiCD20 was performed using NK cells purified from PBMCs from healthy donors, using a 9-dose series and at an effector:target ratio of 2.5. Data were normalized to untreated controls and plotted in Prism.

[0040] FIG. 7 shows representative cytotoxicity dose-response curves (% killing relative to untreated controls) demonstrating safety and specificity of an engager containing NKp80 clone 87-2. A fully functional tri-specific engager containing NKp80 clone 87-2 antibody (antiHER2- antiNKp80-Fc), alongside Trastuzumab was tested for cytotoxicity against HER2-positive normal fetal lung fibroblasts (MRC-5 and WI-38, top and middle). An isotype control of an engager containing NKp80 clone 87-2 (where antiHER2 was replaced by an untargeted IgG) was added to HER2-positive colorectal cancer cells alongside Trastuzumab (positive control) (HCT116, bottom). Experiments for each cell line was performed using PBMCs from healthy donors, using a 9-dose series and at identical effector:target ratios. Data were normalized to untreated controls and plotted in Prism. [0041] FIG. 8 shows clusters for the four activating engagers containing different clones of NKp80. Clones 94-1 and 101-1 are distanced from clone 45-2 and 87-2. Both clone 45-2 and 87- 2 are in the same cluster, with 94-1 and 101-1 are in a different cluster. The cluster with silhouette value for each clone was constructed using input of binding indices generated by tandem binning with bio-layer interferometry (BLI).

[0042] FIG. 9 shows clustering analysis for all the 20 NKp80-activating clones. Clusters are generated from sequence identity matrices of heavy chain CDR3. This sequence-based analysis suggests that clones 87-2 and 45-2 are in the same cluster (cluster 2), while clones 101-1 and 94-2 are distanced in separate clusters (cluster 5 and 3), consistent with BLI binding shown in FIG. 8. This analysis suggests that the 20 NKp80-activating clones are sequentially diverse.

[0043] FIG. 10 shows the amino acid sequences for variable heavy chain (VH) and variable light chain (VL) complementarity determining regions (CDRs) of the NKp80-activating clones.

[0044] FIG. 11 shows the amino acid sequences for variable heavy chain (VH) and variable light chain (VL) framework regions (FRs) of the NKp80-activating clones.

[0045] FIG. 12 shows the amino acid sequences for the NKp80 NK cell receptor, for the wild-type Fc domain and the silent Fc domain, and the VH, VL, CH, and CL domains of the antigen targeting domains binding HER2 (Trastuzumab), EGFR (Cetuximab), and CD20 (Rituximab).

[0046] FIG. 13 shows the amino acid sequences for the variable light chains (VL) (FIG. 13A) and variable heavy chains (VH) (FIG. 13B) of the 20 claimed antiNKp80 clones targeting the HER2 antigen. FIG. 13C shows the VH, CL, CH, CL, CDR, and FR sequences of the humanized antiNKp80 clone 87-2. FIG. 13D shows the amino acid sequence of 4 exemplary EGFR targeting polypeptide constructs, in which the NK cell engagers and the antigen targeting domain are arranged in 4 different permutations. FIG. 13E shows representative cytotoxicity dose-response curve (% killing relative to untreated controls) of the various permutations of the tri-specific engagers containing the domains antiEGFR (Cetuximab), antiNKp80 and wild type Fc, relative to Cetuximab. Experiments were performed using MDA-MB-231 as target cells, PBMCs isolated from healthy donors and in a 9-dose series at an E:T of 28. Data taken at 48 hours were normalized to untreated controls and plotted in Prism.

[0047] FIG. 14 shows the amino acid percent (%) identity matrix for the variable heavy chain complementarity determining region 3 (VHCDR3) for each of the 20 NKp80-binding polypeptide construct clones. [0048] FIG. 15 shows a schematic of how the trispecific engager works: it engages CD16 and NKp80 on the innate immune cell, and the target antigen on the target cell. This trispecific binding triggers antibody-dependent cellular cytotoxicity (ADCC) by the innate immune cell to kill the target cell bearing the target antigen of interest.

[0049] FIG. 16 shows representative cytotoxicity dose-response curves (% killing relative to untreated controls) of an engager targeting anti-NKp80 (87-2). Tri-specific engagers containing NKp80 clone 87-2 antibody with variants of the Fc region (antiHER2-antiNKp80-Fc(variant), where the Fc (variant) may be the wildtype Fc (WT), an inactivated Fc mutant (LALA) or enhanced (E) Fc were tested for cytotoxicity against a HER2-positive breast cancer cell line. Experiments for each cell line was performed using PBMCs from healthy donors, using a 9-dose series and at identical effectortarget ratios. Data at 24 hours were normalized to untreated controls and plotted in Prism.

DETAILED DESCRIPTION

I. INTRODUCTION

[0050] Antibody enhanced Innate cell Modulator (AIM) is a first-in-class, next-generation NK cell engager (NKCE)-based molecule with application in various indications, including cancer, infectious and autoimmune diseases. In cancer immunotherapy, treatment approaches face limitations, for instance monoclonal antibodies are limited to patients with high target expression. The benefit of checkpoint inhibitors is restricted to a small patient population, and high risks of cytokine storm release are associated with T cell bi-specific antibodies and CART cell therapy.

[0051] Natural killer (NK) cells are an essential part of tumor immunosurveillance, evidenced by higher cancer susceptibility and metastasis in association with diminished NK activity in mouse models and clinical studies. Using an array of germline-encoded surface receptors, NK cells recognize and rapidly act against malignant cells without prior sensitization. Upon activation, NK cells release cytotoxic granules containing perforin and granzymes to directly lyse tumor cells, in a similar fashion to activated cytotoxic T cells. NK cells are also potent producers of chemokines and cytokines such as interferon gamma (IFN-y) and tumor necrosis factor alpha (TNF-a) and thereby are essential in modulating adaptive immune responses. Due to their innate ability to eliminate tumor cells, NK cell-based immunotherapies against cancer have been investigated for decades. Early clinical trials demonstrated the overall safety of NK cell infusion, even in the allogeneic setting. The feasibility of utilizing allogeneic NK cells, the established safety profiles, and the fast-acting nature of NK cells largely have led to the emerging effort to develop “off-the- shelf” NK cell-based cancer immunotherapy. However, there are many challenges to overcome, such as difficulty to meet clinical-grade ex vivo expansion, limited in vivo persistence, limited infiltration to solid tumors, and tumor editing to evade NK cell activity.

[0052] In some examples, AIM NKCE boosts innate immune cell responses for improved antitumor effects and minimal side effects. This multi-specific polypeptide construct can be used as a single agent or in combination with existing disease-targeting therapies such as NK cell therapy, T-cell checkpoint inhibitors, or small molecules.

II. DEFINITIONS

[0053] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2^nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3^rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0055] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, is understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. It is understood that wherever aspects are described herein with the22cab22agee “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.

[0056] The terms “about” or “consisting essentially of’ refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, in some examples, “about” or “consisting essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “consisting essentially of’ can mean a range of up to 10% (i.e., +/-10%).

[0057] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0058] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

[0059] Ranges: throughout this disclosure, various aspects of the disclosure are presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range is considered herein to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 is considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0060] In some examples, the multi-specific polypeptide construct comprises domains binding to one or more innate immune cell modulators. In some examples, these domains are described as “targeting domains” or “binding domains” in relation to innate immune cells. As used herein, the term “innate immune cell modulator’, “NK modulator”, or “modulator” refers to immunomodulating molecules (such as receptors) expressed on immune cells modifying the activity of the cells when bound, and effecting a change in overall immune response. In some examples, changes in immune responses triggered by modulators help the body fight cancer, infections, or other diseases.

[0061] In some examples, the multi-specific polypeptide construct comprises domains comprising antibodies or fragments thereof. When used herein, the term “antibody” includes intact antibodies and binding fragments thereof. The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region, means a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. A constant region can include any or all of a CH1 region, hinge region, CH2 region and CH3 region. It will be appreciated that sequence amendments of the constant region domains may also be used. For example, 1 or more amino acid, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 amino acid substitutions, additions and/or deletions may also be made to the antibody constant domains without significantly altering the ability of the antibody to bind to a target antigen.

[0062] In some examples, the multi-specific polypeptide construct comprises domains comprising monoclonal antibodies or fragments thereof. The term “monoclonal antibody” as used herein refers to antibodies that are substantially identical to amino acid sequence or are derived from the same genetic source. A monoclonal antibody composition displays a binding specificity and affinity for a particular epitope, or binding specificities and affinities for specific epitopes.

[0063] In some examples, the multi-specific polypeptide construct comprises domains comprising chimeric antibodies or fragments thereof. The term “chimeric antibody” (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e. g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in human as compared to the original mouse antibody.

[0064] In some examples, the multi-specific polypeptide construct comprises domains comprising humanized antibodies or fragments thereof. The term “humanized antibody” (or antigen-binding fragment thereof), as used herein, is intended to include antibodies (and antigen-binding fragments thereof) having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into the classes: IgA, I g D, IgE, IgG and IgM, and several of these may be further divided into subclasses (subtypes), e.g., lgG1 , lgG2, lgG3, and lgG4, IgAI, and lgA2. Therefore, human IgG constant region domains may be used, especially of the lgG1 and lgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, lgG2 and lgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences. A humanized antibody (or antigen-binding fragment thereof) retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i. e., the constant region as well as the framework portions of the variable region). Additional framework region modifications may be made within the human framework sequences as well as within the CDR sequences derived from the germline of another mammalian species. The humanized antibodies of the present disclosure may include amino acid residues not encoded by human sequences (e. g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing). This definition of a humanized antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries, administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e. g., immunized xenomice via a human B-cell hybridoma technology.

[0065] In some examples, the multi-specific polypeptide construct comprises domains comprising recombinant humanized antibodies or fragments thereof. The term “recombinant humanized antibody” as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transformed to express the humanized antibody, e. g., from a transfectoma, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.

[0066] In some examples, the multi-specific polypeptide construct comprises domains comprising antibodies or fragments thereof. An “isolated antibody” refers to an antibody that is substantially free of other cellular material and/or chemicals. [0067] In some examples, the multi-specific polypeptide construct comprises binding domains. As used herein, a “binding domain” refers to, but is not limited to, any of the following: a “Fab fragment,” i.e. a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a “F(ab)2 fragment”, i.e. a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an “Fd fragment,” i.e. consisting of the VH and CH1 domains; an “Fv fragment,” i.e. consisting of the VL and VH domains of a single arm of an antibody; a “single domain antibody (dAb) fragment,” which consists of a VH domain; an isolated “complementarity determining region” (CDR); a “single-chain Fv”; a “disulfide-stabilized variable fragment (dsFv);” a “singlechain antibody fragment (scab);” STAB, a “single domain antibody (sdAb or dAb);” a “single domain heavy chain antibody (sdCH);” a “single domain light chain antibody (sdCL);” a “nanobody” or a “single variable domain on a heavy chain (VHH);” a “variable new antigen receptor (VNAR)” from shark; a single domain antibodies based on VNAR structure; as well as binding domains based on alternative scaffolds, including but limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies (e. g. f-star technology (F-star’s Modular Antibody TechnologyTM)).

[0068] In some examples, the multi-specific polypeptide construct comprises linkers. As used herein, the term “linker” refers an intervening peptide sequence connecting any two components within the multi-specific polypeptide construct and comprises primary repetitions of residues like glycine (G) and serine (S). Such linkers are broadly categorized as flexible linkers, rigid linkers, and cleavable linkers. The G4S linker refers to a poly-Glycine-Serine linker with four glycine and one serine. The [(G4S)n] linker as described herein refers to the number (n) of consecutively repeated blocks of G4S.

[0069] In some examples, the multi-specific polypeptide construct comprises domains binding to antigens. As used herein, the term “antigen” refers to a structure on a target cell surface, generally acknowledged to be associated with a particular disease state, and to which a polypeptide construct of the disclosure binds. The term “epitope”, as used herein, defines the antigenic determinant to which an antibody, antibody fragment, or other binding domain specifically binds. “Antigen” and “epitope” can be used interchangeably within the context of this disclosure and refer to a target molecule on the surface of a target cell.

[0070] In some examples, the multi-specific polypeptide construct comprises domains binding to bacterial antigens. As used herein, the term “bacterial antigen” includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (such as about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens.

[0071] In some examples, the multi-specific polypeptide construct comprises domains binding to viral antigens. As used herein, the term “viral antigen” includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (for example about 8 amino acids or longer) to be antigenic.

[0072] In some examples, the multi-specific polypeptide construct comprises different regions or domains. As used herein, the terms “region” and “domain” are understood to describe the same component and may therefore be used interchangeably.

[0073] In some examples, the multi-specific polypeptide construct comprises Complementarity Determining Regions. The term “Complementarity Determining Regions” (“CDRs”) refers to amino acid sequences with boundaries determined using any number of well-known schemes, including those described by Kabat (i.e., “Kabat” numbering scheme); Al-Lazikani (“Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (“IMGT” numbering scheme); and the like. For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (VHCDR1), 50-65 (VHCDR2), and 95-102 (VHCDR3); and the CDR amino acid residues in the light chain variable domain” (VL) are numbered 24-34 (VLCDR1), 50-56 (VLCDR2), and 89-97 (VLCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (VHCDR1), 52-56 (VHCDR2), and 95-102 (VHCDR3); and the amino acid residues in VL are numbered 24-34 (VLCDR1), 50-56 (VLCDR2), and 89-97 (VLCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (VHCDR1), 50-65 (VHCDR2), and 95-102 (VHCDR3) in human VH and amino acid residues 24-34 (VLCDR1), 50-56 (LVCDR2), and 89-97 (VLCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (VHCDR1), SI- 57 (VHCDR2) and 93-102 (VHCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (VLCDR1), 50-52 (VLCDR2), and 89-97 (VLCDR3) (numbering according to “Kabat”). Under IMGT, the CDRs of an antibody can be determined using the program IMGT/DomainGap Align.

[0074] In some examples, the multi-specific polypeptide construct comprises light chains and heavy chains. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon. The heavy chains of an antibody define the antibody’s isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. As used herein, the terms “variable light chain CDR1”, “variable light chain CDR2”, “variable light chain CDR3”, “variable heavy chain CDR1”, “variable heavy chain CDR2”, “variable heavy chain CDR3” refers to VLCDR1 , VLCDR2, VLCDR3, VHCDR1 , VHCDR2 and VHCDR3 respectively.

[0075] In some examples, when the multi-specific polypeptide construct is a multi-specific antigen binding polypeptide, the multi-specific polypeptide construct, as described herein, comprises multiple binding domains. These domains bind to or recognize a group selected from a NK modulator or a target antigen. In some examples, each binding domain of the multi-specific polypeptide construct comprises at least one CDR, or at least two CDRs, or at least three CDRs, or at least four CDRs, or at least five CDRs, or all six CDRs as described herein. In some examples, the multi-specific polypeptide construct comprises a combination of one or more CDRs as described herein.

[0076] In some examples, the multi-specific polypeptide construct comprises domains binding to NKp80. As used herein, the term “NKp80” refers to an 80kDa protein reported as a dimer expressed on natural killer (NK) cells and is also known as a killer cell lectin-like receptor subfamily F, member 1 (KLRF1). This receptor is known as a type II transmembrane protein with a C-type lectin domain exposed in the extracellular compartment. This receptor is predominantly expressed on NK cells and is also present in a small subset of T cells. NKp80 induces NK activation and mediates cytotoxicity.

[0077] In some examples, the multi-specific polypeptide construct comprises NKp80 engagers. The term “NKp80 engager” refers to a molecule capable of binding to NKp80, such as an antibody that bind to NKp80 expressed on NK cells. In some examples, the NKp80 engager is a human NKp80 binder (huNKp80 binder) and/or a cynomolgus NKp80 binder (cyNKp80 binder).

[0078] In some examples, the multi-specific polypeptide construct comprises Fc domains. As used herein, the term “Fc domain” refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Also considered to be part of the present disclosure are Fc polypeptides that include the polypeptides making up an Fc domain, e.g., a monomeric Fc. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as human lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. An Fc polypeptide may be obtained from human or any other non-human mammals. The Fc domain comprises the carboxy- terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc domain; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0079] Also included in the multi-specific polypeptide constructs disclosed herein are functional Fc domains which possess an “effector function” of a native / wild type sequence Fc region (FcE). In some examples, “effector functions” are selected from CD16 binding; C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as known in the art. In some examples, the Fc domain includes a functional antibody dependent cell cytotoxicity (ADCC) (e.g., via CD16 binding), a diminished ADCC (FcX) (e.g., via Fc mutations thereby providing either an Fc diminished domain or via antibody configuration to achieve diminished ADCC), a Fc silent domain / inactivated mutant Fc domain (FcLALA) achieved via Fc mutations, or an enhanced ADCC (FcE) (e.g., via Fc mutations thereby providing an enhanced activity of the Fc domain).

[0080] Also considered to be part of the present disclosure are multi-specific polypeptide constructs comprising native I wild type Fc domains and/or variant Fc domains. Variant Fc domains (or Fc mutated domains) comprise an amino acid sequence which differs from that of a native / wild type Fc domain sequence by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). In some examples, the variant Fc domain has at least one amino acid substitution compared to a native / wild type Fc domain sequence or to the Fc domain of a parent polypeptide. In some examples, the variant Fc region (or Fc mutated region) comprises from about 1 to about 10 amino acid substitutions in a native / wild type sequence Fc region. In some examples, the variant Fc region shares at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, homology, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology with a native I wild type Fc domain sequence.

[0081] Also included in exemplary multi-specific polypeptide constructs is an “Fc component” that may include a hinge domain, a CH2 domain or a CH3 domain of an Fc domain.

[0082] In some examples, the multi-specific polypeptide construct comprises framework regions. As used herein, the term “framework region (FR)” is intended to mean each domain of the variable light or heavy chain that separates the CDRs. [0083] As used herein, the term “variable light chain FR1 ,” “variable light chain FR2,” “variable light chain FR3,” “variable light chain FR4,” “variable heavy chain FR1 ,” “variable heavy chain FR2,” “variable heavy chain FR3,” and “variable heavy chain FR4” refers to VLFR1 , VLFR2, VLFR3, VLFR4, VHFR1 , VHFR2, VHFR3, and VHFR4, respectively.

[0084] In some examples, the multi-specific polypeptide construct is a bi-specific antigen binding polypeptide construct. The term “bi-specific antigen binding polypeptide construct”, as used herein, refers to a multi-specific polypeptide construct comprising two binding domains, e.g., an antibody domain, but other binding domains can also be employed. When the binding domain comprises an antibody domain, each domain comprises at least 3 CDRs and framework, for example a VHH comprises 3 CDRs whilst a Fab comprises 6 CDRs.

[0085] In some examples, the multi-specific polypeptide construct is a tri-specific antigen binding polypeptide construct. The term “tri-specific antigen binding polypeptide construct”, as used herein, refers to a multi-specific polypeptide construct binding three different epitopes on three different targets or three different binding sites.

[0086] In some examples, the multi-specific polypeptide construct is multi-specific antigen binding polypeptide constructs. The term “multi-specific antigen binding polypeptide construct”, as used herein, refers to a multi-specific polypeptide construct with two or more binding domains, the construct binding two or more different epitopes on at least two or more different targets. The term “multi-specific antigen binding polypeptide construct” includes, but is not limited to, bi-specific, tri- specific, tetra-specific, penta-specific hexa-specific, and the like.

[0087] In some examples, the multi-specific polypeptide constructs of the disclosure exhibit a synergistic function in their cytotoxicity. The term “synergistic function” or “synergistic biological function” as used herein refers to a biological activity or level of biological activity or an effect on a biological function or activity that: 1) is not observed with individual polypeptide components of the multi-specific polypeptide construct; 2) is observed when the two (or more) binding domains are linked in a specific format); or 3) higher or lower activity in comparison to the activity observed when individual polypeptide components of the multi-specific construct of the present disclosure are employed individually, for example and enhanced activity which is only observed in a bi- specific polypeptide construct.

[0088] Therefore, “synergistic” includes novel biological function or novel activity. Synergistic function as employed herein does not generally include simple targeting i.e., based only on binding but will generally involve some inhibition, activation, signaling or similar after binding. [0089] In some examples, the multi-specific polypeptide construct is a fusion protein. The term “fusion proteins” as used herein is used interchangeably with the term “recombinant protein” and comprises a protein component A or B fused to a binding partnerX or Y (as appropriate). In some examples, the fusion protein is a translational polypeptide construct expressed by recombinant techniques from a genetic construct. In some examples, the fusion protein is expressed in a host from a DNA construct. In the context of the present disclosure one of the key characteristics of a fusion protein is that it is expressed as a “single polypeptide” from a cell.

