NK cell engagers that bind to NKP80 and their applications

Abstract

(a) one or more antigen-targeting domains that bind to one or more cancer-associated antigens; and (b) one or more NK cell-targeting domains, wherein binding to NK cells can stimulate and / or suppress the function of innate immune cells. Also provided are antigen-binding proteins or antigen-binding fragments thereof; nucleic acid sequences; vectors; host cells; methods for producing multispecific polypeptide constructs or antibodies; methods for screening and / or identifying the multispecific polypeptide constructs or antibodies disclosed herein; pharmaceutical compositions; and methods for treating cancer.

Description

[Technical Field]

[0001] This disclosure relates to the field of multispecific polypeptide constructs that are engineered to attract NK cells and bind to cell surface antigens, thereby inducing desired immune responses in various disease adaptations. [Background technology]

[0002] Natural killer (NK) cells are part of the innate immune system and make up 5-15% of circulating lymphocytes. NK cells perform innate immune surveillance against stressed cells such as tumor cells and virus-infected cells, and when they are found, they trigger their lysis. Normally, NK cell activity is mediated by a delicate balance between activating and inhibitory receptors expressed on their cell surface. Normal, healthy cells express HLA class I molecules, which suppress NK cell activity by binding to killer cell immunoglobulin-like receptors (KIRs) on the surface of NK cells. In contrast, ligands expressed by stressed cells bind to activating receptors.

[0003] Both activating and inhibitory receptors are expressed on the surface of NK cells and contribute to the performance of their functions. Inhibitory receptors specific to MHC-I (major histocompatibility complex class I) antigens strictly regulate NK cell-mediated cytotoxicity and lymphokine production. Inhibitory signals from MHC-I specific receptors are essential for hematopoietic target cells to avoid destruction by NK cells. This concept is called the "missing self" and was proposed by Ljunggren and Karre. Such MHC-I-recognizing inhibitory receptors form three families of NK cell surface receptors: KIR (killer cell immunoglobulin-like receptor), LIR (leukocyte immunoglobulin-like receptor), and NKG2A (natural killer group 2A). KIR is a member of the immunoglobulin superfamily and is a type I transmembrane molecule that recognizes classical human leukocyte antigens A, B, and C (HLA class Ia). LIR, also known as ILT (immunoglobin-like transcript), forms a second set of receptors and primarily recognizes non-classical HLA-G (class Ib) molecules in addition to HLA class Ia. LIR belongs to the same Ig superfamily as KIR. NKG2A is a member of the NKG2 group, which consists of seven receptors: A, B, C, D, E, F, and H. It dimerizes with CD94 to form the NKG2A / CD94 receptor. This receptor belongs to the C-type lectin family and recognizes non-classical HLA-E class I molecules as ligands.

[0004] NK cell-mediated disruption requires not only the detection of MHC-I molecules on transformed cells by inhibitory receptors, but also activation of NK cells by activating receptors. Natural cytotoxic receptors (NCRs) represent a group of natural killer cell surface activating receptors, including NKp46, NKp30, and NKp44. These receptors, like NKG2D, DNAM-1 (DNAX accessory molecule-1), and NKp80, recognize ligands expressed on the surface of virus-infected or malignant cells. CD16 (or FcγRIII) is also an activating receptor, mainly expressed by the CD56dim NK cell subset, and is essential for antibody-dependent cytotoxicity (ADCC) against IgG-coated target cells.

[0005] As a promising alternative platform to cellular immunotherapy, NK cells have recently attracted attention as an important type of innate immune regulatory cell. NK cells can rapidly kill multiple adjacent cancer cells through their non-MHC-restrictive action. While tumors may develop multiple resistance mechanisms to endogenous NK cell attack, activating, amplifying, and genetically modifying NK cells in vitro can significantly enhance their antitumor 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 yielded many promising results. However, many challenges remain, including meeting clinical-grade ex vivo amplification requirements, limitations in in vivo persistence, limitations in solid tumor invasion, and tumor editing to evade NK cell activity. Therefore, there is a need to provide alternative multispecific polypeptide constructs. [Overview of the project]

[0006] In one aspect, a multispecific polypeptide construct is provided, which includes the following: (a) One or more antigen-targeting domains that bind to one or more cancer-related antigens; and (b) One or more NK cell targeting domains that can stimulate and / or suppress innate immune cell function by binding to NK cells.

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

[0008] In some examples, the NKp80 targeting domain includes: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with their amino acid sequences; or heavy chain variable domains (VH) containing one, two, or three complementarity-determining regions (CDRs) selected from two or three amino acid substitutions thereof, and / or (2) VLCDR1 of SEQ ID NOs: 1-16, 247; VLCDR2 of SEQ ID NOs: 17-31, 248; and / or VLCDR3 of SEQ ID NOs: 32-50, 249; or having at least about 80% sequence identity with their amino acid sequences; or light chain variable domains (VLs) containing one, two, or three CDRs selected from two or three amino acid substitutions of those.

[0009] In some examples, the NKp80 targeting domain includes: (1) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (2) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with their amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from the 2 or 3 amino acid substitutions thereof.

[0010] In some examples, the NKp80 targeting domain includes: (1) VHCDR1 of sequence numbers 51-67, 250; VHCDR2 of sequence numbers 68-85, 251; and / or VHCDR3 of sequence numbers 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions therein; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions; and / or (4) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from 2 or 3 amino acid substitutions thereof.

[0011] In some examples, the multispecific polypeptide constructs disclosed herein further include a functional Fc domain.

[0012] In some examples, the Fc domain is (i) Native / wild-type Fc domain (FcWT) or reduced-function Fc(FcX) domain of Sequence ID: 224; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (ii) Enhanced Fc domain (FcE) of Sequence ID No. 226; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (iii) Silent Fc domain / inactivated mutant Fc domain (FcLALA) of Sequence ID No. 225; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof. That is the case.

[0013] In some examples, the multispecific polypeptide constructs disclosed herein include: (a) A first domain targeting NKp80; (b) A second domain targeting CD16; (c) One or more antigen-targeting domains that bind to one or more tumor-associated antigens.

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

[0015] In some examples, one or more antigen-targeting domains include: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or having at least about 80% sequence identity with those amino acids; or two or three amino acid substitutions thereof.

[0016] In some examples, the multispecific polypeptide constructs disclosed herein include: (A) NKp80 targeting domains including the following: (1) VHCDR1 of sequence numbers 51-67, 250; VHCDR2 of sequence numbers 68-85, 251; and / or VHCDR3 of sequence numbers 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions therein; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (4) SEQ ID NOs: 105 - 118, 253 of FR1; SEQ ID NOs: 119 - 121, 254 of a; SEQ ID NOs: 122 - 137, 255 of VLFR3; and / or SEQ ID NOs: 138 - 140, 256 of VLFR4; or those having at least about 80% sequence identity with their amino acid sequences; or 1, 2, 3 or 4 VL FRs selected from 2 or 3 amino acid substitutions thereof; and, (B) One or more antigen - targeting domains comprising: (1) VH (VH cetuximab) of SEQ ID NO: 231, VL (VL cetuximab) of SEQ ID NO: 232, CH of SEQ ID NO: 233, and / or CL of SEQ ID NO: 234; or those having at least about 80% sequence identity with their amino acid sequences; or 2 or 3 amino acid substitutions thereof; (2) VH (VH trastuzumab) of SEQ ID NO: 227, VL (VL trastuzumab) of SEQ ID NO: 228, CH of SEQ ID NO: 229, and / or CL of SEQ ID NO: 230; or those having at least about 80% sequence identity with their amino acid sequences; or 2 or 3 amino acid substitutions thereof; and / or (3) VH (VH rituximab) of SEQ ID NO: 244, VL (VL rituximab) of SEQ ID NO: 243, CH (CH rituximab) of SEQ ID NO: 246, and / or CL (CL rituximab) of SEQ ID NO: 245; or those having at least about 80% sequence identity with their amino acid sequences; or 2 or 3 amino acid substitutions thereof.

[0017] In some examples, the multispecific polypeptide constructs disclosed herein comprise: (A) An NKp80 - targeting domain comprising: (1) VH CDR1 with SEQ ID NOs: 51 - 67, 250; VH CDR2 with SEQ ID NOs: 68 - 85, 251; and / or VH CDR3 with SEQ ID NOs: 86 - 104, 252; or those having at least about 80% sequence identity with their amino acid sequences; or VH containing 1, 2, or 3 CDRs selected from 2 or 3 amino acid substitutions of them; (2) VL CDR1 with SEQ ID NOs: 1 - 16, 247; VL CDR2 with SEQ ID NOs: 17 - 31, 248; and VL CDR3 with SEQ ID NOs: 32 - 50, 249; or those having at least about 80% sequence identity with their amino acid sequences; or VL containing 1, 2, or 3 CDRs selected from 2 or 3 amino acid substitutions of them; (3) VH FR1 with SEQ ID NOs: 141 - 155, 257; VH FR2 with SEQ ID NOs: 156 - 162, 258; VH FR3 with SEQ ID NOs: 163 - 179, 259; and / or VH FR4 with SEQ ID NOs: 180 - 182, 260; or those having at least about 80% sequence identity with their amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FR) selected from 2 or 3 amino acid substitutions of them; and / or (4) FR1 with SEQ ID NOs: 105 - 118, 253; a with SEQ ID NOs: 119 - 121, 254; VL FR3 with SEQ ID NOs: 122 - 137, 255; and / or VL FR4 with SEQ ID NOs: 138 - 140, 256; or those having at least about 80% sequence identity with their amino acid sequences; or 1, 2, 3, or 4 VL FR selected from 2 or 3 amino acid substitutions of them; and (B) One or more antigen - targeting domains comprising: (1) VH (VH cetuximab) with SEQ ID NO: 231 in amino acid sequence, VL (VL cetuximab) with SEQ ID NO: 232 in amino acid sequence, CH with SEQ ID NO: 233 in amino acid sequence, and / or CL with SEQ ID NO: 234 in amino acid sequence; or those having at least about 80% sequence identity with their amino acid sequences; or 2 or 3 amino acid substitutions of them; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and (C) Sequence IDs: 224-226; or sequences having at least approximately 80% sequence identity with those sequences; or Fc domains having an amino acid sequence selected from two or three of those amino acid substitutions.