[0090] In some examples, the multi-specific polypeptide construct comprises antigen binding domains, or binding domains, or antigen binding fragments, or antigen targeting domains. The terms “antigen binding portion,” or “binding domain,” or “antigen binding fragment,” or “antigen targeting domain” thereof of a multi-specific polypeptide construct, as used herein, refer to one or more peptide sequences within the multi-specific polypeptide constructs with the ability to specifically bind to a given antigen.

[0091] In some examples, the multi-specific polypeptide construct is an NKcell engager. As used herein, the term “engager” or “Natural Killer cell engager” or “NK cell engager,” or “NK engager” refers to synthetic polypeptides, or multifunction antibodies that are able to bring tumor cells and NK cells together and trigger tumor cell destruction by NK cells. In some examples, the multispecific polypeptide construct is described as a “binder” instead of an engager. As used herein, the term “binder” includes both activating and non-activating binders, occurring in contexts where some multi-specific polypeptide constructs are found to be non-activating during the screening process.

[0092] In some examples, the multi-specific polypeptide construct is evaluated for its binding capacity and/or specificity. As use herein, the term “binds to” or “binding” refers to measurable and reproducible interactions such as binding between a target and an antigen binding polypeptide construct, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. It refers to the ability of an individual antibody to react with one antigenic determinant and not with a different antigenic determinant.

[0093] In some examples, the multi-specific polypeptide construct comprises activating binder domains. As used herein, an “activating binder” refers to a polypeptide construct that shows cytotoxicity towards target cells above a defined baseline (i.e., higher than the median cytotoxicity level of all screened clone) in a cell cytotoxicity assay and is capable of mediating NK cytotoxicity to lyse target cells. [0094] In some examples, the multi-specific polypeptide construct comprises non-binder domains, or non-activating binder domains. As used herein, a “non-binder” or “non-activating binder” is a polypeptide construct that does not show appreciable cytotoxicity towards a target cell and display activity below a defined median baseline in a cell cytotoxicity assay. In some examples, a non-binder determines baseline activity together with a target antigen antibody (such as anti-HER2 antibody: antiHER2-FcX).

[0095] In some examples, the multi-specific polypeptide construct is evaluated through half- maximal effective concentration, or EC50. The term “half-maximal effective concentration” or “EC50,” as used herein, refers to the concentration of an antibody or a multi-specific polypeptide construct/portion thereof, inducing a response, either in an in vivo or an in vitro assay, which is 50% of the maximal response (i.e., halfway between the maximal response and the baseline). The term “mean of EC50 fold change potency” refers to the EC50 fold change of a multi-specific polypeptide construct relative to a control drug. As the standard of care in the treatment of HER2- positive early and advanced breast cancer, Trastuzumab is an exemplary control for exemplary HER2-specific multi-specific polypeptide constructs. Cetuximab is used as a control in the data presented for the EGFR-specific multi-specific polypeptide constructs.

[0096] In some examples, the multi-specific polypeptide construct is put in contact with peripheral blood mononuclear cells (PBMCs). The term “peripheral blood mononuclear cells” or “PBMC” refers to mononuclear blood cells harvested from healthy subjects that are subsequently cultured and used for various bioassays, such as cytotoxicity assays.

[0097] In some examples, the multi-specific polypeptide construct cytotoxicity potential is evaluated in the presence of target cells and effector cells. As used herein, “effector cells” refer to cells that perform a specific function in response to a stimulus, in the present case NK cells. As used herein, “target cells” refer to cells expressing a specific receptor and/or antigen and/or epitope to which an antibody or fragment thereof specifically binds to. In some examples, the cytotoxicity assay contacts the multi-specific polypeptide construct to specific ratios of effector-to- target cells, termed “effector-to-target ratio,” or “effectortarget ratio,”, E:T ratio” or “E/T ratio”.

[0098] In some examples, the term “subject” as used herein includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition, while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and/or an individual free from the medical condition. The term “subject” includes humans and animals. The terms “subject” and “patient” are used interchangeably herein. [0099] In some examples, the multi-specific polypeptide construct or a formulation containing said construct is used to administer a treatment to a subject in need thereof. The term “administering” refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of ad-ministration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, sub-cutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some examples, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[00100] Parenteral” administration of composition of the disclosure includes, e.g., subcutaneous (s.c ), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[00101] In some examples, the multi-specific polypeptide construct is used to treat a disease in a subject. As used herein, “treating” or “treatment” refers to an approach for obtaining beneficial or de-sired results, including and preferably clinical results. Treatment can refer to either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. Treatment is often effective by administering to a subject in need of such treatment a therapeutically effective amount of multi-specific polypeptide constructs.

[00102] In some examples, the multi-specific polypeptide construct or a formulation comprising said construct is administered to a subject in need thereof in a therapeutically effective amount, or an effective amount, or a therapeutically effective dosage. A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a multispecific polypeptide construct is any amount of the construct that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a multi-specific polypeptide construct to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the multi-specific polypeptide construct in in vitro assays.

[00103] In some examples, the multi-specific polypeptide construct is used to treat a cancer. A “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

[00104] In some examples, the multi-specific polypeptide construct exhibits an anti-tumor effect. An “anti-tumor effect”, as used herein, refers to a biological effect presenting as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti-tumor effect can also refer to the prevention of the occurrence of a tumor.

[00105] In some examples, treatment of a subject with a multi-specific polypeptide construct aids in achieving progression free survival. The term “progression-free survival,” abbreviated as PFS, as used herein refers to the time from the treatment date to the date of disease progression.

[00106] In some examples, treatment of a subject with a multi-specific polypeptide construct aids in preventing or slowing disease progression. “Disease progression” or “progressive disease,” abbreviated as PD, as used herein, refers to a worsening of one or more symptom associated with a particular disease. For example, disease progression for a subject afflicted with a cancer can include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[00107] The “duration of response,” abbreviated as DOR, as used herein refers to the time between a subject’s first objective response to the date of confirmed disease progression, per the revised IWG Response Criteria for Malignant Lymphoma, or death.

[00108] In some examples, treatment of a subject with a multi-specific polypeptide construct aids in preventing and/or reducing the severity of symptoms. The term “preventing” and/or “reducing the severity of symptoms” refers to delaying the onset, reducing the severity of symptoms, reducing and/or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and/or ameliorating at least one sign or symptom of a disease.

[00109] In some examples, treatment of a subject with a multi-specific polypeptide construct induces an immune response in the subject. An “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate’s body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of auto-immunity or pathological inflammation, normal human cells or tissues.

[00110] In some examples, the multi-specific polypeptide construct is encoded by nucleic acids. As used herein, the term “nucleic acid” refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). “Nucleic acid” includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single- stranded, double-stranded or triple- stranded. In some examples, nucleic acid molecules can be modified. In the case of a double-stranded polymer, “nucleic acid” can refer to either or both strands of the molecule.

[00111] In some examples, the multi-specific polypeptide construct is encoded by nucleotide sequences. The term “nucleotide sequence,” in reference to a nucleic acid, refers to a contiguous series of nucleotides that are joined by covalent linkages, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl-phosphonate, phosphorothioate, phosphotriester bonds), and/or non-phosphorus linkages (e.g., peptide and/or sulfamate bonds). In some examples, the nucleotide sequence encoding, e.g., a target-binding molecule linked to a localizing domain is a heterologous sequence (e.g., a gene that is of a different species or cell type origin).

[00112] As used herein, the term “sequence identity” or “homology” refers to the percentage sequence identities that are determined with polypeptide sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject polypeptide region (e.g., the entire mature variable region of a heavy or light chain of an antibody) is being compared with the same region of a reference polypeptide, the percentage sequence identity between the subject and reference polypeptide regions is the number of positions occupied by the same amino acid in both the subject and reference polypeptide region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

[00113] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’L Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al. 2000, Current Protocols in Molecular Biology). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly accessible through the National Institutes of Health NCBI internet server). Typically, default program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation € of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[00114] In some examples, the multi-specific polypeptide construct, or any domain or fragment thereof, comprises substitutions. “Conservative substitutions” may be made, for instance, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[00115] As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. [00116] As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[00117] In some examples, the substitutions also include non-classical amino acids. Illustrative non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N- formylmethionine p-alanine, GABA and O-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general.

[00118] In another aspect, there is provided a host cell comprising the vector of the present disclosure.

[00119] In some examples, the host cell as disclosed herein comprises a cloning vector or an expression vector configured to express the multi-specific polypeptide, or the antibody as disclosed herein.

[00120] In some examples, nucleic acids encoding the multi-specific polypeptide construct are comprised into a vector. A “vector” is any molecule or composition having the ability to carry a nucleic acid sequence into a suitable host cell where e.g., synthesis of the encoded polypeptide can take place. Typically, and preferably, a vector is a nucleic acid being engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the present disclosure). Expression vectors typically contain one or more of the following components (if they are not already provided by the nucleic acid molecules): a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[00121] In some examples, the various domains of the multi-specific polypeptide constructs are operably linked. As used herein, the term “operably linked” may refer to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect. For example, a promoter may be operably linked to a coding sequence if it controls the transcription of the coding sequence.

[00122] In some examples, nucleic acids encoding the multi-specific polypeptide construct are inserted in an expression vector. “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising ex-pression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression are supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[00123] In some examples, the multi-specific polypeptide construct, or a fragment or domain thereof, is isolated. The term “isolated” refers to a composition, compound, substance, or molecule altered by the hand of man from the natural state. For example, a composition or substance that occurs in nature is isolated if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is isolated, as the term is employed herein.

[00124] In some examples, the multi-specific polypeptide construct is encoded by a nucleic acid. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[00125] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. In some examples, the nucleotide sequence encoding a protein, or an RNA includes introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more intron(s). [00126] In some examples, the vector comprising the nucleic acid encoding the multi-specific polypeptide construct comprises a promoter. The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[00127] As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence required for expression of a gene product operably linked to the promoter/regulatory sequence. In some examples, this sequence is the core promoter sequence, as in some examples, this sequence also includes an enhancer sequence and other regulatory elements required forexpression of the gene product. In some examples, the promoter/regulatory sequence expresses the gene product in a tissue-specific manner.

[00128] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[00129] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[00130] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[00131] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, being one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[00132] In some examples, the multi-specific polypeptide construct comprises peptides, polypeptides, proteins, and/or fragments thereof. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, ora combination thereof.

[00133] In some examples, the sequence encoding the multi-specific polypeptide constructs, or any domain thereof, comprises conservative sequence modifications. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that may or may not significantly alter the binding characteristics of the anti-body containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. In some examples, the modifications are introduced into a sequence of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the sequence can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using recognized functional assays.

[00134] In some examples, the exogenous nucleic acid encoding the multi-specific polypeptide construct, or any domain thereof, is used to transfect, or transform, or transduce one or more host cells. The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one having been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[00135] This disclosure is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular examples only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[00136] In some examples, the disclosure includes one or more of the features defined hereinabove.

III. DETAILED DESCRIPTION OF THE EMBODIMENTS

[00137] In some examples, the multi-specific polypeptide construct has a first polypeptide domain specifically binding to one or more innate immune cell modulators and a second polypeptide domain binding to one or more target cell antigens.

[00138] In some examples, the multi-specific polypeptide construct target cell antigen binding domain specifically binds the target cell antigen. In its most general form (and when no defined reference is mentioned), “specific binding” is referring to the ability of the multi-specific polypeptide construct to discriminate between the target of interest and a non-target molecule/moiety, as determined, for example, in accordance with a specificity assay known in the art. Such assays comprise, but are not limited to, Western blots, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), Surface plasmon resonance (SPR) tests and peptide scans.

[00139] In some examples, the multi-specific polypeptide construct specifically binds to one or more innate immune cell modulator(s) and/or a target cell antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens.

[00140] In some examples, the multi-specific polypeptide construct comprises one or more innate immune cell targeting domains and/or one or more antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises one innate immune cell targeting domain and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises two innate immune cell targeting domains and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises three innate immune cell targeting domains and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises four innate immune cell targeting domains and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises more than one antigen targeting domain.

[00141] In some examples, the multi-specific polypeptide construct is a bi-specific, a tri-specific, a tetra-specific, a penta-specific or a hexa-specific antigen binding polypeptide. In some examples, the multi-specific polypeptide construct comprises one or more NK cell targeting domains and/or one or more an antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises one NK cell targeting domain and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises two NK cell targeting domains and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises three NK cell targeting domains and an antigen targeting domain. In some examples, the multi-specific polypeptide construct comprises four NK cell targeting domains and an antigen targeting domain.

[00142] In one aspect, there is provided a multi-specific polypeptide construct comprising:

(b) one or more NK cell- targeting domains, wherein binding to NK cells may stimulate and/or suppress innate immune cell functions.

[00143] In some examples, the multi-specific polypeptide construct further comprises one antigen targeting domain, two antigen targeting domains, three antigen targeting domains, four antigen targeting domains, five antigen targeting domains, six antigen targeting domains, or more. In some examples, the multi-specific polypeptide construct comprises one innate immune cell targeting domain and two antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises one innate immune cell targeting domain and three antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises two innate immune cell targeting domains and two antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises two innate immune cell targeting domains and three antigen targeting domains. Other combinations of number of innate immune cell targeting domains and number of antigen targeting domains are also within the purview of the disclosure herein.

[00144] In some examples, the multi-specific polypeptide construct comprises more than one antigen targeting domains. In some examples, the multi-specific polypeptide construct further comprises one antigen targeting domain, or two antigen targeting domains, or three antigen targeting domains, or four antigen targeting domains, or five antigen targeting domains, or six target antigen targeting domains, or more.

[00145] In some examples, the multi-specific polypeptide construct comprises one NK cell targeting domain and two antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises one NK cell targeting domain and three antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises two NK cell targeting domains and two antigen targeting domains. In some examples, the multi-specific polypeptide construct comprises two NK cell targeting domains and three antigen targeting domains. Other combinations of number of NK cell targeting domains and number antigen targeting domains are also within the purview of the disclosure herein.

[00146] In some examples, the NK cell targeting domain is an NKp80 targeting domain, or an anti-NKp80 domain. In some examples, the NKp80 targeting domain is selected from, but not limited to, an NKp80-binding Fab fragment, an NKp80-binding Fd fragment, a NKp80-binding F(ab)2 fragment, an NKp80-binding Fv fragment, an NKp80-binding single domain antibody fragment, an NKp80-binding CDR, an NKp80-binding single chain Fv, an NKp80-binding dsFv, an NKp80-binding scab, an NKp80-binding STAb, an NKp80-binding single domain heavy chain antibody, an NKp80-binding single domain light chain antibody, an NKp80-binding VHH, an NKp80-binding VNAR, and other NKp80-binding domains based on alternative scaffolds.

[00147] In some examples, one of the NK cell-targeting domains is an NKp80-targeting domain.

[00148] In some examples, the NKp80-targeting domain comprises:

[00149] In some examples, the NKp80-targeting domain comprises:

(1) a heavy chain variable domain (VH) comprising 1 , 2, or 3 complementarity determining region (CDR) selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252; and/or

(2) a light chain variable domain (VL) comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO:17-31 ; 248, and/or VLCDR3 of SEQ ID NQ:32-50; 249. [00150] In some examples, the NKp80-targeting domain comprises a VHCDR1 , a VHCDR2, a VHCDR3, a VLCDR1 , a VLCDR2, and/or a VLCDR3 share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 1-104; 247-252.

[00151] In some examples, the VHCDR1 , the VHCDR2, the VHCDR3, the VLCDR1 , the VLCDR2, and/or the VLCDR3 have an amino acid sequence selected from SEQ ID NO: 1-104 comprising 2, or 3 amino acid substitutions.

[00152] In some examples, the NKp80-targeting domain comprises:

[00153] In some examples, the NKp80-targeting domain comprises:

(1) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141- 155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NQ:180-182; 260; and/or

(2) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253, a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO: 138-140; 256.

[00154] In some examples, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and/or the VLFR4 share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:105-182; 253-260. [00155] In some examples, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and/or the VLFR4 have an amino acid sequence selected from SEQ ID NO: 105-182; 253-260 comprising 2, or 3 amino acid substitutions.

[00156] In some examples, the NKp80-targeting domain comprises:

(1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(4) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253 a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO:138- 140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[00157] In some examples, the NKp80-targeting domain comprises:

(1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252;

(2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO: 17-31; 248, and VLCDR3 of SEQ ID NO:32-50; 249;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO: 141- 155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO:180-182; 260; and/or

(4) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253 a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO: 138-140; 256. [00158] In some examples, the VHCDR1 , the VHCDR2, the VHCDR3, the VLCDR1 , the VLCDR2, the VLCDR3, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and/or the VLFR4 share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:1-182; 247-260.

[00159] In some examples, the VHCDR1 , the VHCDR2, the VHCDR3, the VLCDR1 , the VLCDR2, the VLCDR3, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and/or the VLFR4 have an amino acid sequence selected from SEQ ID NO:1-182; 247-260 comprising 2, or 3 amino acid substitutions.

[00160] FIG. 1 summarizes exemplary NKp80 activating binders identified from antibody discovery to functional characterization. FcX denotes an Fc region with diminished ADCC function (see Example 1).

[00161] The present disclosure circumvents many of the difficulties with NK infusion and describes a novel series of NKp80-targeting domains capable of binding to and activating innate immune cells. In some examples, these domains are single innate immune cell-targeting domains used in combinations comprising 1 domain, or 2 domains, or 3 domains, or 4 domains, or 5 domains, or 6 domains. In some examples, these domains are used in multi-specific polypeptide modular constructs comprising components targeting innate immune cells and/or specific antigens. In some examples, the multi-specific polypeptide construct is a multi-specific antigen binding polypeptide construct. In some examples, the multi-specific polypeptide construct is a bispecific, a tri-specific, a tetra-specific, a penta-specific or a hexa-specific multi-specific polypeptide construct. In some examples, the multi-specific polypeptide construct further binds to a target cell antigen. In some examples, the multi-specific polypeptide construct binds both an innate immune cell modulator and a target cell antigen. In some examples, the multi-specific polypeptide construct specifically binds to one or more innate immune cell modulator(s) and to one or more target cell antigen(s).

A. Natural Killer (NK) cells

[00162] Natural killer (NK) cells are specialized immune effector cells playing a critical role in immune activation against abnormal cells. Different from events required for T cell activation, NK cell activation is governed by the interaction of NK receptors with target cells, independent of antigen processing and presentation. Due to relatively unsophisticated cues for activation, NK cells have gained significant attention in the field of cancer immunotherapy. Many efforts from studies in the art are emerging for developing and engineering NK cell-based cancer immunotherapy.

[00163] Various immunoregulatory molecules, including receptors involved in missing and induced self-recognition, influence NK reactivity. The main activating receptors expressed on human NK cells include FcyRllla (CD16), NKG2D, DNAM-1, and the natural cytotoxicity receptors containing the receptors NKp30, NKp44, NKp65, NKp80, and NKp46. Like NKG2D, NKp80 stimulates NK cell cytotoxicity and induces calcium influx in human NK cells after being triggered by the appropriate antibodies.

/. ADCC

[00164] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a potent cytotoxic mechanism that is mainly mediated in humans by natural killer (NK) cells. ADCC mediates the clinical benefit of several widely used cytolytic monoclonal antibodies (mAbs) and increasing its efficacy would improve cancer immunotherapy. CD16a is a receptor for the Fc portion of IgGs and is responsible to trigger NK cell mediated ADCC. The knowledge of the mechanism of action of CD16a gave rise to several strategies to improve ADCC, by working on either the mAbs or the NK cell.

[00165] In some examples, the multi-specific polypeptide construct comprises an Fc domain variant. In some examples, the Fc domain variant shows diminished activity or binding compared to a native / wild type Fc domain. In some examples, the diminished Fc domain is represented as FcX in the present disclosure. In some examples, the diminished Fc domain is constructed in accordance with methods known in the art. In some examples, a component of the multi-specific polypeptide constructs is a native / wild type Fc domain and/or variant Fc domain. An exemplary Fc domain variant (or Fc mutated domain) comprises an amino acid sequence differing from that of a native / wild type Fc region sequence by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). In some examples, the variant Fc region has at least one amino acid substitution compared to a native / wild type sequence Fc region or to the Fc region of a parent polypeptide. In some examples, the variant Fc region (or Fc mutated region) comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in a native I wild type Fc region sequence. In some examples, the multi-specific construct includes a variant Fc region possessing at least about 80% homology, or at least about 85%, or at least about 90% , or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology with a native / wild type sequence Fc region.