[0018] In some examples, the polypeptide construct is a triple-specific antigen-binding construct that includes: (a) First targeting domain that binds to NKp80 (b) A second targeting domain that binds to CD16; and (c) A third targeting domain that binds to the target antigen, Here, the targeting domain is selected from Fab fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-domain Ab(dAb) fragments, isolated CDRs, single-chain Fv(scFv), disulfide-stabilized Fv(dsFv), single-chain Ab(scAb), secretory T cell bispecific Ab(STAb), single-domain Ab(sdAb), single-domain CH antibody, single-domain CL antibody, VHH, variable domains (VNARs) of novel antigen receptors, sdAb based on shark-derived VNAR structures, and binding domains based on alternative scaffolds including, but not limited to, ankyrin-based domains, finomers, avimers, anticarin, fibronectin, and binding sites incorporated into the constant region of antibodies.

[0019] In some examples, the polypeptide construct is a triple-specific antigen-binding construct that includes the following: (a) A first targeting domain that binds to NKp80, the targeting domain being selected from Fab fragment, Fv fragment; sdAb fragment, isolated CDR, scFv, dsFv, scAb, STAb, sdAb, single-domain CH antibody, single-domain CL antibody, VHH, VNAR, and sdAb based on the VNAR structure from shark; (b) A first targeting domain that binds to CD16, the targeting domain being a functional Fc domain selected from FcWT (SEQ ID NO: 224), FcX (SEQ ID NO: 224), silent Fc / Fc inactivation variant (FcLALA) (SEQ ID NO: 225), or FcE (SEQ ID NO: 226); and (c) A third targeting domain that binds to a tumor-associated antigen, optionally to HER2, EGFR, or CD20, the targeting domain being selected from Fab fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-domain Ab(dsAb) fragments, isolated CDRs, single-chain Fv(scFv), disulfide-stabilized Fv(dsFv), single-chain Ab(scAb), secretory T cell bispecific Ab(STAb), single-domain Ab(sdAb), single-domain CH antibody, single-domain CL antibody, VHH, a variable domain (VNAR) of a novel antigen receptor, sdAb based on a shark-derived VNAR structure, and binding domains based on alternative scaffolds including, but not limited to, ankyrin-based domains, finomers, avimers, antikalin, fibronectin, and binding sites incorporated into the constant region of an antibody.

[0020] In some examples, the NKp80 targeting domain includes the following: (1) VH containing an amino acid sequence selected from sequence numbers 203-222, 236; or sequences having at least approximately 80% sequence identity with those sequences; or sequences with two or three amino acid substitutions; (2) Sequence ID: VL containing an amino acid sequence selected from 183-202, 235; Here, VH and VL pair up 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 those having at least about 80% sequence identity with their amino acid sequences; or two or three amino acid substitutions thereof.

[0021] In some examples, the multispecific polypeptide constructs disclosed herein include: (i) an antigen-targeting domain consisting of an Fd fragment or a Fab fragment; a first NK cell-targeting domain consisting of an Fc domain; a first [(G4S)n] linker; and a second NK cell-targeting domain consisting of scFv including VH, a second [(G4S)n] linker, and VL; (ii) A first NK cell targeting domain consisting of an Fd fragment or a Fab fragment; a second NK cell targeting domain consisting of an Fc domain; a first [(G4S)n] linker; and an antigen targeting domain consisting of scFv including VH, a second [(G4S)n] linker, and VL; (iii) A first NK cell targeting domain consisting of an Fd fragment or a Fab fragment; a first [(G4s)n] linker; an antigen targeting domain consisting of scFv including VH, a second [(G4S)n] linker and VL; and a second NK cell targeting domain consisting of an Fc domain including CH2 and CH3; or (iv) An antigen-targeting domain comprising an Fd fragment (containing VH and CH1) or a Fab fragment; a first [(G4S)n] linker; a first NK cell-targeting domain comprising scFv containing VH, a second [(G4S)n] linker and VL; and a second NK cell-targeting domain comprising an Fc domain containing CH2 and CH3.

[0022] In some examples, the NKp80 targeting domain includes members selected from the following: (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 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 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 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 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 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 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); (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); (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 context, an antigen-binding protein, or an antigen-binding fragment thereof, comprising a CDR sequence selected from the following, is provided: (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 (sequence number: 9), VLCDR2 (sequence number: 25), VLCDR3 (sequence number: 41), VHCDR1 (sequence number: 59), VHCDR2 (sequence number: 76), and VHCDR3 (sequence number: 95) (clone 79); (12) VLCDR1 (SEQ ID NO: 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 NO: 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), Here, the CDR sequence shares at least approximately 90% homology with an amino acid sequence selected from SEQ ID NOs: 1-104, 247-252, and / or Here, the CDR sequence selected from sequence numbers 1-104 and 247-252 contains two or three amino acid substitutions.

[0024] In yet another context, an antigen-binding protein, or an antigen-binding fragment thereof, comprising CDR and FR sequences selected from the following, is provided: (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 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 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 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 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 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 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); (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); (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), Here, the FR and CDR sequences share at least approximately 90% homology with amino acid sequences selected from SEQ ID NOs: 1-104, 247-260, and / or Here, the FR and CDR sequences selected from sequence numbers 1-104 and 247-260 contain two or three amino acid substitutions.

[0025] In another aspect, the Specified Classified Multispecific polypeptide constructs or nucleic acid sequences encoding antibodies are provided.

[0026] In yet another aspect, vectors comprising a multispecific polypeptide construct or antibody sequence disclosed herein are provided.

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

[0028] In another aspect, a method for producing a multispecific polypeptide construct or antibody disclosed herein is provided, comprising culturing host cells and optionally isolating a multispecific polypeptide construct from the host cells and / or culture medium.

[0029] In yet another aspect, a method for screening and / or identifying multispecific polypeptide constructs or antibodies disclosed herein, in which the NK cell targeting domain is anti-NKp80, is provided.

[0030] In another aspect, pharmaceutical compositions comprising multispecific polypeptide constructs or antibodies disclosed herein are provided.

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

[0032] In some cases, the subjects have cancer cells that express HER2, CD20, and / or EGFR.