[00166] In some examples, a diminished Fc domain (FcX) confers a diminished ADCC, which refers to a reduction in measurable ADCC response of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% of a control. In some examples, an Fc silent domain / Fc inactivated mutant (FcLALA) confers little to no measurable ADCC, which refers to substantially complete silencing of measurable ADCC response of at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of a control, or substantially complete silencing of ADCC such that no measurable ADCC is detected. In some examples, an enhanced Fc domain confers an enhanced ADCC. Said enhanced ADCC refers to the improvement or increase or multiplication of measurable ADCC response of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150% or more of a control. ii. Immunotherapy

[00167] Current preclinical development of NK cell-based therapy was largely inspired by early clinical studies. With the understanding of how NK cells are activated, the initial NK cell-based therapy was pioneered in the clinical setting of hematopoietic stem cell transplants (HSCTs) whereby NK cells were shown to have the capacity to exert a graft versus leukemia effect. It is currently believed that the success of adoptive transfer requires the creation of a lymphopenic environment to provide a niche for donor cells to survive and proliferate.

[00168] In some examples, the disclosure comprises a multi-specific polypeptide construct or composition or pharmaceutical composition or use or method as described herein, wherein the polypeptide or composition or pharmaceutical composition is administered to the subject through one or more routes of administration including, but not limited to, topical, intravascular, intravenous, oral, subcutaneous, intraarterial, intrathecal, intraperitoneal, intranasal, intradermal, intramuscular, and the like.

[00169] In another aspect, there is provided a pharmaceutical composition comprising the multispecific polypeptide construct, or the antibody as disclosed herein. [00170] In some examples, there is provided a pharmaceutical composition for use in treating cancer, wherein the multi-specific polypeptide construct, or the antibody is to be administered to a subject in need thereof, in an effective amount to treat the cancer in the subject.

[00171] In some examples, there is provided use of a pharmaceutical composition in the manufacture of a medicament for treating cancer, wherein the multi-specific polypeptide construct, or the antibody is to be administered to a subject in need thereof, in an effective amount to treat the cancer in the subject.

[00172] In another aspect, there is provided a method for treating cancer comprising administering to a subject in need thereof the pharmaceutical composition as disclosed herein, wherein the multi-specific polypeptide construct, or the antibody, is administered in an effective amount to treat the cancer in the subject.

[00173] In some examples, the subject has cancer cells that express HER2, CD20 and/or EGFR.

[00174] In some examples, the method as disclosed herein, wherein:

(1) the cancer is a solid cancer;

(2) the cancer is selected from the group consisting of breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and stomach cancer; and/or

(3) the cancer is selected from the group consisting of lung adenocarcinoma, conventional glioblastoma multiforme, glioblastoma, colon adenocarcinoma, and non-small cell carcinoma.

[00175] In some examples, the method as disclosed herein further comprises administering a second therapeutic treatment, wherein the second therapeutic treatment comprises a chemotherapeutic agent, a biologic agent, hormonal therapy, radiation, or surgery.

B. NK cell receptors

[00176] In some examples, the multi-specific polypeptide construct comprises one innate immune cell targeting domain, two innate immune cell targeting domains, three innate immune cell targeting domains, four innate immune cell targeting domains, five innate immune cell targeting domains, or six innate immune cell targeting domains, or more. In some examples, the multi-specific polypeptide construct comprises one or more innate immune cell targeting domains, wherein the innate immune cell targeting domain is a NK cell targeting domain (i.e., an NK targeting domain). In some examples, the NK cell targeting domain is selected from an NKp80- binding Fab fragment, an NKp80-binding Fd fragment, a NKp80-binding F(ab)2 fragment, an NKp80-binding Fv fragment, an NKp80-binding single domain antibody fragment, an NKp80- binding CDR, an NKp80-binding single chain Fv, an NKp80-binding dsFv, an NKp80-binding scab, an NKp80-binding STAb, an NKp80-binding single domain heavy chain antibody, an NKp80-binding single domain light chain antibody, an NKp80-binding VHH, an NKp80-binding VNAR, and other NKp80-binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies. In some examples, the multi-specific polypeptide construct further comprises a second NK targeting domain. In some examples, the second NK targeting domain is an Fc domain.

[00177] In humans, NK cell receptors include killer cell immunoglobulin-like receptors (KIR), C- type lectins (CD94/NKG2A/NKG2C, NKG2D), natural cytotoxicity receptors (NCR; NKp44, NKp30, NKp65, NKp80, and NKp46), CD16/FcyRllla, and integrin/adhesion molecules. When deciding whether to respond to a target cell or not, these signals are combined, including integration of the NK cell activation status influenced by cytokine priming or other events, such as latent viral infection. Once appropriately triggered, the NK cell responds by killing the target and producing cytokines including IFN-y, TNF-a, GM-CSF, Ml P-1 a, and others.

[00178] In some examples, the multi-specific polypeptide construct comprises a second NK cell targeting domain capable of binding to a NK cell. An exemplary second targeting domain is selected from a sequence binding to NKp80, CD16, NKp46, NKp30, NKp44, NKG2D, NKp65, DNAM, CD94, NKG2A, TIGIT, interleukin receptors and the like. In some examples, the multispecific polypeptide construct described herein comprises two NK targeting domains, wherein the first NK targeting domain is a NKp80 targeting domain and the second NK targeting domain is a CD16 targeting domain.

[00179] In some examples, the multi-specific polypeptide construct further comprises a second NK cell targeting domain, wherein the second targeting domain is selected from, but is not limited to, a domain targeting CD16, NKp46, NKp30, NKp44, NKG2D, NKp65, DNAM, CD94, NKG2A, TIGIT, and interleukin receptors.

/. NCR

[00180] In humans, NCRs NKp46, NKp80 and NKp30 are expressed on activated and resting NK cells, but NKp44 is upregulated upon interleukin-2 stimulation of some NK cells. Reported ligands for NKp46 and NKp44 include viral hemagglutinins. Cellular ligands probably exist, given that anti-NCR antibodies abrogate NK cell-mediated lysis of many tumor cell types. Other ligands of NCRs include nuclear factor HLA-B-associated transcript 3, which can be released from tumor cells and binds NKp30. NKp46 and NKp30 have also been shown to bind heparin sulfate proteoglycans and NKp80 binds activation-induced C-type lectin (AICL). More recently, NKp30 has also been shown to bind the B7-H6 tumor antigen. The NCRs have been suggested to be one of the main mechanisms by which NK cells kill tumor targets. (Pegram et al. , Activating and inhibitory receptors of natural killer cells, Immunol and Cell Biol 89(2):216-224 (2010)).

[00181] NKp80, an activating homodimeric C-type lectin-like receptor (CTLR), is expressed on essentially all human natural killer (NK) cells and stimulates their cytotoxicity and cytokine release. The ligand for NKp80 is the myeloid-specific CTLR activation-induced C-type lectin (AICL), which is encoded in the natural killer gene complex (NKC) adjacent to NKp80. In some examples, the NKp80 expressed on NK cells has accession number Q9NZS2. In some examples, the NKp80 receptor comprises the human sequence:

MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTL ISLILLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSAD QTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQ KNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIF SETCSSVFKWICQY (huNKp80 sequence; SEQ ID NO:223).

[00182] In some examples, the NKp80 receptor comprises the cynomolgus sequence:

VLLKCQKGSHSNTTEHEDIGDLKMNNGTRRNTSNKDLCVSRSADQTVLCQSE WLKYRGKCYWFSNEMKSWSDSYVYCLERKSHLLIIQDELEMAFIQKNLRQSNY VWMGLNFTSLKMTWTWVDGSPLDPKIFFIKGPAKENSCAAIKESKIYSETCSSV FKWICQY (cyNKp80 sequence; SEQ ID NO:261).

[00183] In some examples, the NK targeting domain is a domain targeting NKp80.

[00184] In some examples, the multi-specific polypeptide construct as described herein comprises a variable light chain amino acid sequence for the NKp80-targeting domain selected from:

AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKL ASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTE WVK (VL sequence of antiNKp80(13); SEQ ID NO: 183); DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTL ASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVWK (VL sequence of antiNKp80(28)-FcX); SEQ ID NO:184);

DWMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYGASTL ESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTTRSSSIYWPFGGGTEV WK (VL sequence of antiNKp80(36); SEQ ID NO:185);

DWMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYTAYTL ESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEV WK (VL sequence of antiNKp80(37); SEQ ID NO:186);

DWMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTL ESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEV WK (VL sequence of antiNKp80(45); SEQ ID NO:187);

AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTL ESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEV WK (VL sequence of antiNKp80(50); SEQ ID NO:188);

ALVMTQTPSSVSAAVGGTVTIKCQASQSIGNDLAWYQQKPGQPPKLLIYAASNL ESGVPSRFRGSGSGTKFTLTISDLECADAATYYCQGTYRGSSISWPFGGGTEV WK (VL sequence of antiNKp80(51); SEQ ID NO:189);

QIVVTQTPASVSAAVGGTVTISCQSSQNVYGNNELSWYQQKPGQPPKLLIYKA STLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGYSGGMRSFGGGT EWLV (VL sequence of antiNKp80(71); SEQ ID NO: 190);

QIVVTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLAS TLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTE WVK (VL sequence of antiNKp80(74); SEQ ID NO: 191);

AQVLTQTASSVSAAVGGTVTISCQSSQSVYNYNWLGWYQQKPGQPPKLLIYEA SKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSVDCNVFG GGTEVWK (VL sequence of antiNKp80(78); SEQ ID NO:192);

ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDL ASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTK WVK (VL sequence of antiNKp80(79); SEQ ID NO: 193); AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLA SGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEV WK (VL sequence of antiNKp80(81); SEQ ID NO:194);

DWMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTL ESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSKCVFPFGGG TEWVK (VL sequence of antiNKp80(82); SEQ ID NO:195);

DIVMTQTPASVEAAVGGTVTIKCQASQSIGRDLAWYQQKPGQPPKLLIYGASILE SGVPSRFKGNGSGTQFTLTISDLECADAATYYCQGADRSSTPSWPFGGGTEV WK (VL sequence of antiNKp80(87); SEQ ID NO:196);

AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKT SSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTE WVK (VL sequence of antiNKp80(94); SEQ ID NO: 197);

AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTL ESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTE WVK (VL sequence of antiNKp80(101); SEQ ID NO: 198);

DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASD LASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTE

WVK (VL sequence of antiNKp80(102); SEQ ID NO: 199);

DWMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTL ESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTE

WVK (VL sequence of antiNKp80(106); SEQ ID N0:200);

DIVMTQTPASVSAAAGGTVTINCQASQSISNELSWYQQKSGQPPKLLIYGASNL ESGVPSRFKGSGSGTDFTLTISDLECADGATYYCQSNYYDSSSPDFAFGGGTE

WVK (VL sequence of antiNKp80(63); SEQ ID NO:201); and/or

DWMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTL ESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSNCVFPFGGG TEWVK (VL sequence of antiNKp80(83); SEQ ID NO:202).

[00185] In some examples, the multi-specific polypeptide construct as described herein has a sequence comprising a variable heavy chain domain of amino acid sequence for the NKp80- targeting domain selected from: QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIYTGGGS ADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGYGDATDIWGPG TLVTV (VH sequence of antiNKp80(13); SEQ ID NO:203);

QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYAGSSG STHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDYFKIWGPGTL VTV (VH sequence of antiNKp80(28); SEQ ID NO:204);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTGSSGS TYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRYNIWGPGTLVT

V (VH sequence of antiNKp80(36); SEQ ID NO:205);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTGSSGST YYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRYNIWGPGTLVT

V (VH sequence of antiNKp80(37); SEQ ID NO:206;

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSG STYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWRYNIWGPGTLV TV (VH sequence of antiNKp80(45); SEQ ID NO:207);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSG STYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWRYNIWGPGTL VTV (VH sequence of antiNKp80(50); SEQ ID NO:208);

QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYSDSGNT YYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRYNIWGPGTLVT

V (VH sequence of antiNKp80(51); SEQ ID NO:209);

QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSPGVAYY ASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWGQGTLVTV (VH sequence of antiNKp80(71); SEQ ID NO:210);

QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSGGSAYY ASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWGQGTLVTV (VH sequence of antiNKp80(74); SEQ ID NO:211);

QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNGGATYY ASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLWGQGTLVTV (VH sequence of antiNKp80(78); SEQ ID NO:212); QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPSGSIWY ANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWGQGTLVTV (VH sequence of antiNKp80 (79); SEQ ID NO:213);

QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIADKIYYA SWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQGTLVTV (VH sequence of antiNKp80 (81); SEQ ID NO:214);

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYF ASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLWGQGTLVTV (VH sequence of antiNKp80(82); SEQ ID NO:215);

QSLEEYGGDWQPEGSLTLTCTASGFSFSGNYWICVWRQAPGKGLEWIGCIYAGSSG STCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWKYNIWGPGTL VTV (VH sequence of antiNKp80(87); SEQ ID NO:216);

QSVEESGGRLVTPGTPLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTGGSAYY ANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWGQGTLVTV (VH sequence of antiNKp80(94); SEQ ID NO:217);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISWGGNTY YASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDPWGPGTLLTV (VH sequence of antiNKp80(101); SEQ ID NO:218);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSGGDTSY ATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLWGQGTLVTV (VH sequence of antiNKp80(102); SEQ ID NO:219);

QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDGSAYYA SWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWGQGTLVTV (VH sequence of antiNKp80(106); SEQ ID NO:220);

QSLEESGGRLVKPDETLTITCTVSGIDLSSYIISWVRQAPGEGLEYIGFINTDGSAYYAT WAKGRFTISRTSATVDLKMTSLTTEDTATYFCARDAGHRYLFYFKLWGQGTLVTV (VH sequence of antiNKp80(63); SEQ ID NO:221); and

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYY ASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLWGQGTLVTV (VH sequence of antiNKp80(83); SEQ ID NO:222).

[00186] In some examples, the NKp80-targeting domain comprises: (1) a VH comprising an amino acid sequence selected from SEQ ID NO:203-222; 236; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(2) a VL comprising an amino acid sequence selected from SEQ ID NO: 183-202; 235; wherein the VH and the VL are paired to produce clone 13, clone 28, clone 36, clone 37, clone 45, clone 50, clone 51 , clone 63, clone 71 , clone 74, clone 78, clone 79, clone 81 , clone 82, clone 83, clone 87, clone 94, clone 101 , clone 102, clone 106, or humanized clone 87-2; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;.

[00187] In some examples, the NKp80-targeting domain comprises:

(1) a VH comprising an amino acid sequence selected from SEQ ID NQ:203-222; 236;

(2) a VL comprising an amino acid sequence selected from SEQ ID NO: 183-202; 235; wherein the VH and the VL are paired to produce clone 13, clone 28, clone 36, clone 37, clone 45, clone 50, clone 51 , clone 63, clone 71 , clone 74, clone 78, clone 79, clone 81 , clone 82, clone 83, clone 87, clone 94, clone 101 , clone 102, clone 106, or humanized clone 87-2.

[00188] In some examples, the VH, and/or the VL share at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:203-222; 236, and/or selected from SEQ ID NO:183-202; 235, respectively.

[00189] In some examples, the VH, and/or the VL have an amino acid sequence selected from SEQ ID NQ:203-222; 236 and/or selected from SEQ ID NQ:183-202; 235, respectively, comprising 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

[00190] In some examples, the NKp80-targeting domain comprises a member selected from:

(1) VLFR1 (SEQ ID NO:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO:180) (clone 13); (2) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NO:180) (clone 28);

(3) VLFR1 (SEQ ID NQ:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 36);

(4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 37);

(5) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NO:180) (clone 45);

(6) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:36), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:71), VHFR3 (SEQ ID NO:168), VHCDR3 (SEQ ID NO:90), and VHFR4 (SEQ ID NQ:180) (clone 50);

(9) VLFR1 (SEQ ID NO:110), VLCDR1 (SEQ ID NO:7), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:23), VLFR3 (SEQ ID NO:128), VLCDR3 (SEQ ID NO:39), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:146), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:159), VHCDR2 (SEQ ID NO:74), VHFR3 (SEQ ID NO:171), VHCDR3 (SEQ ID NO:93), and VHFR4 (SEQ ID NO:181) (clone 74);

(11) VLFR1 (SEQ ID NO:112), VLCDR1 (SEQ ID NO:9), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:25), VLFR3 (SEQ ID NQ:130), VLCDR3 (SEQ ID NO:41), VLFR4 (SEQ ID NO:140), (VHFR1 (SEQ ID NO:148), VHCDR1 (SEQ ID NO:59), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:76), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:95), and VHFR4 (SEQ ID NO:181) (clone 79);

(12) VLFR1 (SEQ ID NO:113), VLCDR1 (SEQ ID NO:10), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:26), VLFR3 (SEQ ID NO:131), VLCDR3 (SEQ ID NO:42), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:149), VHCDR1 (SEQ ID NQ:60), VHFR2 (SEQ ID NO:160), VHCDR2 (SEQ ID NO:77), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:96), and VHFR4 (SEQ ID NO:181) (clone 81);

(14) VLFR1 (SEQ ID NQ:106), VLCDR1 (SEQ ID NO:12), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:27), VLFR3 (SEQ ID NO:133), VLCDR3 (SEQ ID NO:44), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:151), VHCDR1 (SEQ ID NO:62), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:79), VHFR3 (SEQ ID NO:175), VHCDR3 (SEQ ID NO:98), and VHFR4 (SEQ ID NO:180) (clone 87);

(20) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:50), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:85), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:104), and VHFR4 (SEQ ID NO:181) (clone 83); or (21) VLFR1 (SEQ ID NO:253), VLCDR1 (SEQ ID NO:247), VLFR2 (SEQ ID NO:254), VLCDR2 (SEQ ID NO:248), VLFR3 (SEQ ID NO:255), VLCDR3 (SEQ ID NO:249), VLFR4 (SEQ ID NO:256), (VHFR1 (SEQ ID NO:257), VHCDR1 (SEQ ID NO:250), VHFR2 (SEQ ID NO:258), VHCDR2 (SEQ ID NO:251), VHFR3 (SEQ ID NO:259), VHCDR3 (SEQ ID NO:252), and VHFR4 (SEQ ID NO:260) (humanized clone 87-2).

[00191] In some examples, the multi-specific polypeptide construct as described herein comprises an antigen binding fragment sequence comprising 1 , or 2, or 3, or 4, or 5, or 6 CDRs selected from SEQ ID NO: 1-104; 247-252.

[00192] NKp46 has been established as a critical activating receptor since it is expressed almost exclusively by NK cells and is the only NCR with a mouse orthologue, denoted Ncr1. Its ligand repertoire ranges from viral ligands, such as, hemagglutinin (HA) and hemagglutininneuraminidase (HN) of influenza virus, Sendai virus, Newcastle disease virus, and poxvirus, to fungal ligands, to unknown ligands found on tumors, adipose cells, human pancreatic beta cells, hepatic stellate cells, and bacteria such as Fusobacterium nucleatum. Recently, a soluble NKp46 ligand was identified. The identification of the unknown membrane-bound ligands, in particularly the tumor ligands of NKp46, has been intensely investigated for over two decades. In some examples, the NK targeting domain is a domain targeting NKp46.

[00193] Like NKp80, NKp65 triggers NK cell cytotoxicity in assays of redirected cytolysis. But unlike NKp80, NKp65 is not detectably expressed on human peripheral blood NK or T cells, although NKp65 cDNA was originally cloned from IL-2/IL-12-stimulated peripheral blood NK cells. So far, significant surface expression of NKp65 has been noted only for the NK cell line NK92 and its derivative NK92MI, and hence, cells physiologically expressing NKp65 and the determining factors of NKp65 expression in vivo await elucidation. In some examples, the NK targeting domain is a domain targeting NKp65. ii. C-type lectins

[00194] NK cells recognize ‘stressed’ cells through the activating receptor NKG2D, which is expressed on almost all mouse NK cells. This receptor has been shown to be important in the NK cell-mediated control of some cancers. The NKG2D molecule recognizes several different ligands. This ability is thought to be due to a single binding site in the receptor, with side chains that show a limited flexibility resulting in a rigid body interaction model of ligand binding. NKG2D ligands include MHC class l-related proteins whose expression is regulated by both the DNA damage and heat shock response pathways, which are often activated in tumors. Given the immune-stimulatory nature of NK cells, NKG2D-mediated recognition of tumor cells is integral for an optimal immune response to some tumors. In some examples, the NK targeting domain is a domain targeting NKG2D.

[00195] Another C-type lectin family of receptors is the CD94-NKG2A/C/E heterodimers. These receptors react to the level of non-classical MHC class I on the surface of potential target cells and are thought to be important in the prevention of inappropriate NK cell activation. Heterodimers, CD94-NKG2C and CD94-NKG2E, have been shown to associate with DAP-12 and are thought to be activating receptors. In humans, the inhibitory, ITIM-containing CD94- NKG2A receptor and activating, DAP-12-associating CD94-NKG2C receptor both bind HLA-E, a non-classical HLA class I molecule. The reason for having one activating and one inhibitory receptor specific for the same molecule remains unclear. This phenomenon may allow more specific discrimination between normal and distressed or infected tissue, as expression of this ligand may not necessarily lead to NK cell activation. In some examples, the NK targeting domain is a domain targeting CD94.

Hi. Co-stimulatory receptors

[00196] There are several other NK cell receptors, which are viewed as co-stimulatory. These receptors provide further stimulation to the cell, although alone are not sufficient to trigger NK cell activation. Hence, not only do they provide an alternate mechanism of activation, but also ensure that the NK cells are not activated to respond to normal or healthy tissue. These receptors include DNAM-1 , the NKR-P1 receptors and the PILR receptor.

[00197] DNAM-1 receptor (also known as CD226) is a member of the Ig-superfamily and is constitutively expressed upon approximately 50% of NK cells. The ligands for this co-stimulatory activating receptor are CD155 (also referred to as Polio virus receptor, PVR or Necl-5) and CD112 (Nectin-2), and these ligands can be upregulated on some tumor cells, implicating DNAM-1 in some NK cell-mediated anti-tumor responses. In some examples, the NK targeting domain is a domain targeting DNAM-1 . iv. FcyR

[00198] In humans, there are three classes of Fc receptors that bind IgG (FcyRs): FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). FcyR expression by leukocytes induces their activation (CD64, CD32A, CD32C, CD16A, and CD16B) or inhibition (CD32B) to regulate immune responses and signal thresholds. [00199] CD16 represents a prototype NK cell-activating receptor since its engagement is, by itself, sufficient to trigger cytotoxic activity and production of pro-inflammatory cytokines and chemokines, thus unleashing NK cell antitumor functions. Human CD16, also expressed by macrophages and some circulating monocytes, consists of two extracellular Ig domains, a short cytoplasmic tail and a transmembrane domain that, in NK cells, allows its association with CD3 and FcyRI chains; these immunoreceptor tyrosine-based activation motif (ITAM)-containing subunits connect the receptor to intracellular signal transduction pathways, which coordinate the reorganization of the actin and microtubule cytoskeleton, and the activation of several transcription factors. In some examples, the NK targeting domain is a domain targeting CD16.

[00200] Also included in the present disclosures are functional Fc domain which possess an “effector function” of a native / wild type sequence Fc region. Examples of “effector functions” include but are not limited to CD16 binding; C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require the Fc domain to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as known in the art. In some examples, the Fc domain possesses functional antibody dependent cell cytotoxicity (e.g., via CD16 binding), diminished ADCC (FcX) (e.g., via a configuration change of a functional Fc domain), no ADCC (Fc silent / inactivated mutant Fc domain (FcLALA) (e.g., via specific Fc mutations), or enhanced ADCC (FcE).

[00201] In some examples, the multi-specific polypeptide construct binds to the second modulator through an Fc domain or Fc component. In some examples, the multi-specific polypeptide construct includes, but is not limited to, an Fc domain, a native / wild type Fc domain, an Fc enhanced domain, an Fc diminished domain, an Fc silent domain I an Fc inactivated domain, an Fc mutated domain, a heterodimer Fc domain, and the like. In some examples, the antigen binding protein comprises a native / wild type Fc domain. In some examples, the native / wild type Fc domain is represented as FcWT. In some examples, the antigen binding protein comprises a variant Fc domain. In some examples, the variant Fc domain is a diminished Fc domain. In some examples, the diminished Fc domain is represented as FcX. In some examples, the variant Fc domain is a Fc silent domain I inactivated mutant Fc domain (FcLAI_A). In some examples, the diminished Fc domain is constructed in accordance with methods known in the art. In some examples, the variant Fc domain is an enhanced Fc domain. In some examples, the enhanced Fc domain is represented as FcE. [00202] In some examples, the multi-specific polypeptide construct as disclosed herein further comprises a functional Fc domain.

[00203] In some examples, the Fc domain comprises an amino acid sequence selected from SEQ ID NO:224-226.

[00204] In some examples, the multi-specific polypeptide construct comprises a native I wild type Fc domain. In some examples, the native / wild type Fc domain comprises the sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

(native I wild type Fc domain; SEQ ID NO:224).

[00205] In some examples, the multi-specific polypeptide construct comprises a native I wild type Fc domain sequence configured to have a diminished ADCC function. In some examples, the diminished Fc domain comprises the sequence:

(diminished Fc domain (FcX); SEQ ID NO:224).

[00206] In some examples, the variant Fc domain is a silent Fc domain. In some examples, the silent Fc domain comprises the sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (silent Fc domain / inactivated mutant Fc domain (FcLALA); SEQ ID NO:225).

[00207] In some examples, the variant Fc domain is an enhanced Fc domain (FcE). In some examples, the enhanced Fc domain comprises the sequence: DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(enhanced Fc domain (FcE); SEQ ID NO:226).

[00208] In some examples, the Fc domain is

[00209] In some examples, the Fc domain is:

(i) a native / wild type Fc domain (FcWT) or a diminished Fc (FcX) domain of SEQ ID NO:224;

(ii) an enhanced Fc domain (FcE) of SEQ ID NO:226;

(iii) a silent Fc domain I inactivated mutant Fc domain (FcLALA) of SEQ ID NO:225.

[00210] In some examples, the Fc domain has an amino acid sequence share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:224-226.

[00211] In some examples, the Fc domain has an amino acid sequence selected from SEQ ID NO:224-226 comprising 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

[00212] In some examples, the multi-specific polypeptide construct as disclosed herein, comprises:

(i) an antigen targeting domain consisting of a Fd fragment (comprising of a VH and a CH1) or a Fab fragment (comprising a VH, a CH1 , a CL1 , and a VL);a first NK cell targeting domain consisting of a Fc domain (comprising a CH2 and a CH3); a first [(G4S)n] linker; and a second NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(ii) a first NK cell targeting domain consisting of a Fd fragment (comprising a VH and a CH1) or a Fab fragment (comprising a VH, a CH1 , a CL1 , and a VL); a second NK targeting domain consisting of a Fc domain (comprising a CH2 and a CH3); a first [(G4S)n] linker; and an antigen targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(iii) a first NK cell targeting domain consisting of a Fd fragment (comprising a VH and a CH1) or a Fab fragment (comprising a VH, a CH1 , a CL1 , and a VL); a first [(G4s)n] linker; an antigen targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL; and a second NK cell targeting domain consisting of a Fc domain comprising a CH2 and a CH3; or

(iv) an antigen targeting domain consisting of a Fd fragment (comprising of a VH and a CH1) or a Fab fragment (comprising a VH, a CH1 , a CL1 , and a VL); a first [(G4S)n] linker; a first NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL; and a second NK cell targeting domain consisting of a Fc domain comprising a CH2 and a CH3.

[00213] In some examples, the multi-specific polypeptide construct as disclosed herein comprises:

(a) a first domain targeting NKp80;

(b) a second domain targeting CD16;

[00214] In some examples, the one or more antigen targeting domains comprise:

(2) a VH of amino acid sequence SEQ ID NO:227 (VH Trastuzumab), a VL amino acid sequence SEQ ID NO:228 (VL Trastuzumab), a CH of amino acid sequence SEQ ID NO:229, and/or a CL of amino acid sequence SEQ ID NO:230; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or (3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[00215] In some examples, the one or more antigen binding domains comprise:

(1) a VH of amino acid sequence SEQ ID NO:231 (VH Cetuximab), a VL of amino acid sequence SEQ ID NO:232 (VL Cetuximab), a CH of amino acid sequence SEQ ID NO:233, and/or a CL of amino acid sequence SEQ ID NO:234;

(2) a VH of amino acid sequence SEQ ID NO:227 (VH Trastuzumab), a VL amino acid sequence SEQ ID NO:228 (VL Trastuzumab), a CH of amino acid sequence SEQ ID NO:229, and/or a CL of amino acid sequence SEQ ID NO:230; and/or

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab).

[00216] In some examples, the antigen targeting domain VH, VL, CH, and/or CL share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:227-238; 243-246.

[00217] In some examples, the antigen targeting domain VH, VL, CH, and/or CL have an amino acid sequence selected from SEQ ID NO:227-238; 243-246 comprising 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

[00218] In some examples, the multi-specific polypeptide construct as disclosed herein comprises:

(A) an NKp80-targeting domain comprising:

(1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NQ:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; (2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO: 17-31 ; 248, and VLCDR3 of SEQ ID NQ:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(B) one or more antigen targeting domains comprising:

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;.

[00219] In some examples, he multi-specific polypeptide construct as disclosed herein, comprises:

(A) an NKp80 targeting domain comprising:

(1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; (2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO: 17-31 ; 248, and VLCDR3 of SEQ ID NQ:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141-155; 257, a VHFR2 of SEQ ID NO: 156-162; 258, a VHFR3 of SEQ ID NO: 163-179; 259 and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(B) one or more antigen targeting domains comprising:

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and

(C) a Fc domain having an amino acid sequence selected from SEQ ID:224-226; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

[00220] In some examples, the multi-specific polypeptide construct as disclosed herein, comprises:

(A) an NKp80 targeting domain comprising: (1) a VH comprising 1 , 2, or 3 CDR selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68-85; 251 , and/or a VHCDR3 of SEQ ID NO:86-104; 252;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO: 141- 155; 257, a VHFR2 of SEQ ID NO: 156-162; 258, a VHFR3 of SEQ ID NO: 163-179; 259 and/or a VHFR4 of SEQ ID NO: 180-182; 260; and/or

(4) 1 , 2, 3, or 4 VL FR selected from a FR1 of SEQ ID NO:105-118; 253, a of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO:138- 140; 256; and

(B) one or more antigen targeting domains comprising:

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); and

(C) a Fc domain having an amino acid sequence selected from SEQ ID:224-226.

[00221] In some examples, the VHCDR1 , the VHCDR2, the VHCDR3, the VLCDR1 , the VLCDR2, the VLCDR3, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and the VLFR4 at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:1- 182; 247-260; wherein the VH and VL share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:183-222; 235-236; and/or wherein the Fc domain has an amino acid sequence share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity to an amino acid sequence selected from SEQ ID NO:224-226.

[00222] In some examples, the VHCDR1 , the VHCDR2, the VHCDR3, the VLCDR1 , the VLCDR2, the VLCDR3, the VHFR1 , the VHFR2, the VHFR3, the VHFR4, the VLFR1 , the VLFR2, the VLFR3, and/or the VLFR4 have an amino acid sequence selected from SEQ ID NO:1-182; 247-260 comprising 2, or 3 amino acid substitutions; wherein the VH and VL of amino acid sequence selected from SEQ ID NO: 183-222; 235-236 comprise 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions within; and/or wherein the Fc has an amino acid sequence selected from SEQ ID NO:224-226 comprising 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

[00223] In some examples, the multi-specific polypeptide construct is a tri-specific antigen binding construct comprising:

(a) first targeting domain binding NKp80;

(b) a second targeting domain binding CD16; and

(c) a third targeting domain binding to a target antigen, wherein the targeting domains are selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, tynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies (e g. f-star technology (F-star's Modular Antibody TechnologyTM)). [00224] In some examples, the multi-specific polypeptide construct is a tri-specific antigen binding construct comprising:

(a) a first targeting domain binding to NKp80, wherein the targeting domain is selected from a Fab fragment, a Fv fragment; a sdAb fragment, an isolated CDR, a scFv, a dsFv, a scAb, a ST Ab, a sdAb, a single domain CH antibody, a single domain CL antibody, a VHH, a VNAR, and a sdAb based on the VNAR structure from shark;

(b) a first targeting domain binding to CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO:224), FcX (SEQ ID NO:224), silent Fc I inactivated mutant Fc domain (FcLALA) (SEQ ID NO:225), or FcE (SEQ ID NO:226); and

(c) a third targeting domain binding to a tumor-associated antigen, optionally HER2, EGFR or CD20, wherein the targeting domain is selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, tynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies (e. g. f-star technology (F-star's Modular Antibody TechnologyTM)).

C. Antigen targets

[00225] Exemplary polypeptides of the disclosure include at least one antigen binder. The polypeptides are not limited by the identity of the antigen binder or its binding target. Polypeptides are exemplified by reference to binders to antigens of HER2, EGFR, and/or CD20, but are not limited to these antigen binders.

[00226] In some examples, the one or more antigen targeting domains bind to a member selected from HER-2, EGFR, and CD20.

[00227] In some examples, the multi-specific polypeptide construct binds one or more of these tumor antigens. In some examples, the multi-specific antigen binding polypeptide construct is a bi-specific antigen binding polypeptide binding NKp80 and HER2 (i.e., antiNKp80-antiHER2) as disclosed herein. In some examples, the bi-specific antigen binding polypeptide construct binding NKp80 and EGFR is antiNKp80-antiEGFR as disclosed herein. In some examples, the bi-specific antigen binding polypeptide construct binding NKp80 and CD20 is antiNKp80- antiCD20 as disclosed herein.

/. HER2

[00228] NK cell-mediated ADCC plays an important role in anti-HER2 therapy. However, the cytotoxicity of NK cells decreases with the altered activation receptor phenotype in breast cancer patients. Compared with healthy donors, NK cells express lower levels of NKp30, NKp46, and NKG2D in breast cancer patients. Therefore, the enhancement of NK cells and their ADCC effect is an effective way to improve the efficacy and sensitivity of trastuzumab.

[00229] In some examples, the multi-specific polypeptide construct comprises a binding fragment that is known in the art to bind to HER2. In some examples, the multi-specific polypeptide construct binding to HER2 comprises a heavy chain variable (VH) domain encoded by a sequence comprising:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSS (Trastuzumab VH; SEQ ID NO:227).

[00230] In some examples, the multi-specific polypeptide construct binding to HER2 comprises a light chain variable (VL) domain encoded by a sequence comprising:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Trastuzumab VL; SEQ ID NO:228).

[00231] In some examples, the multi-specific polypeptide construct binding to HER2 comprises a constant heavy chain (CH) domain encoded by a sequence comprising:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSG LYSLSSWTVPSSSLGTQTYI CN VN H KPSNTKVDKKVE PKSC (Trastuzumab CH1 ; SEQ ID NO:229).

[00232] In some examples, the multi-specific polypeptide construct binding to HER2 comprises a constant light chain (CL) domain encoded by a sequence comprising:

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (Trastuzumab CL; SEQ ID NO:230). [00233] In some examples, unless specified otherwise, the representation of the order of the domain as exemplified in the present disclosure does not limit the general structure of the multispecific polypeptide construct of the present disclosure. Therefore, where a multi-specific polypeptide construct is presented as antiNKp80-antiHER2, this disclosure refers to an example where the antiNKp80 arm of the multi-specific polypeptide construct is at either one of the N- terminus or C-terminus and that the antiHER2 is at the other end of the antiNKp80 arm of the multi-specific polypeptide construct (i.e. when antiNKp80 is at N terminus, antiHER2 is at C terminus, or when antiNKp80 is at C terminus, antiHER2 is at N terminus). These permutations are exemplified with EGFR-targeting multi-specific polypeptide constructs in FIG. 13C.

[00234] In some examples, the tri-specific multi-specific polypeptide construct binding NKp80, CD16 and HER2, wherein the Fc domain is functional, is antiHER2-antiNKp80-Fc as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and HER2, wherein the Fcdomain has diminished binding to CD16 is antiHER2-antiNKp80- FcX as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and HER2, wherein the Fc domain has enhanced binding to CD16 is antiHER2-antiNKp80-FcE as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and HER2, wherein the Fc domain is an inactivated mutant / silent domain (FcLALA), is antiHER2-antiNKp80-FcLALA as disclosed herein.

[00235] In some examples, the humanized antibody clones antiHER2-antiNKp80(45-2)-Fc, antiHER2-antiNKp80(101-1)-Fc, antiHER2-antiNKp80(94-1)-Fc, antiHER2-antiNKp80(87-2)-Fc originate from the mother clones of antiHER2-antiNKp80(45)-Fc, antiHER2-antiNKp80(101)-Fc, antiHER2-antiNKp80(94)-Fc, antiHER2-antiNKp80(87)-Fc respectively (FIG. 3).

[00236] In some examples, the tri-specific antigen binding polypeptide constructs (antiHER2- antiNKp80(94)-Fc, antiHER2-antiNKp80(101)-Fc, antiHER2-antiNKp80(45)-Fc and/or antiHER2- antiNKp80(87)-Fc) co-engage both NKp80 and CD16 with improved potency as compared to Trastuzumab (antiHER2-antiNKp80-Fc vs antiHER2-Fc) in OVCAR3 (FIG. 3A). The clone antiHER2-antiNKp80(87)-Fc showed improved potency in the four different cell lines tested (MKN1 , OVCAR3, HCT116, MDA-MB-231) (FIG. 4). In some examples, the selected humanized clones of the multi-specific polypeptide constructs show improved binding and/or cytotoxic potential towards cancer cells compared to the parental polypeptide construct, while showing low immunogenicity.

[00237] In the experimental data provided in the present disclosure, MRC-5, a lung fibroblasts cell line with low HER2 expression was used as a control against the tumor cell lines. The antigen binding polypeptide construct, antiHER2-Fc (Trastuzumab) bound to MRC-5 suggesting that the antiHER2 arm of the antigen binding polypeptide construct as described herein recognizes HER2 on MRC-5. However, the tri-specific antigen binding polypeptide construct (antiHER2-antiNKp80- Fc) as disclosed herein showed specificity of cytotoxicity only in cancer cells, with no killing of the MRC-5. In some examples, the multi-specific polypeptide construct specifically kills cancer cells while showing no cytotoxicity to non-cancerous cells.

[00238] In some examples, the co-engagement of NKp80 and CD16 in the tri-specific antigen binding polypeptide construct antiHER2-antiNKp80 (87-2)-Fc increased the potentiation of NK cell function, with no effect on T cell activation (FIG. 5). Therefore, the antigen binding polypeptide construct as disclosed herein is specific to NK cells. In some examples, the antigen binding polypeptide construct as disclosed herein does not cause T cell activation.

[00239] As shown in the experimental data of the present disclosure, clone HER2-NKp80(87-2)- CD16 consistently showed improved potency (mean EC50) compared to Trastuzumab (FIG. 4; TABLE 1 , Col. 4), with increase in fold change potency as the target antigen expression is decreased in target cells (TABLE 1, Col. 4)

[00240] In some examples, the NKp80 engager includes clone antiHER2-antiNKp80(87)-Fc. In some examples, the antigen binding polypeptide construct has a mean EC50 fold change of about 1 to about 1000. In some examples, the antigen binding polypeptide construct has a mean EC50 fold change potency of about 1 , about 5, about 10, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, and about 1000.

//. EGFR

[00241] In some examples, the tri-specific multi-specific polypeptide construct binding NKp80, CD16 and EGFR, wherein the Fc domain is functional, is antiEGFR-antiNKp80-Fc as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and EGFR, wherein the Fc domain has diminished binding to CD16 is antiEGFR- antiNKp80-FcX as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and EGFR, wherein the Fc domain has enhanced binding to CD16 is antiEGFR-antiNKp80-FcE as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and EGFR, wherein the Fc domain is an inactivated mutant I silent domain (FcLALA), is antiEGFR-antiNKp80-FcLALA as disclosed herein. In some examples, the multi-specific polypeptide construct as described herein comprises a variable light chain amino acid sequence selected from SEQ ID NO:183-202 as above, wherein the Trastuzumab VL sequence (SEQ ID NO:228) is replaced with the Cetuximab VL sequence:

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (Cetuximab VL; SEQ ID NO:232).