[0033] To better understand the various embodiments described, please refer to the following detailed description in conjunction with the following drawings, where similar reference numbers indicate corresponding parts throughout the figures. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows an overview of the NKp80 activating binders identified from antibody discovery to functional characterization. FcX is shown as an Fc region with reduced ADCC function. [Figure 2] Figure 2 shows representative plots of cytotoxicity (killing rate compared to untreated control) at appropriate effector-to-target ratios (ET ratios) for NKp80 binder clones against the HER2-positive tumor cell line N87. Screening was performed in two non-overlapping batches. The NKp80 binder used in this experiment is a triple-specific format containing an Fc region (anti-HER2-anti-NKp80-FcX) that reduces ADCC function. An activated clone is defined as a clone that consistently shows cytotoxicity above the median cutoff in at least two replicates. Twenty activated binders in this representative assay are highlighted with triangles. FcX: Reduced ADCC function. Triangle: Activated binder; Circle: Inactivated binder. [Figure 3]Figure 3A shows representative cytotoxicity dose-response curves (killing rate compared to untreated control) for four NKp80-activated engagers against HER2-positive tumor cells OVCAR3. These four NKp80 engagers are humanized and expressed in a triplicate format containing a fully functional Fc region (anti-HER2-anti-NKp80-Fc vs anti-HER2-Fc), while the trastuzumab control does not contain anti-NKp80 (and therefore anti-HER2-Fc). Figure 3B shows the cytotoxicity (killing rate compared to untreated control) of the four NKp80-activated engagers against HER2-positive cells N87. The NKp80 engagers used in this experiment are in a triplicate format containing an Fc region with reduced ADCC activity (anti-HER2-anti-NKp80-FcX). The plots show mean cytotoxicity values ​​calculated from three independent assays (n=3). These clones exhibit higher cytotoxicity than trastuzumab containing a reduced Fc region (anti-HER2-FcX), but lower cytotoxicity than trastuzumab containing fully functional Fc (anti-HER2-Fc), indicating that functional Fc is necessary for enhanced performance, as demonstrated in Figure 3A. This is thought to be due to the low abundance of NKp80 relative to CD16 on NK cells (4,000 copies / cell of NKp80 compared to 70,000 copies / cell of CD16), as reported in previous studies. FcX: Fc with reduced ADCC function. [Figure 4] Figure 4 shows representative cytotoxic dose-response curves (killing rate compared to untreated control) for engagers, including NKp80 clone 87-2 (triple specific, anti-HER2-anti-NKp80-Fc), to trastuzumab. Experiments for each cell line were performed using PBMCs from healthy donors in a 9-dose series with the same effector:target ratio. Data were normalized to untreated control and plotted in Prism. [Figure 5]Figure 5 shows representative flow cytometry analyses of CD25- and CD137- expression populations on NK cells and T cells in the presence of the antibody (0.08 nM). Secreted IFN-γ levels were also measured in the presence of the antibody (0.1 nM). The HER2-positive tumor cell line HCT116 was used as the target cell, and an appropriate ET ratio was used in the experiment (PBMC: HCT116 cells). [Figure 6] Figure 6 shows representative cytotoxic dose-response curves (killing rate compared to untreated control) for engagers including NKp80 clone 87-2 (triple specificity, anti-EGFR-anti-NKp80-Fc) to cetuximab, tested in two cell lines (HCT116 and MDA-MB-231, top and middle panels). Experiments including anti-EGFR were performed using healthy donor PBMCs in a 9-dose series with the same effector:target ratio. Data were normalized to untreated control and plotted in Prism. The bottom panel shows representative cytotoxic dose-response curves (killing rate compared to untreated control) for engagers including NKp80 clone 87-2 (triple specificity, anti-CD20-anti-NKp80-Fc) to rituximab tested in RAJI cells. This experiment including anti-CD20 was performed using NK cells purified from healthy donor PBMCs in a 9-dose series with an effector:target ratio of 2.5. The data was normalized relative to the untreated control and plotted using Prism. [Figure 7]Figure 7 shows representative cytotoxic dose-response curves (killing rate compared to untreated control) illustrating the safety and specificity of engagers containing NKp80 clone 87-2. Fully functional, trispecific engagers containing the NKp80 clone 87-2 antibody (anti-HER2-anti-NKp80-Fc) were tested for cytotoxicity against HER2-positive normal fetal lung fibroblasts (MRC-5 and WI-38, top and middle panels) together with trastuzumab. Isotype controls of engagers containing NKp80 clone 87-2 (anti-HER2 replaced with untargeted IgG) were added to HER2-positive colorectal cancer cells (HCT116, bottom panel) together with trastuzumab (positive control). Experiments for each cell line were performed using healthy donor PBMCs in a 9-dose series with the same effector:target ratio. Data were normalized to untreated control and plotted in Prism. [Figure 8] Figure 8 shows clusters of four activated engagers containing different clones of NKp80. Clones 94-1 and 101-1 are separated from clones 45-2 and 87-2. Clones 45-2 and 87-2 are in the same cluster, while 94-1 and 101-1 are in different clusters. Clusters with silhouette values ​​for each clone were constructed using binding index inputs generated by tandem binning with biolayer interferometry (BLI). [Figure 9] Figure 9 shows the clustering analysis for all 20 NKp80-activated clones. Clusters are generated from the sequence identity matrix of the heavy chain CDR3. According to this sequence-based analysis, clones 87-2 and 45-2 are in the same cluster (cluster 2), while clones 101-1 and 94-2 are separated into different clusters (clusters 5 and 3), which is consistent with the BLI binding shown in Figure 8. This analysis suggests that the 20 NKp80-activated clones are sequentially diverse. [Figure 10] Figure 10 shows the amino acid sequences of the variable heavy chain (VH) and variable light chain (VL) complementarity-determining regions (CDR) of the NKp80 activating clone. [Figure 11]Figure 11 shows the amino acid sequences of the variable heavy chain (VH) and variable light chain (VL) framework region (FR) of the NKp80 activated clone. [Figure 12] Figure 12 shows the amino acid sequences of the NKp80 NK cell receptor, the wild-type Fc domain and the silent Fc domain, as well as the VH, VL, CH, and CL domains of the antigen-targeting domains that bind HER2 (trastuzumab), EGFR (cetuximab), and CD20 (rituximab). [Figure 13] Figure 13 shows the amino acid sequences of the variable light chain (VL) (Figure 13A) and variable heavy chain (VH) (Figure 13B) of 20 claimed anti-NKp80 clones targeting the HER2 antigen. Figure 13C shows the VH, CL, CH, CL, CDR, and FR sequences of humanized anti-NKp80 clone 87-2. Figure 13D shows the amino acid sequences of four exemplary EGFR-targeted polypeptide constructs in which the NK cell engager and antigen-targeting domain are arranged in four different permutations. Figure 13E shows representative cytotoxic dose-response curves (killing rate compared to untreated control) for various permutations of a trispecific engager containing anti-EGFR (cetuximab), anti-NKp80, and wild-type Fc domains against cetuximab. Experiments were performed using MDA-MB-231, PBMCs isolated from a healthy donor, as the target cells, in a 9-dose series with an E:T of 28. The data after 48 hours was normalized to the untreated control and plotted using Prism. [Figure 14] Figure 14 shows the amino acid percentage (%) identity matrix of variable heavy chain complementarity determination region 3 (VHCDR3) for each of the 20 NKp80-binding polypeptide construct clones. [Figure 15] Figure 15 is a schematic diagram illustrating how a triple-specific engager functions. This engager binds to CD16 and NKp80 on innate immune cells, and to the target antigen on target cells. This triple-specific binding induces antibody-dependent cell-mediated cytotoxicity (ADCC) by innate immune cells, killing target cells that possess the desired target antigen. [Figure 16]Figure 16 shows representative cytotoxic dose-response curves (killing rate compared to untreated control) for engagers targeting anti-NKp80 (87-2). Triple-specific engagers containing NKp80 clone 87-2 antibody with an Fc region variant (anti-HER2-anti-NKp80-Fc(variant), where Fc(variant) may be wild-type Fc(WT), inactivated Fc mutant (LALA), or enhanced (E)Fc) were tested for cytotoxicity against HER2-positive breast cancer cell lines. Experiments against each cell line were performed using PBMCs from healthy donors in a 9-dose series with the same effector:target ratio. Data after 24 hours were normalized to untreated control and plotted in Prism. [Modes for carrying out the invention]

[0035] I. Introduction Antibody-enhanced innate immune cell modulators (AIMs) are first-in-class, next-generation NK cell engager (NKCE)-based molecules with applications in various indications, including cancer, infectious diseases, and autoimmune diseases. In cancer immunotherapy, monoclonal antibodies have limitations in treatment approaches, such as being limited to patients with high levels of target expression. Checkpoint inhibitors are limited to small patient populations, and T cell bispecific antibodies and CART cell therapies carry a high risk of cytokine storm release.

[0036] Natural killer (NK) cells are essential for immune surveillance of tumors, and decreased NK activity has been associated with increased cancer susceptibility and metastasis in mouse models and clinical studies. NK cells recognize malignant cells without prior sensitization and act rapidly using a set of germline-encoded surface receptors. When activated, NK cells release cytotoxic granules containing perforin and granzymes, directly lysing tumor cells in a similar manner to activated cytotoxic T cells. NK cells are also potent producers of chemokines and cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which are essential for regulating adaptive immune responses. Due to their innate ability to eliminate tumor cells, NK cell-based immunotherapy for cancer has been studied for decades. Early clinical trials demonstrated the overall safety of NK cell infusion, even in allogeneic transplantation. The availability of allogeneic NK cells, the established safety profile, and the rapid action of NK cells have led to efforts to develop "off-the-shelf" NK cell-based cancer immunotherapies. However, there are many challenges to overcome, including the difficulty of achieving clinical-grade ex vivo amplification, limitations in in vivo persistence, limitations in invasion into solid tumors, and tumor editing to avoid NK cell activation.

[0037] In several cases, AIM NKCE enhances the response of innate immune cells, improves antitumor effects, and minimizes side effects. This multispecific polypeptide construct can be used as a monotherapy or in combination with existing disease-targeted therapies such as NK cell therapy, T cell checkpoint inhibitors, and small molecule compounds.

[0038] II. Definition To facilitate understanding of this disclosure, certain terms are defined first. Where used in this application, unless expressly provided herein, each of the following terms shall have the meanings set forth below. Additional definitions are provided throughout this application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. For example, see 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 The *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press, ed. 1999, Aca-demic Press, and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide a general dictionary of many of the terms used in this disclosure.

[0040] Throughout this specification and the subsequent claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” are understood to mean including the integer or step, or group of integers or steps, described herein, but not to mean excluding other integers or steps, or groups of integers or steps. Where used herein, the term “comprising” may be replaced by the terms “containing” or “including,” or where used herein, the term “having.” Where an aspect is described as “comprising” as described herein, it is understood that other similar aspects are also provided, described in terms of “consisting of” and / or “consisting essentially of.”

[0041] The terms “approximately” or “substantially” refer to a value or composition that falls within an acceptable margin of error for a particular value or composition, as determined by those skilled in the art, and this depends in part on how the value or composition is measured or determined, i.e., the limits of the measuring system. For example, in some cases, “approximately” or “substantially” may mean within or above one standard deviation, according to practice in the art. Alternatively, “approximately” or “substantially” may mean a range of up to 10% (i.e., ±10%).

[0042] As used herein, “consisting of” excludes any element, step, or component not specified in the elements of the claim. As used herein, “substantially consisting of” does not exclude any material or step that does not materially affect the basic and novel features of the claim.

[0043] The use of alternatives (e.g., "or") should be understood to mean either one, both, or a combination thereof. In this specification, the indefinite article "a" or "an" should be understood to mean "one or more" of the components mentioned or enumerated.