[00242] In some examples, the multi-specific polypeptide construct binding to EGFR comprises a constant light chain (CL) domain encoded by a sequence comprising:

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA EC (Cetuximab CL; SEQ ID NO:234).

[00243] In some examples, the multi-specific polypeptide construct comprises a binding fragment that is known in the art to bind to EGFR. In some examples, the multi-specific polypeptide construct binding to EGFR comprises a heavy chain variable (VH) domain encoded by a sequence comprising:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEF AYWGQGTLVTVSA (Cetuximab VH; SEQ ID NO:231).

[00244] In some examples, the multi-specific polypeptide construct binding to EGFR comprises a constant heavy chain (CH) domain encoded by a sequence comprising:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSG LYSLSSWTVPSSSLGTQTYI CN VN H KPSNTKVDKKVE PKSC (Cetuximab CH1 ; SEQ ID NO:233).

[00245] In the experimental data of the present disclosure, an isotype was used as an untargeted control not recognizing the target antigen.

[00246] As shown in the experimental data of the present disclosure, clone antiEGFR- antiNKp80(87)-Fc was more potent than cetuximab (antiEGFR-Fc) in the cytotoxicity killing assay (FIG. 6). The control of the cytotoxicity killing assay (isotype-antiNKp80(87)-Fc) showed no NK cytotoxicity killing against HER2-positive cell lines, suggesting that the cytotoxicity is antigen dependent (FIG. 7). Hi. CD20

[00247] In some examples, the tri-specific multi-specific polypeptide construct binding NKp80, CD16 and CD20, wherein the Fc domain is functional, is antiCD20-antiNKp80-Fc as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and CD20, wherein the Fc domain has diminished binding to CD16 is antiCD20-antiNKp80- FcX as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and CD20, wherein the Fc domain has enhanced binding to CD16 is antiCD20-antiNKp80-FcE as disclosed herein. In some examples, the tri-specific multi-specific polypeptide construct that binds to NKp80, CD16 and CD20, wherein the Fc domain is an inactivated mutant / silent domain (FcLALA), is antiCD20-antiNKp80-FcLALA as disclosed herein.

In some examples, the multi-specific polypeptide construct as described herein comprises a variable light chain amino acid sequence selected from SEQ ID NO:183-202 as above, wherein the Trastuzumab VL sequence (SEQ ID NO:228) is replaced with the Rituximab VL sequence:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLA SGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (Rituximab VL; SEQ ID NO:243).

[00248] In some examples, the multi-specific polypeptide construct binding to CD20 comprises a constant light chain (CL) domain encoded by a sequence comprising:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (Rituximab CL; SEQ ID NO245)

[00249] In some examples, the multi-specific polypeptide construct comprises a binding fragment that is known in the art to bind to CD20. In some examples, the multi-specific polypeptide construct binding to CD20 comprises a heavy chain variable (VH) domain encoded by a sequence comprising:

Q VQ LQQPG AELVKPG ASVKMSCKASG YTFTSYN M H WVKQTPG RG LEWI GAI Y PGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGG DWYFNVWGAGTTVTVSA (Rituximab VH; SEQ ID NO:244).

[00250] In some examples, the multi-specific polypeptide construct binding to CD20 comprises a constant heavy chain (CH) domain encoded by a sequence comprising: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC (Rituximab CH1 ; SEQ ID NO:246).

D. Innate immune cell engagers

[00251] In some examples, the multi-specific polypeptide construct comprises an innate immune cell engager, wherein the innate immune cell includes but is not limited to a natural killer cell (NK cell), a macrophage, a dendritic cell, an eosinophil, a basophil, a neutrophil, a mast cell, a natural killer T cell (NKT cell), and the like. In some examples, when the multi-specific polypeptide construct is a multi-specific antigen binding polypeptide, the multi-specific polypeptide construct as described herein comprises multiple antigen targeting domains. In some examples, each of these targeting domains binds or recognizes an innate immune cell modulator or a target antigen. In some examples, each targeting domain of the multi-specific polypeptide construct comprises at least one CDR, or at least two CDRs, or at least three CDRs, or at least four CDRs, or at least five CDRs, or all six CDRs as described herein. In some examples, the multi-specific polypeptide construct comprises a combination of one or more CDRs as described herein.

[00252] In some examples, the antigen binding polypeptide construct as described herein comprises multiple antigen targeting domains. Each of these targeting domains binds or recognizes an NK modulator or a target antigen. Therefore, each targeting domain of the multispecific polypeptide construct comprises at least one CDR, or at least two CDRs, or at least three CDRs, or at least four CDRs, or at least five CDRs, or all six CDRs as described herein. In some examples, the multi-specific polypeptide construct comprises a combination of one or more CDRs as described herein. In some examples, the multi-specific polypeptide construct binds to one NK modulator, for example NKp80. In some examples, the multi-specific polypeptide construct is an antigen binding polypeptide comprising one to six CDRs binding NKp80 as described herein.

[00253] In another aspect, there is provided a method of producing the multi-specific polypeptide construct, or the antibody as disclosed herein comprising culturing the host cell, and optionally isolating the multi-specific polypeptide construct from said host cell and/or the culture media.

[00254] In another aspect, there is provided a method of screening and/or identifying the multi-specific polypeptide construct, or the antibody as disclosed herein wherein the NK celltargeting domain is antiNKp80. [00255] In some examples, the disclosure is a method of screening and/or identifying NKp80 binders, non-binders, non-activating binders, activating binders, and/or engagers. In some examples, the screening and/or identifying of NKp80 engagers includes using the bio-layer interferometry (BLI) and Xcelligence® cytotoxicity killing assay.

[00256] In some examples, the multi-specific polypeptide construct comprises a light chain variable region (VL) having one or more CDRs selected from the group consisting of SEQ ID NO:1-50 (FIG. 10), or fragments thereof, or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having 2 or 3 amino acids substitutions. In some examples, the multi-specific polypeptide construct comprises a heavy chain variable region (VH) having one or more CDRs selected from the group consisting of SEQ ID NO:51-104 (FIG. 10), or fragment or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto and/or having 2 or 3 amino acids substitutions. In some examples, the multi-specific polypeptide construct comprises a light chain variable region (VL) having one or more framework regions (FRs) selected from the group consisting of SEQ ID NO: 105-140 (FIG. 11), or fragment or a sequence sharing at least 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto and/or having 2 or 3 amino acids substitutions. In some examples, the multi-specific polypeptide construct comprises a heavy chain variable region (VH) having one or more framework regions (FRs) selected from the group consisting of SEQ ID NO:141-182 (FIG. 11), or fragment or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, homology thereto and/or having 2 or 3 amino acids substitutions.

[00257] In some examples, the disclosure is a method of screening and/or identifying the multispecific polypeptide construct, wherein the multi-specific polypeptide construct includes NKp80 engagers. In some examples, the screening and/or identifying of NKp80 engagers includes using the bio-layer interferometry (BLI) and Xcelligence® cytotoxicity killing assay. In some examples, the multi-specific polypeptide construct binds to one NK modulator, for example NKp80. In some examples, the multi-specific polypeptide construct is an antigen binding polypeptide comprising one to six CDRs binding NKp80 as described herein. In some examples, the multi-specific polypeptide construct comprises a light chain variable region (VL) having one or more CDRs selected from the group consisting of SEQ ID NO: 1-50 (FIG. 10), or fragments thereof, or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least bout 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having 2 or 3 amino acids substitutions, combined to a heavy chain variable region (VH) having one or more CDRs selected from the group consisting of SEQ ID NO:51-104 (FIG. 10), or fragments thereof, or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least bout 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having 2 or 3 amino acids substitutions.

[00258] In some examples, the multi-specific polypeptide construct comprises a sequence that is at least 80% identical to any one of the sequences disclosed herein. In some examples, the multi-specific polypeptide construct comprises a target binding site or CDR comprising a sequence that shares at least about at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to any of the sequences disclosed herein. In some examples, the sequences as disclosed herein have 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

[00259] In some examples, the multi-specific polypeptide construct comprises an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the multi-specific polypeptide construct comprises an amino acid sequence having 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 15, or 20 amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the one or more amino acid mutations are independently selected from substitutions, insertions, deletions, and truncations. In some examples, the amino acid mutations are amino acid substitutions, and include conservative and/or non-conservative substitutions. [00260] In some examples, the substitutions include non-classical amino acids. In some examples, non-classical amino acids are selected from selenocysteine, pyrrolysine, N- formylmethionine β-alanine, GABA and O-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general.

[00261] In some examples, modification of the amino acid sequences is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. In some examples, the mutations do not substantially reduce the antigen binding polypeptide construct's capability to specifically bind to a target. In some examples, the mutations do not substantially reduce the antigen binding polypeptide construct’s capability to specifically bind to a target and without functionally modulating (e.g., partially or fully neutralizing) the target.

[00262] In some examples, the binding affinity of the multi-specific polypeptide construct of the disclosure for the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or monomeric and/or dimeric forms and/or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and/or dimer forms) of said multispecific polypeptide construct is described by the equilibrium dissociation constant (K_d). In some examples, the multi-specific polypeptide construct binds to the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and/or dimeric forms) of said multispecific polypeptide construct with a Kd of less than about 1pM, about 900nM, about 800nM, about 700nM, about 600nM, about 500nM, about 400nM, about 300nM, about 200nM, about 100nM, about 90nM, about 80nM, about 70nM, about 60nM, about 50nM, about 40nM, about 30nM, about 20nM, about 10nM, or about 5nM, or about 1nM.

[00263] In a preferred embodiment, the co-engagement of NKp80 and CD16 in the tri-specific multi-specific polypeptide construct antiHER2-antiNKp80 (87-2)-Fc increased the potentiation of NK cell function, with no effect on T cell activation (FIG. 5). Therefore, the multi-specific polypeptide construct as disclosed herein is specific to NK cells. In some examples, the multispecific polypeptide construct as disclosed herein does not cause T cell activation. [00264] In some examples, the tri-specific multi-specific polypeptide constructs comprising an

NKp80 engager include clone antiHER2-antiNKp80(13)-FcX, antiHER2-antiNKp80(28)-FcX, antiHER2-antiNKp80(36)-FcX, antiHER2-antiNKp80(37)-FcX, antiHER2-antiNKp80(45)-FcX, antiHER2-antiNKp80(50)-FcX, antiHER2-antiNKp80(51)-FcX, antiHER2-antiNKp80(63)-FcX, antiHER2-antiNKp80(71)-FcX, antiHER2-antiNKp80(74)-FcX, antiHER2-antiNKp80(78)-FcX, antiHER2-antiNKp80(79)-FcX, antiHER2-antiNKp80(81)-FcX, antiHER2-antiNKp80(82)-FcX, antiHER2-antiNKp80(83)-FcX, antiHER2-antiNKp80(87)-FcX, antiHER2-antiNKp80(94)-FcX, antiHER2-antiNKp80(101)-FcX, antiHER2-antiNKp80(102)-f cX, and/or antiHER2- antiNKp80(106)-FcX.

[00265] As shown by the experimental data of the present disclosure, the tri-specific multispecific polypeptide constructs (antiHER2-antiNKp80(94)-Fc, antiHER2-antiNKp80(101)-Fc, antiHER2-antiNKp80(45)-Fc and/or antiHER2-antiNKp80(87)-Fc) co-engage both NKp80 and CD16 with improved potency as compared to Trastuzumab (antiHER2-antiNKp80-Fc vs antiHER2-Fc) in OVCAR3 (FIG. 3A). The clone antiHER2-antiNKp80(87)-Fc shows improved potency in the four different cell lines tested (MKN1, OVCAR3, HCT116, MDA-MB-231) (FIG. 4) (see Example 3).

/. Immunoglobulins

[00266] A key effector function for IgG antibodies is antibody-dependent cellular cytotoxicity (ADCC) in which antibody coated antigens activate effector cells, such as NK cells or monocytes, to destroy the antibody coated target by binding of the complex to the FcyR. The ADCC activity is significantly dependent on the glycan composition of the IgG and furthermore, the net result of binding to activating and inhibitory FcyR..

[00267] In some examples, Fc polypeptides include the polypeptides that make up an Fc domain, e.g., a monomeric Fc. In some examples, an Fc polypeptide is obtained from any suitable immunoglobulin, such as human lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. In some examples, an Fc polypeptide is obtained from human or any other non-human mammals. In some examples, the Fc domain comprises the carboxy-terminal portions of both H chains held together by disulfides. In some examples, the effector functions of antibodies are determined by sequences in the Fc domain; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[00268] In some examples, a native/ wild type Fc domain (FcX) confers diminished ADCC, which refers to a reduction in measurable ADCC response of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% of a control. In some examples, an Fc silent I inactivated mutant Fc domain (FcLALA) confers little to no measurable ADCC, which refers to substantially complete silencing of measurable ADCC response of at least about 90%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of a control, or substantially complete silencing of ADCC such that no measurable ADCC is detected. In some examples, an enhanced ADCC refers to the improvement or increase or multiplication of measurable ADCC response of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150% or more of a control.

[00269] In some examples, the multi-specific polypeptide construct binds to the second innate immune cell modulator through an Fc domain. In some examples, the multi-specific polypeptide construct second innate immune cell modulator binding domain is selected from a native / wild type Fc domain (FcWT), an Fc enhanced (FcE) domain, an Fc diminished (FcX) domain, an Fc silent domain I an inactivated mutant of Fc domain (FcLALA), an Fc mutated domain, a heterodimer Fc domain, and the like. As used herein, the terms “region” and “domain” are understood to describe the same component and may therefore be used interchangeably.

[00270] In some examples, the multi-specific polypeptide construct comprises an Fc domain. In some examples, the Fc domain comprises a sequence comprising:

(native / wild type Fc domain; SEQ ID NO:224).

[00271] In some examples, the multi-specific polypeptide construct comprises a variant Fc domain. In some examples, the variant Fc domain is a diminished Fc domain (FcX). In some examples, the diminished Fc domain is constructed in accordance with methods known in the art. In some examples, the diminished Fc domain comprises an amino acid sequence comprising: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

(diminished Fc domain (FcX); SEQ ID NO:224).

[00272] In some examples, the variant Fc domain may be a silent Fc domain / inactivated mutant. In some examples, the silent Fc domain / inactivated mutant of Fc domain comprises a sequence comprising:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK (silent Fc domain / inactivated mutant Fc domain (Fc LALA); SEQ ID NO:225)

[00273] In some examples, the variant Fc domain may be an enhanced Fc domain. In some examples, the enhanced Fc domain comprises a sequence comprising:

DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (enhanced Fc domain (FcE): SEQ ID NO:226).

[00274] In some examples of the present disclosure, the NK cell engagers contain native I wild type Fc domains and/or variant Fc domains. Variant Fc domains (or Fc mutated region) comprise an amino acid sequence which differs from that of a native / wild type sequence Fc domain by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). In some examples, the variant Fc domain has at least one amino acid substitution compared to a native I wild type sequence Fc domain or to the Fc domain of a parent polypeptide. For example, the variant Fc domain (or Fc mutated domain) may comprise from about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions in a native / wild type sequence Fc domain. The variant Fc domain herein shares at least about 80% , or at least about 85% homology, or at least about 90% homology, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% or at least about 99% homology with a native I wild type sequence Fc region.

[00275] In some examples, the “Fc domain” includes a hinge region, a CH2 domain or a CH3 domain of an Fc region. ii. CDR and FR

[00276] Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains 3 hypervariable loops known as “complement determining regions” (CDR1 , CDR2, and CDR3) and 4 somewhat invariant “framework regions” (FR1 , FR2, FR3, and FR4). When natural antibodies fold, the FR form the beta sheets providing the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc domain of naturally occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity (U.S Pat. Appl. No. 20220040231 , hereby incorporated by reference in its entirety).

[00277] As used herein, terms “variable light chain CDR1”, “variable light chain CDR2”, “variable light chain CDR3”, “variable heavy chain CDR1”, “variable heavy chain CDR2”, “variable heavy chain CDR3” refers to VLCDR1 , VLCDR2, VLCDR3, VHCDR1 , VHCDR2 and VHCDR3 respectively. In some examples, when the multi-specific polypeptide construct is a multi-specific antigen binding polypeptide, said multi-specific polypeptide construct as described herein comprises multiple antigen targeting domains. Each of these targeting domains bind or recognize an innate immune cell modulator or a target antigen. In some examples, each targeting domain of the multi-specific polypeptide construct comprises at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, and all six CDRs as described herein. In some examples, the multi-specific polypeptide construct comprises a combination of one or more CDRs as described herein.

[00278] In some examples, the multi-specific polypeptide construct binds to one modulator, for example NKp80. In some examples, the multi-specific polypeptide construct is an antigen binding polypeptide construct comprising one to six CDRs that bind to NKp80 as described herein. In some examples, the multi-specific polypeptide construct comprises a light chain variable region (VL) having one or more CDRs selected from the group consisting of sequences from FIG. 10 or fragments thereof, or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having 2 or 3 amino acids substitutions.

[00279] In some examples, the multi-specific polypeptide construct binds to one modulator, for example NKp80. In some examples, the multi-specific polypeptide construct is an antigen binding polypeptide construct comprising one to six CDRs that bind to NKp80 as described herein. In some examples, the multi-specific polypeptide construct comprises a heavy chain variable region (VH) having one or more CDRs selected from the group consisting of sequences from FIG. 10 or fragments thereof, or a sequence sharing at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having 2 or 3 amino acids substitutions.

[00280] In another aspect, there is provided an antigen-binding protein, or an antigenbinding fragment thereof, comprising the CDR sequences selected from:

(7) VLCDR1 (SEQ ID NO:5), VLCDR2 (SEQ ID NO:22), VLCDR3 (SEQ ID NO:37), VHCDR1 (SEQ ID NO:56), VHCDR2 (SEQ ID NO:72), and VHCDR3 (SEQ ID NO:91) (clone 51); (8) VLCDR1 (SEQ ID NO:6), VLCDR2 (SEQ ID NO:18), VLCDR3 (SEQ ID NO:38), VHCDR1 (SEQ ID NO:57), VHCDR2 (SEQ ID NO:73), and VHCDR3 (SEQ ID NO:92) (clone 71);

(10) VLCDR1 (SEQ ID NO:8), VLCDR2 (SEQ ID NO:24), VLCDR3 (SEQ ID NO:40), VHCDR1 (SEQ ID NO:58), VHCDR2 (SEQ ID NO:75), and VHCDR3 (SEQ ID NO:94) (clone 78);

(11) VLCDR1 (SEQ ID NO:9), VLCDR2 (SEQ ID NO:25), VLCDR3 (SEQ ID NO:41), VHCDR1 (SEQ ID NO:59), VHCDR2 (SEQ ID NO:76), and VHCDR3 (SEQ ID NO:95) (clone 79);

(16) VLCDR1 (SEQ ID NO:1), VLCDR2 (SEQ ID NO:29), VLCDR3 (SEQ ID NO:46), VHCDR1 (SEQ ID NO:64), HCDR2 (SEQ ID NO:81), and VHCDR3 (SEQ ID N0:100) (clone 101);

[00281] In some examples, there is provided an antigen-binding protein, or an antigenbinding fragment thereof, comprising the CDR sequences selected from:

(11) VLCDR1 (SEQ ID NO:9), VLCDR2 (SEQ ID NO:25), VLCDR3 (SEQ ID NO:41), VHCDR1 (SEQ ID NO:59), VHCDR2 (SEQ ID NO:76), and VHCDR3 (SEQ ID NO:95) (clone 79); (12) VLCDR1 (SEQ ID NO:10), VLCDR2 (SEQ ID NO:26), VLCDR3 (SEQ ID NO:42), VHCDR1 (SEQ ID NQ:60), VHCDR2 (SEQ ID NO:77), and VHCDR3 (SEQ ID NO:96) (clone 81);

(20) VLCDR1 (SEQ ID NO:11), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NQ:50), VHCDR1 (SEQ ID NO:61), VHCDR2 (SEQ ID NO:85), and VHCDR3 (SEQ ID NO:104) (clone 83); or

(21) VLCDR1 (SEQ ID NO:247), VLCDR2 (SEQ ID NO:248), VLCDR3 (SEQ ID NO:249), VHCDR1 (SEQ ID NO:250), VHCDR2 (SEQ ID NO:251), and VHCDR3 (SEQ ID NO:252) (humanized clone 87-2), wherein the CDR sequences share at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% homology to an amino acid sequence selected from SEQ ID NO: 1-104; 247-252, and/or wherein the CDR sequences selected from SEQ ID NO:1-104; 247-252 comprise 2, or 3 amino acid substitutions.