[0044] Scope: Throughout this disclosure, various aspects of the disclosure are presented in scope form. These scope descriptions are for convenience and brevity only and should not be interpreted as restricting the scope of the disclosure inflexibly. Therefore, in this specification, a scope description is deemed to specifically disclose all possible sub-scopes, not just the individual numbers within that scope. For example, a scope description such as 1 to 6 is deemed to specifically disclose sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, as well as the individual numbers within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0045] In some cases, multispecific polypeptide constructs include domains that bind to one or more innate immune cell modulators. In some cases, these domains are described as “targeting domains” or “binding domains” in relation to innate immune cells. As used herein, the terms “innate immune cell modulator,” “NK modulator,” or “modulator” refer to immunomodulatory molecules (such as receptors) expressed on immune cells that, upon binding, alter cellular activity and alter the overall immune response. In some cases, the alteration of the immune response caused by a modulator helps the body fight cancer, infection, or other diseases.

[0046] In some examples, multispecific polypeptide constructs include domains containing antibodies or fragments thereof. As used herein, the term “antibody” includes intact antibodies and their conjugated fragments. The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two pairs of identical polypeptide chains, each pair having one “light chain” (approximately 25 kDa) and one “heavy chain” (approximately 50–70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100 to 110 amino acids or more, primarily involved in antigen recognition. This variable region is initially expressed ligated to a cleavable signal peptide. A variable region that does not contain a signal peptide is sometimes called a mature variable region. For example, a light chain mature variable region means a light chain variable region that does not contain a light chain signal peptide. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector function. The constant region may include one or all of the CH1, hinge, CH2, and CH3 regions. Sequence modifications of the constant region domain may also be used. Substitution, addition, and / or deletion of one or more amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, can also be performed on the antibody constant domain without significantly altering the antibody's ability to bind to the target antigen.

[0047] In some examples, multispecific polypeptide constructs include a domain containing a monoclonal antibody or a fragment thereof. As used herein, the term "monoclonal antibody" refers to an antibody whose amino acid sequence is substantially identical or derived from the same gene source. Monoclonal antibody compositions exhibit binding specificity and affinity to a specific single epitope, or to a specific group of epitopes.

[0048] In some cases, multispecific polypeptide constructs contain domains comprising a chimeric antibody or a fragment thereof. The term “chimeric antibody” (or its antigen-binding fragment) means an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or part thereof has been modified, substituted, or exchanged so as to be bound to the constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new characteristics to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or part thereof has been modified, substituted, or exchanged to 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 of human immunoglobulin. By replacing it with a human constant region, the chimeric antibody can be made less antigenic in humans compared to the original mouse antibody while retaining its specificity to recognize the antigen.

[0049] In some cases, multispecific polypeptide constructs include domains containing humanized antibodies or fragments thereof. The term “humanized antibody” (or its antigen-binding fragment) as used herein is intended to include antibodies (and their antigen-binding fragments) having a variable region in which both the framework region and the CDR region are derived from human sequences. Antibodies or immunoglobulins are classified into the following classes based on the amino acid sequence of the constant region of their heavy chain: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4, IgAl, and IgA2. Therefore, when an antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, particularly those of the IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes may be used. Furthermore, if the antibody contains a constant region, the constant region also originates from such human sequences. Humanized antibodies (or their antigen-binding fragments) retain the reactivity of non-human antibodies while exhibiting low immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region and replacing the rest of the antibody with a human-compatible portion (i.e., the framework portion of the constant and variable regions). Further modifications of the framework region can be made not only within the human framework sequence but also within the CDR sequence derived from germline cells of other mammalian species. The humanized antibodies of this disclosure may contain amino acid residues not encoded by the human sequence (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or in vivo somatic mutation, or conservative substitutions to enhance stability or production). This definition of a humanized antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries, transgenic animals that have been modified to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been deactivated, such as immunized xenomuse via human B-cell hybridoma technology.

[0050] In some examples, the multispecific polypeptide constructs include domains containing recombinant humanized antibodies or fragments thereof. As used herein, the term “recombinant humanized antibody” includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transformed to express a humanized antibody, such as transfectomas, and antibodies prepared, expressed, produced or isolated by any other means of splicing all or part of the sequence of a human immunoglobulin gene with another DNA sequence.

[0051] In some examples, multispecific polypeptide constructs contain domains comprising antibodies or fragments thereof. “Isolated antibody” refers to an antibody that is substantially free of other cellular material and / or chemical substances.

[0052] In some cases, multispecific polypeptide constructs include a binding domain. As used herein, “binding domain” refers to, but is not limited to, any of the following: “Fab fragment,” i.e., a monovalent fragment consisting of VL, VH, CL, and CH1 domains; “F(ab)2 fragment,” i.e., a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at a hinge region; “Fd fragment,” i.e., consisting of VH and CH1 domains; “Fv fragment,” i.e., an “Fv fragment” consisting of the VL and VH domains of a single arm of the antibody; “single-domain antibody (dAb) fragment” consisting of the VH domain; isolated “complementarity-determining region (CDR)”; single-chain Fv "Disulfide-stabilized variable fragments (dsFv)", "single-chain antibody fragments (scab)", "STAB", "single-domain antibodies (sdAb or dAb)", "single-domain heavy-chain antibodies (sdCH)"; STAB, "single-domain antibodies (sdAb or dAb)", "single-domain heavy-chain antibodies (sdCH)", "single-domain light-chain antibodies (sdCL)", "nanobodies" or "single variable domains on heavy chains (VHH)", shark-derived "variable novel antigen receptors (VNAR)", single-domain antibodies based on VNAR structures, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, finomers, avimers, antikalin, fibronectin, and binding sites incorporated into the constant region of antibodies (e.g., f-star's Modular Antibody Technology™).

[0053] In some cases, multispecific polypeptide constructs contain linkers. As used herein, the term “linker” refers to an intervening peptide sequence that links any two components within a multispecific polypeptide construct, and includes primary repeats of residues such as glycine (G) and serine (S). Such linkers are broadly classified into flexible linkers, rigid linkers, and cleavable linkers. A G4S linker refers to a polyglycine-serine linker having four glycine and one serine. As described herein, a [(G4S)n] linker refers to the number (n) of consecutively repeated blocks of G4S.

[0054] In some examples, multispecific polypeptide constructs include a domain that binds to an antigen. As used herein, the term “antigen” refers to a structure on the surface of a target cell to which the polypeptide constructs of this disclosure bind, and which is generally recognized as being associated with a particular disease state. As used herein, the term “epitope” defines an antigenic determinant to which an antibody, antibody fragment, or other binding domain specifically binds. “Antigen” and “epitope” may be used interchangeably in the context of this disclosure and refer to a target molecule on the surface of a target cell.

[0055] In some examples, multispecific polypeptide constructs include domains that bind to bacterial antigens. As used herein, the term “bacterial antigen” includes, but is not limited to, intact, attenuated, or dead bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein that is long enough to be antigenic (e.g., about 8 amino acids or more). Examples include Gram-positive and Gram-negative bacterial antigens.

[0056] In some examples, the multispecific polypeptide constructs include domains that bind to viral antigens. As used herein, the term “viral antigen” includes, but is not limited to, intact, attenuated, or dead whole viruses, any structural or functional viral protein, or any peptide portion of a viral protein that is long enough to be antigenic (e.g., about 8 amino acids or more).

[0057] In some examples, multispecific polypeptide constructs contain different regions or domains. As used herein, the terms "region" and "domain" are understood to refer to the same component and can therefore be used interchangeably.

[0058] In some cases, multispecific polypeptide constructs include complementarity-determining regions. The term “complementarity-determining region” (“CDR”) refers to an amino acid sequence with a boundary determined using any number of well-known schemes, including those described by Kabat (i.e., the “Kabat” numbering scheme); Al-Lazikani (the “Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (the “IMGT” numbering scheme); etc. For example, in the classical format, Kabat numbers the CDR amino acid residues of the heavy chain variable domain (VH) as 31–35 (VHCDR1), 50–65 (VHCDR2), and 95–102 (VHCDR3), and the CDR amino acid residues of the “light chain variable domain” (VL) as 24–34 (VLCDR1), 50–56 (VLCDR2), and 89–97 (VLCDR3). In Chothia, the CDR amino acids in VH are numbered 26-32 (VHCDR1), 52-56 (VHCDR2), and 95-102 (VHCDR3), while the amino acid residues in VL are numbered 24-34 (VLCDR1), 50-56 (VLCDR2), and 89-97 (VLCDR3). Combining the CDR definitions of both Kabat and Chothia, the CDR in human VH consists of amino acid residues 26-35 (VHCDR1), 50-65 (VHCDR2), and 95-102 (VHCDR3), while in human VL it consists of amino acid residues 24-34 (VLCDR1), 50-56 (LVCDR2), and 89-97 (VLCDR3). Under IMGT, the CDR amino acid residues of VH are numbered approximately 26-35 (VHCDR1), 51-57 (VHCDR2), and 93-102 (VHCDR3), while the CDR amino acid residues of VL are numbered approximately 27-32 (VLCDR1), 50-52 (VLCDR2), and 89-97 (VLCDR3) (numbering according to "Kabat"). In IMGT, the CDR of an antibody can be determined using the IMGT / DomainGap Align program.

[0059] In some examples, multispecific polypeptide constructs include a light chain and a heavy chain. The light chain is classified as κ or λ. The heavy chain is classified as γ, μ, α, δ, or ε. The heavy chain of an antibody defines the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 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," and "variable heavy chain CDR3" refer to VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3, respectively.

[0060] In some examples, when the multispecific polypeptide construct is a multispecific antigen-binding polypeptide, the multispecific polypeptide construct described herein includes multiple binding domains. These domains bind to or recognize a group selected from NK modulators or target antigens. In some examples, each binding domain of the multispecific polypeptide construct includes 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 multispecific polypeptide construct includes a combination of one or more CDRs as described herein.

[0061] In some cases, the multispecific polypeptide constructs include a domain that binds to NKp80. As used herein, "NKp80" refers to an 80 kDa protein reported as a dimer expressed in natural killer (NK) cells, also known as killer cell lectin-like receptor subfamily F, member 1 (KLRF1). This receptor is known as a type II transmembrane protein with a type C lectin domain exposed to the extracellular compartment. It is primarily expressed in NK cells and also present in a small number of T cells. NKp80 induces NK activation and mediates cytotoxicity.

[0062] In some cases, multispecific polypeptide constructs contain NKp80 engagers. The term "NKp80 engager" refers to a molecule that can bind to NKp80, such as an antibody that binds to NKp80 expressed on NK cells. In some cases, the NKp80 engager is a human NKp80 binder (huNKp80 binder) and / or a cynomolgus monkey NKp80 binder (cyNKp80 binder).

[0063] In some examples, multispecific polypeptide constructs include an Fc domain. As used herein, the term “Fc domain” refers to a dimeric complex containing the C-terminal polypeptide sequence of an immunoglobulin heavy chain, which is obtained by papain digestion of an intact antibody. The Fc sequence of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. Polypeptides constituting the Fc domain, such as monomeric Fc polypeptides, are also considered part of this disclosure. Fc polypeptides can be obtained from any suitable immunoglobulin, e.g., human IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. Fc polypeptides may be obtained from humans or other non-human mammals. The Fc domain contains the carboxyl-terminal portions of both H chains linked by disulfide. The effector function of the antibody is determined by the sequence of the Fc domain. This region is also recognized by Fc receptors (FcRs) found in certain cells.

[0064] Furthermore, the multispecific polypeptide constructs disclosed herein include functional Fc domains (FcEs) having “effector functions” of the native / wild-type Fc region. In some examples, the “effector function” is selected from CD16 binding; C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using a variety of assays known in the art. In some cases, the Fc domain may be functional antibody-dependent cytotoxicity (ADCC) (e.g., via CD16 binding), impaired ADCC (FcX) (e.g., via Fc mutations, thereby providing an Fc impaired domain, or via an antibody composition to achieve impaired ADCC), an Fc silent domain / inactivating mutant Fc domain (FcLALA) achieved via Fc mutations, or enhanced ADCC (FcE) (e.g., via Fc mutations, thereby providing enhanced activity of the Fc domain).

[0065] Also considered part of this disclosure are multispecific polypeptide constructs comprising native / wild-type Fc domains and / or mutant Fc domains. The mutant Fc domain (or Fc mutant domain) contains an amino acid sequence different from the native / wild-type Fc domain sequence by at least one amino acid modification, preferably one or more amino acid substitutions. In some examples, the mutant Fc domain has at least one amino acid substitution compared to the native / wild-type Fc domain sequence or the Fc domain of the parent polypeptide. In some examples, the mutant Fc region (or Fc mutant region) contains about 1 to about 10 amino acid substitutions in the native / wild-type sequence Fc region. In some cases, the mutant Fc region shares at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, or at least approximately 94% homology with the native / wild-type Fc domain sequence, or at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% homology.

[0066] Furthermore, the exemplary multispecific polypeptide constructs include an "Fc component" which may contain a hinge domain, CH2 domain, or CH3 domain of the Fc domain.

[0067] In some examples, multispecific polypeptide constructs include a framework region. As used herein, the term "framework region (FR)" is intended to mean each domain of the variable light or heavy chain that isolates the CDR.

[0068] As used herein, the terms "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" mean VLFR1, VLFR2, VLFR3, VLFR4, VHFR1, VHFR2, VHFR3, and VHFR4, respectively.

[0069] In some examples, multispecific polypeptide constructs are bispecific antigen-binding polypeptide constructs. As used herein, the term “bispecific antigen-binding polypeptide construct” refers to a multispecific polypeptide construct containing two binding domains, for example, an antibody domain, although other binding domains may also be employed. If the binding domains include an antibody domain, each domain contains at least three CDRs and a framework; for example, VHH contains three CDRs, while Fab contains six CDRs.

[0070] In some cases, a multispecific polypeptide construct is a triplespecific antigen-binding polypeptide construct. The term “triplespecific antigen-binding polypeptide construct,” as used herein, refers to a multispecific polypeptide construct that binds to three different epitopes or three different binding sites on three different targets.

[0071] In some cases, a multispecific polypeptide construct is a multispecific antigen-binding polypeptide construct. As used herein, the term "multispecific antigen-binding polypeptide construct" refers to a multispecific polypeptide construct having two or more binding domains that bind to two or more different epitopes on at least two or more different targets. The term "multispecific antigen-binding polypeptide construct" includes, but is not limited to, bispecific, triplicate, quadruple, quinticate, hexaspecific, and so on.

[0072] In some cases, the multispecific polypeptide constructs of this disclosure exhibit synergistic function in their cytotoxicity. As used herein, “synergistic function” or “synergistic biological function” means biological activity or level of biological activity, or effect on biological function or activity, such as: 1) activity not observed in the individual polypeptide components of the multispecific polypeptide construct; 2) activity observed when two (or more) binding domains are linked in a particular manner; or 3) activity that is higher or lower than the activity observed when the individual polypeptide components of the multispecific polypeptide construct of this disclosure are adopted individually, such as enhanced activity observed only in the bispecific polypeptide construct.

[0073] Therefore, “synergistic” includes novel biological functions or novel activities. The synergistic functions adopted herein generally do not include simple targeting, i.e., those based solely on binding, but generally include any post-binding inhibition, activation, signal transduction, or similar.

[0074] In some cases, a multispecific polypeptide construct is a fusion protein. As used herein, the term “fusion protein” is used interchangeably with the term “recombinant protein” and includes a protein component A or B fused with a binding partner X or Y (where appropriate). In some cases, a fusion protein is a translation polypeptide construct expressed from a gene construct by recombinant technology. In some cases, a fusion protein is expressed in a host from a DNA construct. In the context of this disclosure, one key characteristic of a fusion protein is that it is expressed from a cell as a “single polypeptide.”

[0075] In some examples, a multispecific polypeptide construct includes an antigen-binding domain, or a binding domain, or an antigen-binding fragment, or an antigen-targeting domain. As used herein, the “antigen-binding portion,” “binding domain,” “antigen-binding fragment,” or “antigen-targeting domain” of a multispecific polypeptide construct refers to one or more peptide sequences within the multispecific polypeptide construct that have the ability to specifically bind to a given antigen.

[0076] In some cases, multispecific polypeptide constructs are NK cell engagers. As used herein, the terms “engager,” “natural killer cell engager,” “NK cell engager,” or “NK engager” refer to synthetic polypeptides or multifunctional antibodies capable of attracting tumor cells to NK cells and inducing tumor cell destruction by NK cells. In some cases, multispecific polypeptide constructs are described as “binders” instead of “engagers.” As used herein, the term “binder” includes both activated and deactivated binders, which may occur if some multispecific polypeptide constructs are found to be deactivated during the screening process.

[0077] In some cases, multispecific polypeptide constructs are evaluated for their binding ability and / or specificity. As used herein, “binding” or “binding” refers to a measurable and reproducible interaction, such as binding between a target and an antigen-binding polypeptide construct, which determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. This refers to the ability of individual antibodies to react with certain antigenic determinants and not with different antigenic determinants.

[0078] In some examples, the polyspecific polypeptide constructs include an activating binder domain. As used herein, “activating binder” refers to a polypeptide construct that exhibits cytotoxicity to target cells above the baseline defined in the cytotoxicity assay (i.e., higher than the median cytotoxicity level of all screened clones) and is capable of mediating NK cell lysis that lyses the target cells.

[0079] In some cases, the multispecific polypeptide constructs include a non-binder domain or an inactivated binder domain. As used herein, "non-binder" or "inactivated binder" refers to a polypeptide construct that does not exhibit evaluable cytotoxicity against target cells and exhibits activity below the baseline median as defined in cytotoxicity assays. In some cases, the inactivated binder determines the baseline activity together with the target antigen antibody (anti-HER2 antibody: anti-HER2-FcX, etc.).

[0080] In some cases, multispecific polypeptide constructs are evaluated by their semi-maximal effective concentration (EC50). As used herein, the terms “semi-maximal effective concentration” or “EC50” refer to the concentration of an antibody or multispecific polypeptide construct / part thereof that induces a response that is 50% of the maximum response (i.e., midway between the maximum response and baseline) in an in vivo or in vitro assay. The term “mean EC50 multiplier change potency” refers to the EC50 multiplier change of a multispecific polypeptide construct relative to a control drug. As a standard of care in the treatment of HER2-positive early and advanced breast cancer, trastuzumab is an exemplary control for exemplary HER2-specific multispecific polypeptide constructs. Cetuximab is used as a control in the data presented for EGFR-specific multispecific polypeptide constructs.

[0081] In some cases, multispecific polypeptide constructs are contacted with peripheral blood mononuclear cells (PBMCs). The term “peripheral blood mononuclear cells” or “PBMCs” refers to mononuclear cells collected from healthy subjects, subsequently cultured, and used in various bioassays, such as cytotoxicity assays.