[00282] In another aspect, there is provided an antigen-binding protein, or an antigenbinding fragment thereof, comprising the CDR and FR sequences selected from: (1) VLFR1 (SEQ ID NO:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO:180) (clone 13);

[00283] In some examples, there is provided an antigen-binding protein, or an antigenbinding fragment thereof, comprising the CDR and FR sequences selected from:

(1) VLFR1 (SEQ ID NQ:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO:180) (clone 13);

(2) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NQ:180) (clone 28);

(3) VLFR1 (SEQ ID NQ:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 36); (4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO:180) (clone 37);

(5) VLFR1 (SEQ ID NQ:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NO:180) (clone 45);

(10) VLFR1 (SEQ ID NO:111), VLCDR1 (SEQ ID NO:8), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:24), VLFR3 (SEQ ID NO:129), VLCDR3 (SEQ ID NO:40), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:147), VHCDR1 (SEQ ID NO:58), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:75), VHFR3 (SEQ ID NO:172), VHCDR3 (SEQ ID NO:94), and VHFR4 (SEQ ID NO:181) (clone 78);

(11) VLFR1 (SEQ ID NO:112), VLCDR1 (SEQ ID NO:9), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:25), VLFR3 (SEQ ID NQ:130), VLCDR3 (SEQ ID NO:41), VLFR4 (SEQ ID NO:140), (VHFR1 (SEQ ID NO:148), VHCDR1 (SEQ ID NO:59), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:76), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:95), and VHFR4 (SEQ ID NO:181) (clone 79);

(12) VLFR1 (SEQ ID NO:113), VLCDR1 (SEQ ID NO:10), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:26), VLFR3 (SEQ ID NO:131), VLCDR3 (SEQ ID NO:42), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:149), VHCDR1 (SEQ ID NO:60), VHFR2 (SEQ ID NO:160), VHCDR2 (SEQ ID NO:77), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:96), and VHFR4 (SEQ ID NO:181) (clone 81);

(13) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:43), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:78), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:97), and VHFR4 (SEQ ID NO:181) (clone 82);

(16) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:29), VLFR3 (SEQ ID NO:134), VLCDR3 (SEQ ID NO:46), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO: 153), VHCDR1 (SEQ ID NO:64), VHFR2 (SEQ ID NO: 158), VHCDR2 (SEQ ID NO:81), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NQ:100), and VHFR4 (SEQ ID NO:182) (clone 101);

(21) VLFR1 (SEQ ID NO:253), VLCDR1 (SEQ ID NO:247), VLFR2 (SEQ ID NO:254), VLCDR2 (SEQ ID NO:248), VLFR3 (SEQ ID NO:255), VLCDR3 (SEQ ID NO:249, VLFR4 (SEQ ID NO:256), (VHFR1 (SEQ ID NO:257), VHCDR1 (SEQ ID NO:250), VHFR2 (SEQ ID NO:258), VHCDR2 (SEQ ID NO:251), VHFR3 (SEQ ID NO:259), VHCDR3 (SEQ ID NO:252), and VHFR4 (SEQ ID NO:260) (humanized clone 87-2), wherein the FR and CDR sequences share at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% homology to an amino acid sequence selected from SEQ ID NO: 1-104; 247-260, and/or wherein the FR and CDR sequences selected from SEQ ID NO:1-104; 247-260 comprise 2, or 3 amino acid substitutions.

Hi. Light chains and heavy chains

[00284] The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in multi-specific or multi-specific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C- terminal, both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Polypeptide constructs of Immunological Interest. Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. On the other hand, in multi-specific antigen binding polypeptide construct, each of the binding site of the multi-specific polypeptide construct is different. That is, in a bifunctional or bi-specific multispecific polypeptide construct, the multi-specific polypeptide construct has two different binding sites, and the like.

[00285] The binding fragments are selected from a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains), a F(ab)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), an Fd fragment (consisting of the VH and CH1 domains), an Fv fragment (consisting of the VL and VH domains of a single arm of an antibody), a single domain antibody (dAb) fragment (consisting of a VH domain), an isolated complementarity determining region (CDR), a single-chain Fv (scFv), a dsFv, a scAb, a STAb, a single domain antibody (sdAb or dAb), a single domain heavy chain antibody, and a single domain light chain antibody, a VHH, a VNAR, a single domain antibody based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies (e. g. f-star technology (F-star's Modular Antibody TechnologyTM)). Single domain antibodies in which one chain is separated from its natural partners are sometimes known as Dabs. The constant regions or parts of constant regions is comprised or not in single domain antibodies. In some examples, the antigen targeting domain is selected from a Fab fragment, a F(ab)2, fragments, an Fd fragment, an Fv fragment, a dAb, an isolated CDR, an scFv, a dsFv, an scAb, a STAb, an sdAb, a CH domain, a CL domain, a VHH, a VNAR, an sdAb from a VNAR, an ankyrin-based domain, a fynomer, an avimer, a fibronectin domain, and an F-star’s Modular Antibody TechnologyTM domain.

[00286] The polypeptide constructs as disclosed herein typically bind to their designated target with an association constant of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹° M. Such binding is specific binding in that it is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding results of bond formation between particular functional groups or particular spatial fit (e g., lock and key type), whereas nonspecific binding usually results from van der Waals forces. Specific binding does not necessarily imply that an antibody binds one and only one target. In some examples, the multi-specific polypeptide construct specifically binds to one or more antigens.

[00287] In some examples, the antigen binding polypeptide construct as described herein comprises a VL binding NKp80 with a sequence selected from the group consisting of SEQ ID NO: 183-202 (FIG. 13A).

AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKL ASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTE WVK (VL sequence of antiNKp80(13); SEQ ID NO:183);

DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTL ASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVWK (VL sequence of antiNKp80(28); SEQ ID NO:184);

DWMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTL ESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEV WK (VL sequence of antiNKp80(45); SEQ ID NO:187); AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTL ESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEV WK (VL sequence of antiNKp80(50); SEQ ID NO:188);

QIWTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLAS TLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTE WVK (VL sequence of antiNKp80(74); SEQ ID NO: 191);

ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDL ASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTK WVK (VL sequence of antiNKp80(79); SEQ ID NO: 193);

AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLA SGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEV WK (VL sequence of antiNKp80(81); SEQ ID NO:194);

AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKT SSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTE WVK (VL sequence of antiNKp80(94); SEQ ID NO: 197); AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTL ESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTE WVK (VL sequence of antiNKp80(101); SEQ ID NO: 198);

DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASD LASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTE WVK (VL sequence of antiNKp80(102); SEQ ID NO: 199);

DWMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTL ESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTE WVK (VL sequence of antiNKp80(106); SEQ ID N0:200);

DIVMTQTPASVSAAAGGTVTINCQASQSISNELSWYQQKSGQPPKLLIYGASNL ESGVPSRFKGSGSGTDFTLTISDLECADGATYYCQSNYYDSSSPDFAFGGGTE WVK (VL sequence of antiNKp80(63); SEQ ID NO:201); and

[00288] In some examples, the antigen binding polypeptide construct as described herein comprises a VH binding to NKp80 with a sequence selected from the group consisting of (SEQ ID NO:203-222) (FIG. 13B).

QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIY TGGGSADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGY GDATDIWGPGTLVTV (VH sequence of antiNKp80(13); SEQ ID NO:203);

QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYA GSSGSTHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDY FKIWGPGTLVTV (VH sequence of antiNKp80(28); SEQ ID NO:204);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTG SSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRY NIWGPGTLVTV (VH sequence of antiNKp80(36); SEQ ID NO:205);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTG SSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRY NIWGPGTLVTV (VH sequence of antiNKp80(37); SEQ ID NO:206); QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYT GSSGSTYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWR YNIWGPGTLVTV (VH sequence of antiNKp80(45); SEQ ID NO:207);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYT GSSGSTYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWR YNIWGPGTLVTV (VH sequence of antiNKp80(50); SEQ ID NO:208);

QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYS DSGNTYYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRY NIWGPGTLVTV (VH sequence of antiNKp80(51); SEQ ID NO:209);

QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSP GVAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWG

QGTLVTV (VH sequence of antiNKp80(71); SEQ ID NO:210);

QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSG GSAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWG QGTLVTV (VH sequence of antiNKp80(74); SEQ ID NO:211);

QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNG GATYYASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLW GQGTLVTV (VH sequence of antiNKp80(78); SEQ ID NO:212);

QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPS GSIWYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWG QGTLVTV (VH sequence of antiNKp80 (79); SEQ ID NO:213);

QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIAD KIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQG TLVTV (VH sequence of antiNKp80 (81); SEQ ID NO:214);

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTD GSAYFASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLW GQGTLVTV (VH sequence of antiNKp80(82); SEQ ID NO:215);

QSLEEYGGDVVQPEGSLTLTCTASGFSFSGNYWICWVRQAPGKGLEWIGCIYA GSSGSTCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWK YNIWGPGTLVTV (VH sequence of antiNKp80(87); SEQ ID NO:216); QSVEESGGRLVTPGTPLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTG GSAYYANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWG QGTLVTV (VH sequence of antiNKp80(94); SEQ ID NO:217);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISW GGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDP WGPGTLLTV (VH sequence of antiNKp80(101); (SEQ ID NO:218);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSG GDTSYATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLW GQGTLVTV (VH sequence of antiNKp80(102); SEQ ID NO:219);

QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDG SAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWG QGTLVTV (VH sequence of antiNKp80(106); SEQ ID NO:220);

QSLEESGGRLVKPDETLTITCTVSGIDLSSYIISWVRQAPGEGLEYIGFINTDGSA YYATWAKGRFTISRTSATVDLKMTSLTTEDTATYFCARDAGHRYLFYFKLWGQ GTLVTV (VH sequence of antiNKp80(63); SEQ ID NO:221); and

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTD GSAYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLW GQGTLVTV (VH sequence of antiNKp80(83); SEQ ID NO:222).

[00289] With respect to each of the sequences set forth hereinabove, in some examples, one or more of the sequences set forth herein share at least about 80%, or at least about 85%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to any of the sequences disclosed herein. In some examples, the sequences as disclosed herein have 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or more amino acid substitutions.

E. Diseases

[00290] In some examples, the multi-specific polypeptide construct binds to a target antigen that is an antigen associated to a disease. In some examples, the target antigen is a molecule associated with a disease. In some examples, the target antigen is an antigen associated with a disease. In some examples, the target antigen molecule is selected from an extracellular molecule, an intracellular molecule, and a transmembrane molecule. In some examples, the molecule is selected from a polypeptide, a polynucleotide, a carbohydrate, and the like. In some examples, the disease is a proliferative disease selected from a proliferative disease (such as a tumor/cancer, an inflammatory disease, and the like), an infectious disease, an autoimmune disease, an autoimmune disorder, and the like. In some examples, the disease may be a tumor/cancer.

[00291] In some examples, the disease is selected from a proliferative disease (such as cancer), an infectious disease, an autoimmune disease, an autoimmune disorder, and the like. In some examples, the disease is a tumor.

[00292] In some examples, the target includes tumor antigens selected from, but not limited to, HER2, and EGFR. In some examples, the multi-specific polypeptide construct binds one or more of these tumor antigens. In some examples, the multi-specific polypeptide construct binds to a target cell, wherein the target cell includes but is not limited to, tumor cells, cancer cells, and the like. In some examples, the tumor cells or cancer cells express HER2 and/or EGFR.

[00293] In some examples, the tumor cells, or cancer cells include, but are not limited to, bladder cancer cells, breast cancer cells, cervical cancer cells, cholangiocarcinoma cells (extrahepatic, or intrahepatic), colorectal cancer cells, esophageal or esophagogastric junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck carcinoma cells, hepatocellular carcinoma cells, intestinal (small) malignancy cells, lung cancer cells (non-small cells), lung adenocarcinoma cells, conventional glioblastoma cells, glioblastoma cells, melanoma cells, ovarian (epithelial) cancer cells, ovarian (non-epithelial) cancer cells, pancreatic adenocarcinoma cells, prostate cancer cells, unknown primary cancer cells, or uterine cancer cells.

[00294] In some examples, the multi-specific polypeptide construct binds to a tumor cell, wherein the tumor cell is a bladder cancer cells, breast cancer cells, cervical cancer cells, cholangiocarcinoma cells (extrahepatic, or intrahepatic), colorectal cancer cells, esophageal or esophagogastric junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck carcinoma cells, hepatocellular carcinoma cells, intestinal (small) malignancy cells, lung cancer cells (non-small cells), lung adenocarcinoma cells, conventional glioblastoma cells, glioblastoma cells, melanoma cells, ovarian (epithelial) cancer cells, ovarian (non-epithelial) cancer cells, pancreatic adenocarcinoma cells, prostate cancer cells, unknown primary cancer cells, or uterine cancer cells. In some examples, the multi-specific polypeptide construct binds to a cell such as, but is not limited to, an immortalized cell line, a primary cell, and the like. In some examples, the multi-specific polypeptide construct binds to a cancer cell line, such as an immortalized cell line. In some examples, the multi-specific polypeptide construct binds to a cancer cell line such as, but is not limited to, MKN1 , OVCAR3, HCT116, MDA-MB-231, N87, RAJI, and the like.

[00295] In some examples, the disease is an infectious disease. In some examples, the infectious disease is caused by a bacterial pathogen and/or a viral pathogen. In some examples, the multi-specific polypeptide construct binds one or more bacterial antigens and/or viral antigens.

[00296] In some examples, the disease is an autoimmune disease or an autoimmune disorder. In some examples, the autoimmune disease/disorder includes any disorder, condition, or disease in which the immune system mounts a reaction against self-cells or tissues, due to a breakdown in the ability to distinguish self from non-self or otherwise.

/. Diagnostics

[00297] In some examples, the disclosure comprises a method of detecting a disease in a subject in need thereof, the method comprising contacting the multi-specific polypeptide construct or composition as described herein to a sample obtained from the subject.

[00298] In some examples, the sample is a biological sample obtained from a biological subject, including a sample of biological tissue or fluid obtained in vivo or in vitro. In some examples, the biological sample is a solid biological sample or a liquid biological sample. In some examples, the solid biological sample includes a tissue specimen or a biopsy. In another exemplary embodiment, the fluid biological sample or liquid biological sample is selected from blood, serum, plasma, sputum, lavage fluid (for example peritoneal lavage), cerebrospinal fluid, urine, semen, sweat, tears, saliva, and the like. As used herein, the terms “blood,” “plasma,” and “serum” encompass fractions or processed portions thereof. Similarly, where a sample is taken from a biopsy, swab, smear, etc., the “sample” encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.

//. Pharmaceutical Compositions

[00299] In some examples, the disclosure comprises a composition comprising the multi-specific polypeptide construct as described herein. In some examples, the disclosure comprises a pharmaceutical composition comprising the multi-specific polypeptide construct as described herein and suitable pharmaceutical composition thereof. In some examples, the disclosure comprises a composition or pharmaceutical composition, wherein the composition is a prophylactic and/or therapeutic composition.

[00300] In some examples, the pharmaceutically acceptable agents for use in the present pharmaceutical compositions are selected from carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

[00301] In some examples, the composition described herein is used in therapy/medicine. In some examples, the composition described herein further comprises an excipient and/or stabilizers. In some examples, the composition described herein is used as a single agent and/or combined the multi-specific polypeptide construct with a disease targeting therapy such as, but not limited to, a NK cell therapy, a T-cell checkpoint inhibitor therapy, small molecules therapy that could stimulate NK cells to enhance anti-tumor responses, and the like.

[00302] In some examples, a method of preventing and/or treating a disease in a subject in need thereof, the method comprises administering to the subject the multi-specific polypeptide construct or composition of any one of the preceding examples. In some examples, the disclosure comprises the use of the multi-specific polypeptide construct as described herein in the manufacture of a medicament for preventing and/or treating a disease.

F. Antibody/engager production

[00303] Monoclonal antibodies (mAbs) are often selected from antigen-specific single B cells derived from different hosts, which are notably short-lived in ex vivo culture conditions and hence, arduous to interrogate. The development of several new techniques and protocols has facilitated the isolation and retrieval of antibody-coding sequences of antigen-specific B cells by also leveraging miniaturization of reaction volumes. Alternatively, mAbs can be generated independently of antigen-specific B cells, comprising display technologies and, more recently, artificial intelligence-driven algorithms. Consequently, a considerable variety of techniques are used, raising the demand for better consolidation.

[00304] In some examples, the NKp80 engager is generated by methods known in the art such as rabbit single B cell cloning, phage library, and the like. In some examples, the nucleic acid (such as DNA) encoding sequence for the NKp80 engager clones are isolated and the NKp80 engagers are identified with methods known in the art such as ELISA screening. 7. Vectors

[00305] Vectors are typically selected to be functional in the host cell in which the vector will be used (the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur. The vector as described herein is an expression vector and/or a cloning vector.

[00306] In some examples, the vector is selected from the group consisting of a plasmid, a viral particle, a phage, a baculovirus, a yeast plasmid, a lipid based vehicle, a polymer microsphere, a liposome, and a cell based vehicle, a colloidal gold particle, lipopolysaccharide, polypeptide, polysaccharide, a viral vehicle, an adenovirus, a retrovirus, a lentivirus, an adeno-associated viruses, a herpesvirus, a vaccinia virus, a foamy virus, a cytomegalovirus, a Semliki forest virus, a poxvirus, a pseudorabies virus, an RNA virus vector, a DNA virus vector and a vector derived from a combination of a plasmid and a phage DNA, further optionally wherein said polynucleotide is operatively linked to an expression control sequence(s) to direct peptide synthesis, even further optionally wherein the vector comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

//. Host cells

[00307] In some examples, the disclosure is a host cell comprising the vector comprising a nucleic acid sequence encoding for the multi-specific polypeptide construct of any one of the preceding examples. In some examples, the host cell comprises cloning or expression vectors as described above and/or nucleic acid sequences encoding for the multi-specific polypeptide construct, antibodies and binding fragments thereof as described above. In some examples, the host cell of the preceding examples comprises a cloning or expression vectors configured to express the multi-specific polypeptide construct as disclosed herein.

[00308] In another aspect, there is provided a nucleic acid encoding the multi-specific polypeptide construct, or the antibody as disclosed herein.

[00309] In some examples, the host cell is any type of cell capable of being transformed or transfected with the nucleic acid or vector to produce a multi-specific polypeptide construct or binding fragment/polypeptide construct thereof encoded thereby. In some embodiment, the host cell comprising the nucleic acid or vector is used to produce the multi-specific polypeptide construct or binding fragment/polypeptide construct thereof, or a portion thereof (e.g., a heavy chain sequence, or a light chain sequence encoded by the nucleic acid or vector). In some examples, after introducing the nucleic acid or vector into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence. In some examples, the antibody, multi-specific polypeptide construct, or fragment, or portion of the antibody then is isolated from the cell.

[00310] In some examples, the host cells are prokaryotic host cells (such as E. coll) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell). In some examples, the host cell, when cultured under appropriate conditions, expresses an antibody or binding fragment thereof which is subsequently collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). In some examples, selection of an appropriate host cell depends upon desired expression levels, polypeptide modifications that are desirable or necessary for activity, such as glycosylation or phosphorylation, and ease of folding into a biologically active molecule, or other factors routinely considered in the art. In some examples, selection of the host cell depends in part on whether the antibody or binding fragment thereof is to be post-transcriptionally modified (e.g., glycosylated and/or phosphorylated). In another embodiment, the host cell comprises a bacterial cell, a yeast cell, an animal cell e g., a mammalian cell and/or a plant cell.

[00311] In some examples, suitable mammalian host cells include CHO, myeloma or hybridoma cells. Many host cell lines are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types of use in expressing antibodies include lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells.