[0082] In some cases, the cytotoxicity of a multispecific polypeptide construct 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, and in this case, NK cells. As used herein, “target cells” refer to cells that express a specific receptor and / or antigen and / or epitope to which an antibody or fragment thereof specifically binds. In some cases, the cytotoxicity assay involves contacting the multispecific polypeptide construct with a specific ratio of effector to target cells, referred to as the “effector-to-target ratio,” or “effector:target ratio,” “E:T ratio,” or “E / T ratio.”

[0083] In some instances, the term “subject” as used herein includes patients and non-patients. The term “patient” refers to an individual who is suffering from or may suffer from a medical condition, while “non-patient” refers to an individual who is not suffering from or may not suffer from a medical condition. “Non-patients” include healthy individuals, disease-free individuals, and / or individuals who do not suffer from a medical condition. The term “subject” includes humans and animals. The terms “subject” and “patient” are used interchangeably herein.

[0084] In some cases, multispecific polypeptide constructs or formulations containing such constructs are used to administer to subjects in need of treatment. The term “administration” means the physical introduction of a drug into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes, such as injection or infusion. As used herein, “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, lymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some cases, formulations are administered via parenteral routes, such as orally. Other parenteral routes of administration include topical, cutaneous, or mucosal routes, such as nasal, vaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over a longer period of time.

[0085] Parenteral administration of the compositions of this disclosure includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0086] In some cases, multispecific polypeptide constructs are used to treat diseases in subjects. As used herein, “treat” or “therapy” refers to an approach to obtain beneficial or desired outcomes, preferably including clinical outcomes. Treatment may refer to either improvement of the symptoms of a disease or condition, or delaying the progression of a disease or condition. Treatment is often effective by administering a therapeutically effective amount of multispecific polypeptide construct to a subject in need of such treatment.

[0087] In some cases, a multispecific polypeptide construct or a formulation containing such construct is administered to a subject in need in a therapeutically effective amount, effective dose, or therapeutically effective dosage. “Therapeutally effective amount,” “effective dose,” “effective amount,” or “therapeutally effective dosage” of a multispecific polypeptide construct means any amount of the construct, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or functional impairment due to the disease. The ability of a multispecific polypeptide construct to promote disease regression can be evaluated using various methods known to those skilled in the art, such as human subjects in clinical trials, animal model systems for predicting efficacy in humans, or assays of the activity of the multispecific polypeptide construct in in vitro assays.

[0088] In some cases, multispecific polypeptide constructs are used to treat cancer. "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and proliferation result in the formation of malignant tumors, which can invade adjacent tissues and, in some cases, metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0089] In some cases, multispecific polypeptide constructs exhibit antitumor effects. As used herein, “antitumor effect” refers to a biological effect manifested as a reduction in tumor volume, a reduction in tumor cell count, a reduction in tumor cell proliferation, a reduction in the number of metastases, an extension of overall survival or progression-free survival, an extension of life expectancy, or an improvement in various physiological symptoms associated with tumor. Antitumor effect may also refer to the prevention of tumor development.

[0090] In several cases, treatment of a target with a multispecific polypeptide construct helps achieve progression-free survival. As used herein, “progression-free survival” (abbreviated as PFS) refers to the period from the date of treatment to the date of disease progression.

[0091] In some cases, treatment of a subject with a multispecific polypeptide construct helps prevent or slow disease progression. As used herein, “disease progression” or “progressive disease” (abbreviated as PD) refers to the worsening of one or more symptoms associated with a particular disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0092] As used herein, the "response period" (abbreviated as DOR) refers to the time from the subject's first objective response to the day on which disease progression according to the revised IWG response criteria for malignant lymphoma was confirmed, or to death.

[0093] In some cases, treatment of a target with a multispecific polypeptide construct helps prevent symptoms and / or reduce their severity. The terms “prevent” and / or “reduce symptoms” mean delaying the onset, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, suppressing exacerbation, suppressing further exacerbation, and / or improving at least one sign or symptom of the disease.

[0094] In some cases, treatment of a target with a multispecific polypeptide construct induces an immune response in the target. "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced by these cells or the liver, which selectively target, bind to, damage, destroy, and / or eliminate from the body of a vertebrate an invading pathogen, a pathogen-infected cell or tissue, a cancer cell or other abnormal cell, or, in the case of autoimmune or pathological inflammation, normal human cells or tissue.

[0095] In some cases, multispecific polypeptide constructs are encoded by nucleic acids. As used herein, the term “nucleic acid” refers to a polymer containing 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 may be naturally occurring, recombinant, or synthetic. Furthermore, nucleic acid molecules may be single-stranded, double-stranded, or triple-stranded. In some cases, nucleic acid molecules can be modified. In the case of double-stranded polymers, “nucleic acid” may refer to either one or both strands of the molecule.

[0096] In some cases, multispecific polypeptide constructs are encoded by nucleotide sequences. The term "nucleotide sequence" in relation to nucleic acids refers to a sequence of nucleotides linked by covalent bonds such as phosphorus bonds (e.g., phosphodiester bonds, alkyl and aryl-phosphonate bonds, phosphorothioate bonds, phosphotriester bonds) and / or non-phosphorus bonds (e.g., peptide bonds and / or sulfamic acid bonds). In some cases, for example, a nucleotide sequence encoding a target-binding molecule linked to a localization domain is a heterologous sequence (e.g., a gene from a different species or cell type).

[0097] As used herein, the terms “sequence identity” or “homology” refer to the percentage sequence identity determined by the most alignable polypeptide sequence according to the Kabat-numbering rules. When, after alignment, a target polypeptide region (e.g., the entire maturation variable region of the heavy or light chain of an antibody) is compared to the same region of a reference polypeptide, the percentage of sequence identity between the target polypeptide region and the reference polypeptide region is calculated by multiplying the number of positions occupied by the same amino acids in both the target polypeptide region and the reference polypeptide region by 100, dividing by the total number of aligned positions in the two regions (without counting gaps), and then multiplying by 100 to obtain a percentage.

[0098] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl. Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol.Biol.48:443 (1970), the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al., 2000, Current Protocols in Molecular Biology). One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analysis is generally available through the National Center for Biotechnology Information (NCBI Internet Server), which is publicly accessible through the National Institutes of Health (NCBI). Sequence comparisons can usually be performed using default program parameters, but customized parameters can also be used. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value of 10, and a BLOSUM62 scoring matrix by default (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0099] In some cases, multispecific polypeptide constructs, or their domains or fragments, include substitutions. “Conservative substitutions” may be made, for example, based on the similarity of the amino acid residues involved in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0100] As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid listed within the same group as the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the thus modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0101] As used herein, "non-conservative substitution" is defined as the exchange 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.

[0102] In some cases, substitutions include non-classical amino acids. Examples of non-classical amino acids include, but are not limited to, designer amino acids such as selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and amino acid analogs in general.

[0103] In another context, host cells containing the vector of this disclosure are provided.

[0104] In some examples, the host cells disclosed herein include a cloning vector or expression vector configured to express a multispecific polypeptide or antibody disclosed herein.

[0105] In some examples, nucleic acids encoding multispecific polypeptide constructs are included in a vector. A “vector” is any molecule or composition having the ability to carry a nucleic acid sequence to a suitable host cell in which the encoded polypeptide can be synthesized. Typically, and preferably, a vector is a nucleic acid manipulated to incorporate a desired nucleic acid sequence (e.g., the nucleic acids of this disclosure) using recombinant DNA techniques known in the art. An expression vector typically comprises one or more of the following components (if not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, 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.

[0106] In some examples, various domains of a multispecific polypeptide construct are operably linked. As used herein, the term “operably linked” may refer to the juxtaposition or arrangement of certain elements that enable them to work together to produce a certain effect. For example, if a promoter controls the transcription of a coding sequence, the promoter may be operably linked to the coding sequence.

[0107] In some cases, nucleic acids encoding multispecific polypeptide constructs are inserted into expression vectors. “Expression vector” refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, with other elements for expression supplied by a host cell or in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate recombinant polynucleotides.

[0108] In some cases, multispecific polypeptide constructs, or fragments or domains thereof, are isolated. The term “isolated” refers to a composition, compound, substance, or molecule that has been altered by human intervention from its natural state. For example, a composition or substance present in nature is isolated if it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated from a naturally occurring coexisting substance is isolated, as the term is used herein.

[0109] In some cases, multispecific polypeptide constructs are encoded by nucleic acids. "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to function as a template for the 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 that arise therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for the transcription of the gene or cDNA) can be said to code for the protein or other product of that gene or cDNA.

[0110] Unless otherwise specified, "nucleotide sequences 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, a protein-coding nucleotide sequence, or RNA, contains introns to the same extent that a protein-coding nucleotide sequence may contain one or more introns in some versions.

[0111] In some examples, vectors containing nucleic acids encoding multispecific polypeptide constructs include promoters. As used herein, the term “promoter” is defined as a DNA sequence recognized by the cellular synthetic machinery, or introduced synthetic machinery, which is necessary to initiate the specific transcription of a polynucleotide sequence.

[0112] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product operably ligated to a promoter / regulatory sequence. In some examples, this sequence is a core promoter sequence, and in some examples, this sequence also includes enhancer sequences and other regulatory elements required for the expression of the gene product. In some examples, the promoter / regulatory sequence expresses the gene product in a tissue-specific manner.

[0113] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene product or a polynucleotide defining a gene product, causes the cell to produce the gene product under almost all physiological conditions.

[0114] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene product or a polynucleotide defining a gene product, substantially causes the gene product to be produced in the cell only if the corresponding inducer is present in the cell.

[0115] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene or a polynucleotide defined by a gene, substantially causes the cell to produce its gene product only if the cell is a tissue type corresponding to the promoter.

[0116] As used herein, "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. Lentiviruses can deliver a significant amount of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.

[0117] In some examples, multispecific polypeptide constructs include peptides, polypeptides, proteins, and / or fragments thereof. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include peptides or proteins containing two or more amino acids linked to each other by peptide bonds. As used herein, the use of the terms refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of which many types exist. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0118] In some examples, the sequence encoding a multispecific polypeptide construct, or any domain thereof, includes conservative sequence modifications. As used herein, “conservative sequence modification” is intended to mean an amino acid modification that significantly alters, or does not significantly alter, the antibody-binding properties, including the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. In some examples, modifications are introduced into the sequences of this disclosure by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in a sequence can be substituted with other amino acid residues from the same side chain family, and the modified antibody can be tested for its ability to bind to an antigen using recognized functional assays.

[0119] In some cases, a multispecific polypeptide construct, or an exogenous nucleic acid encoding any domain thereof, is used to transfect, transform, or transduce one or more host cells. As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is introduced into or into a host cell. A “transfected,” “transformed,” or “transduced” cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary target cells and their progeny cells.

[0120] This disclosure is not limited to, and is therefore subject to change, the specific methodologies, protocols, materials, reagents, substances, etc., described herein. The terms used herein are for illustrative purposes only and are not intended to limit the scope of this disclosure as defined solely by the claims.

[0121] In some instances, this disclosure includes one or more of the features defined herein.

[0122] III. Detailed Description of Embodiments In some examples, the multispecific polypeptide construct comprises a first polypeptide domain that specifically binds to one or more innate immune cell modulators and a second polypeptide domain that binds to one or more target cell antigens.

[0123] In some examples, the target cell antigen-binding domain of a multispecific polypeptide construct specifically binds to the target cell antigen. In its most common form (and unless a defined reference is cited), “specific binding” refers to the ability of a multispecific polypeptide construct to distinguish between a target of interest and a non-target molecule / protein, as determined, for example, according to specificity assays known in the art. Such assays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), electrochemiluminescence (ECL), immunoradiometric assays (IRMA), surface plasmon resonance (SPR) tests, and peptide scans.

[0124] In some cases, multispecific polypeptide constructs specifically bind to one or more innate immune cell modulators and / or target cell antigens with higher affinity, avidity, greater ease, and / or longer duration than they would to other antigens.

[0125] In some examples, the multispecific polypeptide construct includes one or more innate immune cell targeting domains and / or one or more antigen targeting domains. In some examples, the multispecific polypeptide construct includes one innate immune cell targeting domain and one antigen targeting domain. In some examples, the multispecific polypeptide construct includes two innate immune cell targeting domains and one antigen targeting domain. In some examples, the multispecific polypeptide construct includes three innate immune cell targeting domains and one antigen targeting domain. In some examples, the multispecific polypeptide construct includes four innate immune cell targeting domains and one antigen targeting domain. In some examples, the multispecific polypeptide construct includes one or more antigen targeting domains.

[0126] In some examples, the multispecific polypeptide construct is a bispecific, triplicate, quadruplicate, quinticate, or hexaspecific antigen-binding polypeptide. In some examples, the multispecific polypeptide construct contains one or more NK cell targeting domains and / or one or more antigen-targeting domains. In some examples, the multispecific polypeptide construct contains one NK cell targeting domain and one antigen-targeting domain. In some examples, the multispecific polypeptide construct contains two NK cell targeting domains and one antigen-targeting domain. In some examples, the multispecific polypeptide construct contains three NK cell targeting domains and one antigen-targeting domain. In some examples, the multispecific polypeptide construct contains four NK cell targeting domains and one antigen-targeting domain.

[0127] In some cases, a multispecific polypeptide construct is provided that includes the following: (a) One or more antigen-targeting domains that bind to one or more cancer-related antigens; and (b) One or more NK cell targeting domains that can stimulate and / or suppress the function of innate immune cells by binding to NK cells.

[0128] In some examples, the multispecific 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 multispecific polypeptide construct comprises one innate immune cell-targeting domain and two antigen-targeting domains. In some examples, the multispecific polypeptide construct comprises one innate immune cell-targeting domain and three antigen-targeting domains. In some examples, the multispecific polypeptide construct comprises two innate immune cell-targeting domains and two antigen-targeting domains. In some examples, the multispecific polypeptide construct comprises two innate immune cell-targeting domains and three antigen-targeting domains. Other combinations of the number of innate immune cell-targeting domains and the number of antigen-targeting domains are also within the scope of this disclosure.

[0129] In some examples, a multispecific polypeptide construct includes one or more antigen-targeting domains. In some examples, a multispecific polypeptide construct further includes 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 or more target antigen-targeting domains.

[0130] In some examples, the multispecific polypeptide construct comprises one NK cell targeting domain and two antigen targeting domains. In some examples, the multispecific polypeptide construct comprises one NK cell targeting domain and three antigen targeting domains. In some examples, the multispecific polypeptide construct comprises two NK cell targeting domains and two antigen targeting domains. In some examples, the multispecific polypeptide construct comprises two NK cell targeting domains and three antigen targeting domains. Other combinations of the number of NK cell targeting domains and the number of antigen targeting domains are also within the scope of the disclosure herein.

[0131] In some cases, the NK cell targeting domain is an NKp80 targeting domain or an anti-NKp80 domain. In some cases, the NKp80 targeting domain is selected from, but is not limited to, NKp80-binding Fab fragments, NKp80-binding Fd fragments, NKp80-binding F(ab)2 fragments, NKp80-binding Fv fragments, NKp80-binding single-domain antibody fragments, NKp80-binding CDRs, NKp80-binding single-chain Fvs, NKp80-binding dsFvs, NKp80-binding scabs, NKp80-binding STAbs, NKp80-binding single-domain heavy chain antibodies, NKp80-binding single-domain light chain antibodies, NKp80-binding VHHs, NKp80-binding VNARs, and other NKp80-binding domains based on alternative scaffolds.

[0132] In some cases, one of the NK cell targeting domains is the NKp80 targeting domain.

[0133] In some examples, the NKp80 targeting domain includes: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with their amino acid sequences; or heavy chain variable domains (VH) containing one, two, or three complementarity-determining regions (CDRs) selected from two or three amino acid substitutions thereof, and / or (2) VLCDR1 of SEQ ID NOs: 1-16, 247; VLCDR2 of SEQ ID NOs: 17-31, 248; and / or VLCDR3 of SEQ ID NOs: 32-50, 249; or having at least about 80% sequence identity with their amino acid sequences; or light chain variable domains (VLs) containing one, two, or three CDRs selected from two or three amino acid substitutions of those.

[0134] In some examples, the NKp80 targeting domain includes: (1) Heavy chain variable domains (VH) containing one, two, or three complementarity-determining regions (CDRs) selected from VHCDR1 (SEQ ID NOs: 51-67, 250); VHCDR2 (SEQ ID NOs: 68-85, 251); and / or VHCDR3 (SEQ ID NOs: 86-104, 252); and / or (2) A light chain variable domain (VL) comprising one, two, or three CDRs selected from sequence numbers: 1-16, 247 (VLCDR1); 17-31, 258 (VLCDR2); and / or 32-50, 249 (VLCDR3).

[0135] In some cases, the NKp80 targeting domain includes VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3, which 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 with an amino acid sequence selected from SEQ ID NOs: 1-104, 247-252.

[0136] In some cases, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and / or VLCDR3 have amino acid sequences selected from SEQ ID NOs: 1 to 104, which include two or three amino acid substitutions.

[0137] In some examples, the NKp80 targeting domain includes: (1) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (2) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from 2 or 3 amino acid substitutions thereof.

[0138] In some examples, the NKp80 targeting domain includes: (1) One, two, three, or four VH framework regions (FRs) selected from VHFR1 with sequence numbers 141-155, 257; VHFR2 with sequence numbers 156-162, 258; VHFR3 with sequence numbers 163-179, 259; and / or VHFR4 with sequence numbers 180-182, 260, and / or (2) One, two, three, or four VLFRs selected from sequence numbers: 105-118, 253 (VLFR1); 119-121, 254 (VLFR2); 122-137, 255 (VLFR3); and / or 138-140, 256 (VLFR4).

[0139] In some cases, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and / or 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 with amino acid sequences selected from SEQ ID NOs: 105–182, 253–260.

[0140] In some cases, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and / or VLFR4 have amino acid sequences selected from SEQ ID NOs: 105-182, 253-260, which include two or three amino acid substitutions.

[0141] In some examples, the NKp80 targeting domain includes: (1) VHCDR1 of sequence numbers 51-67, 250; VHCDR2 of sequence numbers 68-85, 251; and / or VHCDR3 of sequence numbers 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions therein; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (4) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from 2 or 3 amino acid substitutions thereof.