Hi. Clone selection

[00312] The development and engineering of antibodies for different purposes, such as diagnostics or therapeutics, requires comprehensive characterization to determine affinity, specificity, and mechanism of action. Biolayer interferometry (BLI) is widely used for analyzing interactions between two biomolecules. Thus, it can aid in antibody characterization in a relatively easy and fast manner. In BLI, the binding between a ligand immobilized on the biosensor tip and an analyte in solution produces an increase in optical thickness at the biosensor tip, resulting in a wavelength shift proportional to the extent of binding. The sensor tips collect readings in real time, while immersed in the analyte solution (“dip-and-read”), without the need for continuous flow fluidics. The system therefore allows the measurements of different antibody-antigen interactions, using various sensors, suitable for label-free molecules or widely used tags. [00313] The xCELLigence platform utilizes gold microelectrodes embedded in the bottom of microtiter wells to monitor the status of adherent cells, or suspension cells which have been tethered to the plate bottom. The basic assay principle is based on impedance measurements through the surface of gold electrodes where attached cells act as insulators, impeding the flow of an alternating microampere electric current between electrodes. This impedance signal is measured automatically, at a frequency defined by the user (every 10 seconds, once per hour, etc.), and provides an extremely sensitive readout of cell number, cell size, and cell-substrate attachment strength. In contrast to surface-attached cancer cell targets, immune effector cells are non-adherent and therefore do not directly affect the impedance signal; their cytotoxic activity can however be detected through the reduction of the target cancer cells number. Because of this property, the cytolytic activity of NK cells, T cells, CARTs, oncolytic virus, checkpoint inhibitors, bi-specific antibodies, BiTEs, etc. can be selectively monitored in real-time.

[00314] In some examples, the screening and/or identifying of NKp80 engagers includes using the bio-layer interferometry (BLI) and Xcelligence® cytotoxicity killing assay. In some examples, the selected engagers show the highest binding affinity as determined by BLI. In some examples, the selected NK engagers show the highest cytotoxic profile as determined by xCELLigence. iv. Humanization

[00315] Antibodies and antigen targeting domains have emerged as effective tools in the treatment and diagnosis of different human diseases. Non-human antibodies and antigen targeting domains have been demonstrated to induce human immune responses, which result in neutralization of administered antibody and limits the application of such antibodies in treatment of human diseases. To overcome this problem the technology of antibody humanization has been developed. Antibody humanization is an efficient approach to eliminate or reduce the immunogenicity of these antibodies and antigen targeting domains. So far, various methods have been innovated by researchers for humanization of non-human antibodies and antigen targeting domains and to improve their affinity, specificity, and other properties. Each of these methods has its advantages and disadvantages.

[00316] A common method for humanization of non-human antibodies and antigen targeting domains is complementary determining regions (CDR) grafting in which the CDRs of non-human antibodies or antigen targeting domains are grafted onto the human framework regions. Usually, human framework regions with highest homology to the framework regions of non-human antibody or antigen targeting domain are chosen as an acceptor for CDR grafting. The straightforward grafting of CDR loops from murine antibodies or antigen targeting domains onto human frameworks do not affect the antibody or antigen targeting domain affinity in some cases, while in many more cases it reduces the affinity significantly. Some murine residues in framework regions, referred to as vernier zone residues, have been demonstrated to affect the conformation of CDR loops and affinity of antibody or antigen targeting domains. These residues are localized in the p-sheet framework regions closely underlying the CDRs. Therefore, after the selection of desired human framework regions these residues are retained in humanized antibodies and antigen targeting domains.

[00317] Human germline genes could be used as an alternative source of framework regions for humanization of murine antibodies or antigen targeting domains. Compared with framework regions derived from IgG, the germline genes have less intraclonal somatic hypermutation. Therefore, it is expected that humanized antibodies or antigen targeting domains with germline framework regions show lower immunogenicity than humanized antibodies or antigen targeting domains with IgG framework regions. So, these features encouraged research on application of these sequences in antibody and antigen targeting domain humanization.

[00318] Several approaches have been used by researchers to increase the affinity of humanized antibodies or antigen targeting domains, whereby given residues in framework or CDR regions of engineered antibodies or antigen targeting domains are altered. Among all CDRs, alteration of the heavy-chain CDR3 (VHCDR3) has been used most frequently to increase the affinity or specificity of antibodies or antigen targeting domains. This CDR is the most variable CDR, and its variation arises from somatic mutations and recombination of the variable (V), diversity (D) and joining (J) fragment-encoding sequences.

[00319] Antibody resurfacing method is another strategy for humanization of non-human antibodies or antigen targeting domains. This method involves the replacement of potentially antigenic surface framework residues with the most common human residues at those positions. The basis of this method is that human anti-mouse antibody (HAMA) response to the variable region is caused only by surface residues. Antibodies and antigen targeting domains humanized by this method usually exhibit little change in stability and affinity.

[00320] Humanization based on CDR homology is based on the idea that framework regions of murine and human antibodies or antigen targeting domains with similar CDRs can support CDR structure of each other with high affinity retention. In this method, homology of framework regions is not considered to choose human framework regions, and the critical murine residues (vernier zone residues) are not restored in humanized antibody or antigen targeting domain. Using this method, the formation of motifs that may be recognized as foreign is reduced. The antibodies or antigen targeting domains produced by this method have been found to retain good degrees of affinity, relatively more than those produced by the framework-homology-based humanization method.

[00321] Fully human antibodies or antigen targeting domains from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies or antigen targeting domains of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. In some examples, the selected multi-specific polypeptide construct clones comprise humanized variable regions, humanized CDRs, and/or humanized framework regions.

[00322] In some examples, the NKp80 targeting domains is humanized. In some examples, the humanized NKp80 targeting domain comprises the VL domain sequence (FIG. 13C):

DIQMTQSPSSVSASVGDRVTITCQASQSIGRDLAWYQQKPGKAPKLLIYGASIL ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGADRSSTPSWPFGQGTKV EIK (VL sequence of humanized antiNKp80 domain; SEQ ID NO:235)

[00323] In some examples, the humanized NKp80 targeting domain comprises the humanized VH sequence:

QVQLVESGGGVVQPGGSLRLSCAASGFSFSGNYWICWVRQAPGKGLEWIGCI YAGSSGSTCYATWAKGRFTISKDLSKNTVYLQMNSLRAEDTAVYYCARDTGSG YWKYNIWGRGTLVTVSS (VH sequence of humanized antiNKp80 domain; SEQ ID NO:236)

[00324] In some examples, the humanized NKp80 targeting domain comprises the CL domain sequence:

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (CL sequence of antiNKp80 domain; SEQ ID NO:237)

[00325] In some examples, the humanized NKp80 targeting domain comprises the CH domain sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (CH sequence of antiNKp80 domain; SEQ ID NO:238) [00326] In some examples, the humanized NKp80 targeting comprises 1, or 2, or 3, or 4, or 5, or 6 CDR selected from SEQ ID NO:247-252. In some examples, the humanized NKp80 targeting domain comprises 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8 FR selected from SEQ ID NO:253-260.

G. Gene editing methods

[00327] In some examples, modification of the amino acid sequences is achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis.

/. Site-directed mutagenesis

[00328] Mutagenesis is usually employed to understand the regulatory regions of genes and the relationship between the polypeptide construct structure and its function. Depending on the number of sites to be mutated, site-directed mutagenesis can be divided into two types: simple or multiple mutations. For single mutations, methods are based on the amplification of doublestranded DNA from plasmids using complementary oligonucleotides carrying the mutation of interest. Due to its simplicity, the low number of hours spent, and high efficiency, this is one of the most common strategies to introduce mutations in DNA fragments. For multiple mutations, methods incorporate the desired mutations simultaneously in the same reaction or they are obtained after several rounds of mutations. In some examples, the nucleic acid sequence encoding the multi-specific polypeptide construct, or any domain or fragment thereof, is modified using site-directed mutagenesis.

//. PCR-based mutagenesis

[00329] PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type Ils restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type Ils restriction enzyme. After digestion of two fragments with a type Ils enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. Major strategies of PCR-based mutagenesis include base substitution, deletion, insertion, chimeric gene generation, multiple-site mutagenesis, and random mutagenesis at either a single site or multiple sites. Numerous PCR-based methods have been developed commercially or noncommercially. Among those methods, the overlap extension method, megaprimer method, Quick Change Method (Stratagene, La Jolla, CA), and their modified versions are currently prevalent. In some examples, the nucleic acid sequence of the multi-specific polypeptide construct, or any domain or fragment thereof, is modified using PCR-based mutagenesis.

IV. EXAMPLES

Example 1: Screening and identification novel NKp80 engagers

[00330] New Zealand White (NZW) Rabbits were used for immunization with biolistic DNA delivery containing the gene of interest of NKp80. Titers were monitored during the immunization period. Peripheral whole blood was collected for B-cell isolation. Briefly, B cells with positive antirabbit IgG staining were sorted using flow cytometry for culture. B cell cultivation supernatants were used to identify positive NKp80 binders with ELISA screening, followed by mRNA isolation and cDNA synthesis. The cognate VH and VL gene segments were PCR amplified and verified by sequencing. The DNA encoding sequences of VH and VL were cloned into their respective expression vector for recombinant multi-specific polypeptide construct expression. The multispecific polypeptide construct containing a HER2 targeting domain, a diminished ADCC function of Fc constant region, and a specific NKp80 engager, is hereafter referred as antiHER2- antiNKp80-FcX (FcX: diminished ADCC).

[00331] Multi-specific polypeptide construct clones were recombinantly expressed in a mammalian system via transient transfection. Multi-specific polypeptide construct candidates were purified using polypeptide construct A columns with > 95% purity assessed by SDS-PAGE under non-reducing conditions. Different formats of multi-specific polypeptide construct candidates exemplified in this study, including the tri-specific multi-specific polypeptide construct that contain a HER2 targeting domain, an Fc constant region with ADCC function, and a humanized NKp80 targeting domain, hereafter referred as antiHER2-antiNKp80-Fc, were also recombinantly expressed and purified using the same approach.

[00332] The DNA encoding sequence for NKp80 binder clones were then isolated and cloned into rabbit IgG format for validation using ELISA screening. A total of 108 DNA encoding sequences for NKp80 binders were inserted into mammalian expression vectors to be expressed in tri-specific format (antiHER2-antiNKp80-FcX) in a mammalian system (FIG. 1 , Point 1). The Fc constant region of the bi-specific polypeptide construct, denoted as FcX here, had diminished ADCC function. The inclusion of an Fc domain with diminished function was important in the validation process: it demonstrated that any detected cytotoxicity was due to NKp80 engagement. Example 2: NKp80 engager clones: binding and cytotoxicity killing screening assay

[00333] Potential NKp80 binding clones were determined using a ForteBio Octet Bio-Layer Interferometry system (BLI). The streptavidin biosensors were loaded with different biotinylated antigens including both human and cynomolgus NKp80 to determine the binding kinetic of these candidates.

[00334] Amongst the 108 clones that had diminished ADCC function (tri-specific, antiHER2- antiNKp80-FcX), 78 and 67 clones were verified binders for human and cynomolgus NKp80 respectively using Bio-layer interferometry (BLI) (FIG. 1, Point 2a).

[00335] The ability of NKp80 binders to redirect NK cell cytotoxicity against HER2-positive target cell line N87 was determined by an Xcelligence cytotoxicity killing assay, that tracked cell death in real time. A single dose concentration of the clones of interest was incubated with primary PBMCs (from healthy donors) at the stated effector to target cell ratios. The effectors were primary PBMCs isolated from healthy donors.

[00336] A total of 92 clones were assayed for cytotoxicity in this manner (70 NKp80 binders, 22 non binders). Non-binders were included to determine baseline activity together with anti-HER2 antibody (antiHER2-FcX).

[00337] Activating binders were defined as clones that showed consistent killing activity higher than the median value in 2 independent assays using 2 different PBMC donors. A total of 20 NKp80 clones were identified as activating binders (out of 70 binders) (FIG. 2 shows the killing activity of all 20 activating binders). Importantly, the 22 non-binders did not induce significantly higher cytotoxicity than the baseline (data not shown). Taken together, the data reveals a unique subset of NKp80 binders that engage and potentiate NK cell cytotoxicity.

Example 3: Enhanced cytotoxicity mediated by NKp80 and CD16 coengagement

[00338] Xcelligence real-time assays were performed to assess cytotoxic potential of the various multi-specific polypeptide construct candidates. Briefly, on day 0, target cells were seeded on an Xcelligence microtitre plate. On day 1 , PBMCs from healthy donors were added at pre-determined effectoctarget (ET) ratios, together with the NKp80-binding candidates or engagers under study. Cytolysis of the target cells were tracked over time, based on electric impedance detected by the Xcelligence machine. The output, the cell index, was a measure of the electrical impedance detected and was proportional to the number of adherent target cells. [00339] For hit screening, a single concentration of potential multi-specific NKp80-binding polypeptide construct candidates in tri-specific format (antiHER2-antiNKp80-FcX) was used for identifying activating binders of NKp80 in the presence of N87 cancer cells.

[00340] In some examples, the multi-specific polypeptide construct’s innate immune cell cytotoxicity killing is examined with cell cytotoxicity assays such as but is not limited to Xcelligence® cytotoxicity killing assay, and the like.

[00341] Without wishing to be bound by theory, the real time Xcelligence® cell cytotoxicity killing assay examines the capability of a treatment in redirecting an innate immune cell cytotoxicity against a target antigen positive target cell. For example, it can assess the capability of an NKp80 engager containing an antiHER2 arm in redirecting NK cell cytotoxicity against a HER2 positive target cell like N87.

[00342] As shown in the experimental data of the present disclosure, the NKp80 activating binders showed cytotoxicity greater than the median as compared to the rest of the population in a cell cytotoxicity assay. Amongst the 108 clones containing an Fc domain with diminished ADCC function (tri-specific, antiHER2-antiNKp80-FcX), 78 and 67 clones were verified binders for human and cynomolgus NKp80 respectively using Bio-layer interferometry (BLI) (FIG. 1, point 2a). A total of 92 tri-specific clones (70 NKp80 binders, 22 non binders) were assayed for cytotoxicity and a total of 20 NKp80 activating binder clones were identified.

Out of the 20 activating binders, 13 were humanized and constructed into tri-specific engagers with a fully functional Fc domain (tri-specific antiHER2-antiNKp80-Fc). All clones were again verified as human NKp80 binders using BLI in single point measurements, showing dissociation constants (KD) ranging from low nM to sub pM.

[00343] For validation experiments assessing the synergistic effects of NKp80 and CD16 coengagement, antiNKp80 clones identified as activating binders were humanized and formatted as tri-specific engagers (antiHER2-antiNKp80-Fc), containing fully functional ADCC Fc regions. These clonal engagers were tested for binding again, and 4 of the clones were selected for further studies. These 4 clones were assayed for cytotoxicity in a dose-dependent manner, using OVCAR3 cells as target cells. All clones demonstrated improved cytotoxicity relative to the Trastuzumab control (FIG. 3A). Without the Fc function, these 4 clones were able to induce cytotoxicity, but at a level lower than that induced by Trastuzumab (FIG. 3B), suggesting that the co-engagement of NKp80 and CD16 is necessary for boosting synergistic cytotoxicity of NK cells. Dose response curves and EC50 values were plotted using normalized cell indices (to the point of PBMC and engager addition) in Prism. From these 4 clones, one was selected for further assessment in 4 cell lines of different cancer origins, using 3-4 independent PBMC donors for each cell line. Dose response curves and EC50 values were similarly calculated as described above.

Example 4: AntiNKp80 clone 87-2 potency in multiple target cell lines and PBMC donors

[00344] Of the 4 engagers containing antiNKp80 clones that showed improved potencies with the combined targeting of NKp80 and CD16, the engager containing clone 87-2 was selected for further exemplification (tri-specific antiHER2-antiNKp80(87-2)-Fc). This engager was tested for cytotoxicity using the Xcelligence assay in 4 different cell lines of different cancer origins, using 3-4 independent PBMCs lots from healthy donors (TABLE 1 , Col. 3). These experiments used Trastuzumab as a benchmark. Selected cell lines with their tumour antigen copy numbers/cell, are shown. Selected cell lines with their target copy/cell, are shown (TABLE 1, Col 1, 2).

[00345] EC50 values were used as a measure of potency for these experiments. The foldchange of EC50 values (compared to Trastuzumab) were then calculated for each experiment and averaged across the independent experiments to obtain an overall EC50 fold-change value. The engager containing antiNKp80 clone 87-2 consistently showed improved potency (mean EC50) compared to Trastuzumab across the different cell lines (TABLE 1 , Col. 4; FIG. 3). Interestingly, it also showed increasing fold change potencies as the target antigen copy number expression decreases decreased (TABLE 1 , Col. 4). This was possibly due to the weakening potency of Trastuzumab with decreasing target antigen copy numbers (MKN1 , mean EC50 0.71 nM vs MDA-MB-231 , mean EC50 6.85 nM) (TABLE 1 , Col. 5).

[00346] The increased cytotoxic potencies may be underpinned by increased NK cell activation upon the co-engagement of NKp80 and CD16. (FIG. 5). The engager containing antiNKp80 clone 87-2 increased NK activation marker expression and cytokine secretion (vs Trastuzumab), as measured by flow cytometry. Importantly, this engager did not induce T cell activation.

[00347] Next, antiNKp80 clone 87-2 was cloned into a tri-specific format where antiHER2 was replaced with antiEGFR. This version of the tri-specific engager targeted EGFR instead of HER2 as seen in all prior experiments. Xcelligence cytotoxicity assays confirmed that this engager was able to induce cytotoxicity in EGFR+ cell lines, with greater potencies compared to Cetuximab (antiEGFR-Fc) (FIG. 6). As shown in the experimental data, antiEGFR-antiNKp80(87-2)-Fc was more potent than Cetuximab (antiEGFR-Fc) in the cytotoxicity killing assay. The control for the cytotoxicity killing assay (isotype-anti NKp80(87-2)-Fc) showed no NK cytotoxicity killing against HER2-positive cell lines, suggesting that the cytotoxicity was antigen dependent.

TABLE 1: Mean EC50 fold-change of antiHER2-antiNKp80(87-2)-Fc relative to Trastuzumab.

Example 5: Off target activation of NK cells on antigen target-negative cell line

[00348] To assess the possibility of on-target-off-tumor effects, the tri-specific engager (antiHER2-antiNKp80-Fc) containing clone 87-2 was tested against a normal, healthy fibroblast cell line, MRC-5. No killing of these cells was observed, suggesting no “on-target-off-tumor” effects (FIG. 7). Moreover, without the HER2-targeting antibody arm (isotype-antiNKp80-Fc, where HER2 was replaced by isotype control), no killing of cells was observed compared to the positive control, Trastuzumab, demonstrating that NK cytotoxicity was target antigen-dependent (FIG. 7).

Example 6: Binding site and sequence similarities of identified NKp80 binder clones

[00349] To analyze the epitope binding patterns on NKp80, a BLI epitope binning experiment was performed using the 4 antiNKp80 clones selected for cytotoxicity studies in FIG. 3A: humanized clones 45-2, 87-2, 94-1 and 101-1 (TABLE 2). In these experiments, the target antigen was first immobilized onto the biosensor, and (potentially) competing antibodies added in consecutive steps. If the second antibody generated a signal even after the addition of the first antibody, it meant that the second antibody bound to an epitope distinct from the first. The binding indices of the epitope binning assay were visualized in FIG. 8: clones 45-2 and 87-2 bound to similar epitopes, while clones 94-1 and 101-1 bound distinct epitopes. TABLE 2: Normalized binding indices generated for each clone via tandem binning experiment.

[00350] To assess the overall diversity of the 20 activating binders, including the 4 clones analyzed in FIG. 8 (FIG. 1 , Point 2b), sequence identity matrices were generated using the heavy chain CDR3 region sequences of the 20 NKp80 binders (FIG. 14). A subsequent clustering analysis of the output revealed 5 distinct clusters (FIG. 9), confirming the diversity of the 20 multispecific polypeptide construct. In agreement with the epitope binning data in FIG. 8, clones 45 and 87 fell into the same cluster (C2), while clone 94 fell into cluster 3 (C3) and clone 101 into cluster 5 (C5), suggesting that the similar underlying sequence of clones 45 and 87 accounted for their binding to a similar epitope of NKp80.

Example 7: Domain permutations within the multi-specific polypeptide construct

[00351] FIG. 13D shows different possible permutations of the multi-specific polypeptide construct domains. The selected exemplary constructs were the following:

1) Cetuximab(Fd)/-FcWT/anti-NKp80(87-2) (scFv) (SEQ ID NO:239)

2) Anti-NKp80(87-2)(Fd)/-FcWT/-Cetuximab (scFv) (SEQ ID NO:240)

3) Anti-NKp80(87-2)(Fd)/-Cetuximab(scFv)/-FcWT (SEQ ID NO:241)

4) Cetuximab(Fd)/-anti-NKp80(87-2) (scFv)/-FcWT (SEQ ID NO:242)

As illustrated in FIG. 13E, various permutations of the tri-specific engager containing antiEGFR, wild type Fc and antiNKp80 (87-2) consistently resulted in superior cytotoxicity against breast cancer MDA-MB-231 cells compared to Cetuximab, suggesting that the superior activity conferred by the additional antiNKp80-targeting domain was not limited to a specific format. Dose response curves and EC50 values were plotted using normalized cell indices (to the point of PBMC and engager addition) in Prism. Example 8: Further exemplification of antigen-targeting domain flexibility using CD20

[00352] To further exemplify the flexibility of NKp80-targeting, CD20 are used as another target antigen. Anti-CD20 (the Fab portion of Rituximab) is cloned into a tri-specific format by directly replacing the Trastuzumab Fab in the respective pcDNA-based VL and VH antiHER2-antiNKp80- Fc expression plasmids, keeping everything else identical.

[00353] These plasmids are recombinantly expressed in an EXPI-CHO cell mammalian system. Expression vectors containing different fragments of each multi-specific polypeptide construct (one containing the VH, another containing the VL) are co-transfected into EXPI-CHO cells according to the manufacturer’s manual and purified using protein A column chromatography, eluted with 0.2 M Tris-glycine pH 2.7 and neutralized with 1 M Tris pH 8.0. Protein purity is assessed by SDS-PAGE under non-reducing conditions (ideally, >95% purity). The constructs are buffer exchanged with 1x PBS in ultracentrifugation tubes and have their concentrations measured by the Nanodrop.

[00354] To assess cytotoxic potential, the tri-specific engager antiCD20-antiNKp80-Fc is tested in a cytotoxicity assay such as the Xcelligence® assay or a Calcein AM-based staining assay, alongside a Rituximab control. Processing is identical to those experiments performed with antiHER2-antiNKp80-Fc.

Claims

WHAT IS CLAIMED IS:

1. A multi-specific polypeptide construct comprising:

2. The multi-specific polypeptide construct of claim 1 , wherein one of the NK cell-targeting domains is an NKp80-targeting domain.

3. The multi-specific polypeptide construct of claims 1-2, wherein the NKp80-targeting domain comprises:

(1) a heavy chain variable domain (VH) comprising 1 , 2, or 3 complementarity determining region (CDR) selected from a VHCDR1 of SEQ ID NO:51-67; 250, a VHCDR2 of SEQ ID NO:68- 85; 251, and/or a VHCDR3 of SEQ ID NO:86-104; 252; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(2) a light chain variable domain (VL) comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO: 1-16; 247, a VLCDR2 of SEQ ID NO:17-31; 248, and/or VLCDR3 of SEQ ID NO:32- 50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

4. The multi-specific polypeptide construct of claims 1-3, wherein the NKp80-targeting domain comprises:

(1) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO:141- 155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(2) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253, a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO: 138-140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

5. The multi-specific polypeptide construct of claims 1-4, wherein the NKp80-targeting domain comprises:

(2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO:17-31 ; 248, and VLCDR3 of SEQ ID NQ:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO: 141- 155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(4) 1 , 2, 3, or 4 VL FR selected from a VLFR1 of SEQ ID NO:105-118; 253 a VLFR2 of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO: 138-140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

6. The multi-specific polypeptide construct of claims 1-5, further comprising a functional Fc domain.

7. The multi-specific polypeptide construct of claim 6, wherein the Fc domain is

(i) a native I wild type Fc domain (FcWT) or a diminished Fc (FcX) domain of SEQ ID NO:224; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

8. The multi-specific polypeptide construct of claims 6-7, comprising:

(a) a first domain targeting NKp80;

(b) a second domain targeting CD16; (c) one or more antigen targeting domains binding to one or more tumor-associated antigen.

9. The multi-specific polypeptide construct of claims 1-8, wherein the one or more antigen targeting domains bind to a member selected from HER-2, EGFR, and CD20.

10. The multi-specific polypeptide construct of claims 1-9, wherein the one or more antigen targeting domains comprise:

(3) a VH of amino acid sequence SEQ ID NO:244 (VH Rituximab), a VL of amino acid sequence SEQ ID NO:243 (VL Rituximab), a CH of amino acid sequence SEQ ID NO:246 (CH Rituximab), and/or a CL of amino acid sequence SEQ ID NO:245 (CL Rituximab); or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

11. The multi-specific polypeptide construct of claims 1-10, comprising:

(A) an NKp80-targeting domain comprising:

(2) a VL comprising 1 , 2, or 3 CDR selected from a VLCDR1 of SEQ ID NO:1-16; 247, a VLCDR2 of SEQ ID NO:17-31 ; 248, and VLCDR3 of SEQ ID NO:32-50; 249; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof;

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO: 141- 155; 257, a VHFR2 of SEQ ID NO:156-162; 258, a VHFR3 of SEQ ID NO:163-179; 259, and/or a VHFR4 of SEQ ID NO: 180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or (4) 1 , 2, 3, or 4 VL FR selected from a FR1 of SEQ ID NO:105-118; 253, a of SEQ ID NO:119-121 ; 254, a VLFR3 of SEQ ID NO:122-137; 255, and/or a VLFR4 of SEQ ID NO:138- 140; 256; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and

(B) one or more antigen targeting domains comprising:

12. The multi-specific polypeptide construct of claims 6-11 , comprising:

(A) an NKp80 targeting domain comprising:

(3) 1 , 2, 3, or 4 VH framework region (FR) selected from a VHFR1 of SEQ ID NO: 141- 155; 257, a VHFR2 of SEQ ID NO: 156-162; 258, a VHFR3 of SEQ ID NO: 163-179; 259 and/or a VHFR4 of SEQ ID NO:180-182; 260; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof; and/or

(B) one or more antigen targeting domains comprising:

13. The multi-specific polypeptide construct of claims 7-12, wherein the polypeptide construct is a tri-specific antigen binding construct comprising:

(a) first targeting domain binding NKp80;

(b) a second targeting domain binding CD16; and

(c) a third targeting domain binding to a target antigen, wherein the targeting domains are selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, tynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies.

14. The multi-specific polypeptide construct of claims 7-13, wherein the polypeptide construct is a tri-specific antigen binding construct comprising:

(b) a first targeting domain binding to CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO:224), FcX (SEQ ID NO:224), silent Fc / inactivated mutant Fc domain (FcLALA) (SEQ ID NO:225), or FcE (SEQ ID NO:226); and

(c) a third targeting domain binding to a tumor-associated antigen, optionally HER2, EGFR or CD20, wherein the targeting domain is selected from a Fab fragment, a F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single chain Ab (scAb), secreted T-cell bi-specific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of new antigen receptor (VNAR), a sdAb based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, tynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies.

15. The multi-specific polypeptide construct of claims 7-14, wherein the NKp80-targeting domain comprises:

(2) a VL comprising an amino acid sequence selected from SEQ ID NO:183-202; 235; wherein the VH and the VL are paired to produce clone 13, clone 28, clone 36, clone 37, clone 45, clone 50, clone 51 , clone 63, clone 71 , clone 74, clone 78, clone 79, clone 81 , clone 82, clone 83, clone 87, clone 94, clone 101 , clone 102, clone 106, or humanized clone 87-2; or at least about 80% sequence identity to amino acid sequence thereof; or 2, or 3 amino acid substitutions thereof.

16. The multi-specific polypeptide construct of claims 7-15, comprising: (i) an antigen targeting domain consisting of a Fd fragment or a Fab fragment ; a first NK cell targeting domain consisting of a Fc domain; a first [(G4S)n] linker; and a second NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(II) a first NKcell targeting domain consisting of a Fd fragment ora Fab fragment ; a second NK targeting domain consisting of a Fc domain; a first [(G4S)n] linker; and an antigen targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL;

(iv) an antigen targeting domain consisting of a Fd fragment (comprising of a VH and a CH1) or a Fab fragment; a first [(G4S)n] linker; a first NK cell targeting domain consisting of a scFv comprising a VH, a second [(G4S)n] linker, and a VL; and a second NKcell targeting domain consisting of a Fc domain comprising a CH2 and a CH3.

17. The multi-specific polypeptide construct of claims 1-16, wherein the NKp80-targeting domain comprises a member selected from:

(1) VLFR1 (SEQ ID NO:105), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:119), VLCDR2 (SEQ ID NO:17), VLFR3 (SEQ ID NO:122), VLCDR3 (SEQ ID NO:32), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:141), VHCDR1 (SEQ ID NO:51), VHFR2 (SEQ ID NO:156), VHCDR2 (SEQ ID NO:68), VHFR3 (SEQ ID NO:163), VHCDR3 (SEQ ID NO:86), and VHFR4 (SEQ ID NO: 180) (clone 13);

(2) VLFR1 (SEQ ID NQ:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NO: 180) (clone 28);

(3) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NOTO), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO: 180) (clone 36);

(4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO: 180) (clone 37);

(5) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NQ:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NO: 180) (clone 45);

(6) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:36), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:71), VHFR3 (SEQ ID NO:168), VHCDR3 (SEQ ID NO:90), and VHFR4 (SEQ ID NO: 180) (clone 50);

(9) VLFR1 (SEQ ID NO:110), VLCDR1 (SEQ ID NO:7), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:23), VLFR3 (SEQ ID NO:128), VLCDR3 (SEQ ID NO:39), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:146), VHCDR1 (SEQ ID NO:57), VHFR2 (SEQ ID NO:159), VHCDR2 (SEQ ID NO:74), VHFR3 (SEQ ID NO:171), VHCDR3 (SEQ ID NO:93), and VHFR4 (SEQ ID NO: 181) (clone 74);

(10) VLFR1 (SEQ ID NO:111), VLCDR1 (SEQ ID NO:8), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:24), VLFR3 (SEQ ID NO:129), VLCDR3 (SEQ ID NO:40), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:147), VHCDR1 (SEQ ID NO:58), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:75), VHFR3 (SEQ ID NO:172), VHCDR3 (SEQ ID NO:94), and VHFR4 (SEQ ID NO: 181) (clone 78); (11) VLFR1 (SEQ ID NO:112), VLCDR1 (SEQ ID NO:9), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:25), VLFR3 (SEQ ID NQ:130), VLCDR3 (SEQ ID NO:41), VLFR4 (SEQ ID NO:140), (VHFR1 (SEQ ID NO:148), VHCDR1 (SEQ ID NO:59), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:76), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:95), and VHFR4 (SEQ ID NO: 181) (clone 79);

(13) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NQ:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:43), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NQ:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:78), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:97), and VHFR4 (SEQ ID NO: 181) (clone 82);

(14) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:12), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:27), VLFR3 (SEQ ID NO:133), VLCDR3 (SEQ ID NO:44), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:151), VHCDR1 (SEQ ID NO:62), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:79), VHFR3 (SEQ ID NO:175), VHCDR3 (SEQ ID NO:98), and VHFR4 (SEQ ID NO: 180) (clone 87);

(15) VLFR1 (SEQ ID NO:115), VLCDR1 (SEQ ID NO:13), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:28), VLFR3 (SEQ ID NO:127), VLCDR3 (SEQ ID NO:45), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:152), VHCDR1 (SEQ ID NO:63), VHFR2 (SEQ ID NO:160), VHCDR2 (SEQ ID NO:80), VHFR3 (SEQ ID NO:176), VHCDR3 (SEQ ID NO:99), and VHFR4 (SEQ ID NO: 181) (clone 94);

(16) VLFR1 (SEQ ID NO:108), VLCDR1 (SEQ ID NO:1), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:29), VLFR3 (SEQ ID NO:134), VLCDR3 (SEQ ID NO:46), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:153), VHCDR1 (SEQ ID NO:64), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:81), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID N0:100), and VHFR4 (SEQ ID NO:182) (clone 101);

(17) VLFR1 (SEQ ID NO:116), VLCDR1 (SEQ ID NO:14), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:25). VLFR3 (SEQ ID NO:135), VLCDR3 (SEQ ID NO:47), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:153), VHCDR1 (SEQ ID NO:65), VHFR2 (SEQ ID NO:162), VHCDR2 (SEQ ID NO:82), VHFR3 (SEQ ID NO:177), VHCDR3 (SEQ ID NO:101), and VHFR4 (SEQ ID NO: 181) (clone 102);

(18) VLFR1 (SEQ ID NO:117), VLCDR1 (SEQ ID NO:15), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:30), VLFR3 (SEQ ID NO:136), VLCDR3 (SEQ ID NO:48), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:154), VHCDR1 (SEQ ID NO:66), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:83, VHFR3 (SEQ ID NO:178), VHCDR3 (SEQ ID NO:102), and VHFR4 (SEQ ID NO: 181) (clone 106);

(19) VLFR1 (SEQ ID NO:118), VLCDR1 (SEQ ID NO:16), VLFR2 (SEQ ID NO:121), VLCDR2 (SEQ ID NO:31), VLFR3 (SEQ ID NO:137), VLCDR3 (SEQ ID NO:49), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:155), VHCDR1 (SEQ ID NO:67), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:84), VHFR3 (SEQ ID NO:179), VHCDR3 (SEQ ID NO:103), and VHFR4 (SEQ ID NO: 181) (clone 63);

(20) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:50), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:85), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:104), and VHFR4 (SEQ ID NO: 181) (clone 83); or

18. An antigen-binding protein, or an antigen-binding fragment thereof, comprising the CDR sequences selected from:

(4) VLCDR1 (SEQ ID NO:3), VLCDR2 (SEQ ID NO:20), VLCDR3 (SEQ ID NO:35), VHCDR1 (SEQ ID NO:54), VHCDR2 (SEQ ID NO:70), and VHCDR3 (SEQ ID NO:88) (clone 37); (5) VLCDR1 (SEQ ID NO:4), VLCDR2 (SEQ ID NO:21), VLCDR3 (SEQ ID NO:35), VHCDR1 (SEQ ID NO:55), VHCDR2 (SEQ ID NO:70), and VHCDR3 (SEQ ID NO:89) (clone 45);

(18) VLCDR1 (SEQ ID NO:15), VLCDR2 (SEQ ID NQ:30), VLCDR3 (SEQ ID NO:48), VHCDR1 (SEQ ID NO:66), VHCDR2 (SEQ ID NO:83, and VHCDR3 (SEQ ID NO:102) (clone 106);

(19) VLCDR1 (SEQ ID NO:16), VLCDR2 (SEQ ID NO:31), VLCDR3 (SEQ ID NO:49), VHCDR1 (SEQ ID NO:67), VHCDR2 (SEQ ID NO:84), and VHCDR3 (SEQ ID NO:103) (clone 63); (20) VLCDR1 (SEQ ID NO:11), VLCDR2 (SEQ ID NO:19), VLCDR3 (SEQ ID NO:50), VHCDR1 (SEQ ID NO:61), VHCDR2 (SEQ ID NO:85), and VHCDR3 (SEQ ID NO:104) (clone 83); or

(21) VLCDR1 (SEQ ID NO:247), VLCDR2 (SEQ ID NO:248), VLCDR3 (SEQ ID NO:249), VHCDR1 (SEQ ID NO:250), VHCDR2 (SEQ ID NO:251), and VHCDR3 (SEQ ID NO:252) (humanized clone 87-2), wherein the CDR sequences share at least about 90%homology to an amino acid sequence selected from SEQ ID NQ:1-104; 247-252, and/or wherein the CDR sequences selected from SEQ ID NO: 1-104; 247-252 comprise 2, or 3 amino acid substitutions.

19. An antigen-binding protein, or an antigen-binding fragment thereof, comprising the CDR and FR sequences selected from:

(2) VLFR1 (SEQ ID NO:106), VLCDR1 (SEQ ID NO:2), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:18), VLFR3 (SEQ ID NO:123), VLCDR3 (SEQ ID NO:33), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:142), VHCDR1 (SEQ ID NO:52), VHFR2 (SEQ ID NO:157), VHCDR2 (SEQ ID NO:69), VHFR3 (SEQ ID NO:164), VHCDR3 (SEQ ID NO:87), and VHFR4 (SEQ ID NO: 180) (clone 28);

(3) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:34), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:53), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:165), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO: 180) (clone 36);

(4) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:3), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:20), VLFR3 (SEQ ID NO:124), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:143), VHCDR1 (SEQ ID NO:54), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:70), VHFR3 (SEQ ID NO:166), VHCDR3 (SEQ ID NO:88), and VHFR4 (SEQ ID NO: 180) (clone 37); (5) VLFR1 (SEQ ID NO:107), VLCDR1 (SEQ ID NO:4), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:21), VLFR3 (SEQ ID NO:125), VLCDR3 (SEQ ID NO:35), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:144), VHCDR1 (SEQ ID NO:55), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NQ:70), VHFR3 (SEQ ID NO:167), VHCDR3 (SEQ ID NO:89), and VHFR4 (SEQ ID NO: 180) (clone 45);

(10) VLFR1 (SEQ ID NO:111), VLCDR1 (SEQ ID NO:8), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:24), VLFR3 (SEQ ID NO:129), VLCDR3 (SEQ ID NO:40), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:147), VHCDR1 (SEQ ID NO:58), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:75), VHFR3 (SEQ ID NO:172), VHCDR3 (SEQ ID NO:94), and VHFR4 (SEQ ID NO: 181) (clone 78);

(11) VLFR1 (SEQ ID NO:112), VLCDR1 (SEQ ID NO:9), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:25), VLFR3 (SEQ ID NO:130), VLCDR3 (SEQ ID NO:41), VLFR4 (SEQ ID NO:140), (VHFR1 (SEQ ID NO:148), VHCDR1 (SEQ ID NO:59), VHFR2 (SEQ ID NO:158), VHCDR2 (SEQ ID NO:76), VHFR3 (SEQ ID NO:173), VHCDR3 (SEQ ID NO:95), and VHFR4 (SEQ ID NO: 181) (clone 79);

(13) VLFR1 (SEQ ID NO:114), VLCDR1 (SEQ ID NO:11), VLFR2 (SEQ ID NO:120), VLCDR2 (SEQ ID NO:19), VLFR3 (SEQ ID NO:132), VLCDR3 (SEQ ID NO:43), VLFR4 (SEQ ID NO:138), (VHFR1 (SEQ ID NO:150), VHCDR1 (SEQ ID NO:61), VHFR2 (SEQ ID NO:161), VHCDR2 (SEQ ID NO:78), VHFR3 (SEQ ID NO:174), VHCDR3 (SEQ ID NO:97), and VHFR4 (SEQ ID NO: 181) (clone 82);

(21) VLFR1 (SEQ ID NO:253), VLCDR1 (SEQ ID NO:247), VLFR2 (SEQ ID NO:254), VLCDR2 (SEQ ID NO:248), VLFR3 (SEQ ID NO:255), VLCDR3 (SEQ ID NO:249, VLFR4 (SEQ ID NO:256), (VHFR1 (SEQ ID NO:257), VHCDR1 (SEQ ID NO:250), VHFR2 (SEQ ID NO:258), VHCDR2 (SEQ ID NO:251), VHFR3 (SEQ ID NO:259), VHCDR3 (SEQ ID NO:252), and VHFR4 (SEQ ID NO:260) (humanized clone 87-2), wherein the FR and CDR sequences share at least about 90%homology to an amino acid sequence selected from SEQ ID NO:1-104; 247-260, and/or wherein the FR and CDR sequences selected from SEQ ID NO: 1-104; 247-260 comprise 2, or 3 amino acid substitutions.

20. A nucleic acid sequence encoding the multi-specific polypeptide construct, or the antibody, of any one of claims 1-19.

21. A vector comprising sequences for the multi-specific polypeptide construct, or the antibody, of claims 1-19.

22. A host cell comprising the vector of claim 21 .

23. A method of producing the multi-specific polypeptide construct, or the antibody, of claims 1- 19, comprising culturing the host cell, and optionally isolating the multi-specific polypeptide construct from said host cell and/or the culture media.

24. A method of screening and/or identifying the multi-specific polypeptide construct, or the antibody, of claims 1-19, wherein the NK cell-targeting domain is antiNKp80.

25. A pharmaceutical composition comprising the multi-specific polypeptide construct, or the antibody, of claims 1-19.

26. A method for treating cancer comprising administering to a subject in need thereof the pharmaceutical composition of claim 25, wherein the multi-specific polypeptide construct, or the antibody, is administered in an effective amount to treat the cancer in the subject.

27. The method of claim 26, wherein the subject has cancer cells that express HER2, CD20, and/or EGFR