[0142] In some examples, the NKp80 targeting domain includes: (1) A VH containing one, two, or three CDRs selected from sequence numbers 51-67, 250 (VHCDR1); sequence numbers 68-85, 251 (VHCDR2); and / or sequence numbers 86-104, 252 (VHCDR3); (2) A VL containing one, two, or three CD-Rs selected from VLCDR1 with sequence numbers 1-16 and 247; VLCDR2 with sequence numbers 17-31 and 248; and VLCDR3 with sequence numbers 32-50 and 249; (3) One, two, three or four VH framework regions (FRs) selected from VHFR1 with sequence numbers 141-155, 257; VHFR2 with sequence numbers 156-162, 258; VHFR3 with sequence numbers 163-179, 259; and / or VHFR4 with sequence numbers 180-182, 260; and / or (4) One, two, three, or four VLFRs selected from sequence numbers: 105-118, 253 (VLFR1); 119-121, 254 (VLFR2); 122-137, 255 (VLFR3); and / or 138-140, 256 (VLFR4).

[0143] In some cases, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and / or 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 with amino acid sequences selected from SEQ ID NOs: 1-182, 247-260.

[0144] In some examples, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and / or VLFR4 have an amino acid sequence selected from SEQ ID NOs: 1-182, 247-260, which includes two or three amino acid substitutions.

[0145] Figure 1 summarizes exemplary NKp80-activating binders identified from antibody discovery to functional characterization. FcX indicates an Fc region with reduced ADCC function (see Example 1).

[0146] This disclosure describes a novel set of NKp80 targeting domains that can circumvent many of the difficulties of NK injection and can bind to and activate innate immune cells. In some examples, these domains are single innate immune cell targeting domains used in combinations including one domain, two domains, three domains, four domains, five domains, or six domains. In some examples, these domains are used in multispecific polypeptide modular constructs that include components targeting innate immune cells and / or specific antigens. In some examples, the multispecific polypeptide construct is a multispecific antigen-binding polypeptide construct. In some examples, the multispecific polypeptide construct is a bispecific, triplicate, quadruplicate, quinticate, or hexaspecific multispecific polypeptide construct. In some examples, the multispecific polypeptide construct further binds to target cell antigens. In some examples, the multispecific polypeptide construct binds to both innate immune cell modulators and target cell antigens. In some examples, the multispecific polypeptide construct specifically binds to one or more innate immune cell modulators and one or more target cell antigens.

[0147] A. Natural killer (NK) cells Natural killer (NK) cells are specialized immune effector cells that play a crucial role in immune activation against abnormal cells. Unlike the events required for T cell activation, NK cell activation is governed by the interaction between NK receptors and target cells, independently of antigen processing or presentation. Because the activation cues are relatively simple, NK cells have attracted considerable attention in the field of cancer immunotherapy. Research in this field is giving rise to many efforts toward the development and engineering of cancer immunotherapy using NK cells.

[0148] Various immunomodulatory molecules, including receptors involved in missing or induced self-recognition, influence NK cell reactivity. Major activating receptors expressed on human NK cells include FcγRIIIa(CD16), NKG2D, DNAM-1, and natural cytotoxic receptors such as NKp30, NKp44, NKp65, NKp80, and NKp46. Similar to NKG2D, NKp80 stimulates NK cell cytotoxicity and, after being induced by appropriate antibodies, induces calcium influx in human NK cells.

[0149] i. ADCC Antibody-dependent cell-mediated cytotoxicity (ADCC) is a potent cytotoxic mechanism in humans, primarily mediated by natural killer (NK) cells. ADCC mediates the clinical utility of several widely used cytolytic monoclonal antibodies (mAbs), and enhancing their efficacy leads to improvements in cancer immunotherapy. CD16a is a receptor for the Fc portion of IgG and triggers NK cell-mediated ADCC. Knowledge of the mechanism of action of CD16a has led to several strategies to improve ADCC by acting on either mAbs or NK cells.

[0150] In some examples, the multispecific polypeptide construct includes an Fc domain variant. In some examples, the Fc domain variant exhibits reduced activity or binding compared to the native / wild-type Fc domain. In some examples, the deactivated Fc domain is represented in this disclosure as FcX. In some examples, the deactivated Fc domain is constructed according to methods known in the art. In some examples, the components of the multispecific polypeptide construct are the native / wild-type Fc domain and / or the mutant Fc domain. An exemplary Fc domain variant (or Fc mutant domain) includes an amino acid sequence different from the native / wild-type Fc region sequence by at least one amino acid modification, preferably one or more amino acid substitutions. In some examples, the mutant Fc region has at least one amino acid substitution compared to the native / wild-type sequence Fc region or the Fc region of the parent polypeptide. In some examples, the mutant Fc region (or Fc mutant region) includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in the native / wild-type Fc region sequence. In some examples, the multispecific construct includes a mutant Fc region that has at least approximately 80% homology, or at least approximately 85% homology, or at least approximately 90% homology, or at least approximately 91% homology, or at least approximately 92% homology, or at least approximately 93% homology, or at least approximately 94% homology, or at least approximately 95% homology, or at least approximately 96% homology, or at least approximately 97% homology, or at least approximately 98% homology, or at least approximately 99% homology to the native / wild-type sequence Fc region.

[0151] In some cases, a deactivated Fc domain (FcX) results in a reduction of ADCC, which refers to a reduction in measurable ADCC response of at least approximately 10%, or at least approximately 20%, or at least approximately 30%, or at least approximately 40%, or at least approximately 50%, or at least approximately 60%, or at least approximately 70%, or at least approximately 80%, or at least approximately 90%, or at least approximately 95%, or at least approximately 99% of the control. In some cases, the Fc silent domain / Fc inactivation variant (FcLALA) confers little to no measurable ADCC, which refers to substantially complete silencing of at least approximately 90%, or at least approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% of the measurable ADCC response, or substantially complete silencing of ADCC to the point where no measurable ADCC is detected. In some cases, enhanced Fc domains confer enhanced ADCC. The enhanced ADCC refers to an improvement, increase, or amplification of a 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 compared to the control.

[0152] ii. Immunotherapy Current preclinical development of NK cell-based therapies has been greatly influenced by early clinical research. Understanding how NK cells are activated led to the development of the first NK cell-based treatment in the clinical setting of hematopoietic stem cell transplantation (HSCT), demonstrating the ability of NK cells to exert a graft-versus-leukemia effect. Currently, it is believed that successful adoptive transplantation requires the creation of a lymphocyte environment that provides a niche for donor cells to survive and proliferate.

[0153] In some cases, this disclosure includes multispecific polypeptide constructs or compositions or pharmaceutical compositions or uses or methods described herein, wherein the polypeptides or compositions or pharmaceutical compositions are administered to a subject via one or more routes of administration, including but not limited to topical, intravascular, intravenous, oral, subcutaneous, intra-arterial, intraperitoneal, intranasal, intradermal, intramuscular, etc.

[0154] In another context, pharmaceutical compositions comprising multispecific polypeptide constructs or antibodies disclosed herein are provided.

[0155] In some examples, pharmaceutical compositions are provided for use in treating cancer, in which a multispecific polypeptide construct or antibody is administered to a subject in need in an amount effective for treating cancer in that subject.

[0156] In some examples, the use of pharmaceutical compositions in the manufacture of pharmaceuticals for the treatment of cancer is provided, where a multispecific polypeptide construct or antibody is administered to a subject in need in an amount effective to treat cancer in that subject.

[0157] In another aspect, a method for treating cancer is provided, comprising administering a pharmaceutical composition disclosed herein to a subject in need thereof, wherein the multispecific polypeptide construct or antibody is administered in an amount effective to treat cancer in the subject.

[0158] In some cases, the subjects have cancer cells that express HER2, CD20, and / or EGFR.

[0159] In some examples, the method is as disclosed herein, and here (1) Cancer is a solid tumor; (2) Cancer is selected from the group consisting of breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and gastric cancer; and / or (3) Cancer is selected from the group consisting of lung adenocarcinoma, conventional glioblastoma multiforme, glioblastoma, colorectal cancer, and non-small cell carcinoma.

[0160] In some examples, the methods disclosed herein further include the administration of a second therapeutic treatment, the second therapeutic treatment including chemotherapeutic agents, biological agents, hormone therapy, radiotherapy, or surgery.

[0161] B. NK cell receptor In some examples, the multispecific 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 or more innate immune cell targeting domains. In some examples, the multispecific polypeptide construct comprises one or more innate immune cell targeting domains, where the innate immune cell targeting domain is an NK cell targeting domain (i.e., an NK targeting domain). In some examples, the NK cell targeting domain includes NKp80-binding Fab fragments, NKp80-binding Fd fragments, NKp80-binding F(ab)2 fragments, NKp80-binding Fv fragments, NKp80-binding single-domain antibody fragments, NKp80-binding CDRs, NKp80-binding single-chain Fvs, NKp80-binding dsFvs, NKp80-binding scabs, NKp80-binding STAbs, NKp80-binding single-domain heavy-chain antibodies, NKp80-binding single-domain light-chain antibodies, NKp80-binding VHHs, NKp80-binding VNARs, and other NKp80-binding domains based on alternative scaffolds that include, but are not limited to, ankyrin-based domains, finomers, avimers, anticarin, fibronectin, and binding sites incorporated into the constant region of antibodies. In some examples, the multispecific polypeptide construct further includes a second NK targeting domain. In some examples, the second NK targeting domain is an Fc domain.

[0162] In humans, NK cell receptors include killer cell immunoglobulin-like receptors (KIRs), type C lectins (CD94 / NKG2A / NKG2C, NKG2D), natural cytotoxic receptors (NCRs; NKp44, NKp30, NKp65, NKp80, NKp46), CD16 / FcγRIIIa, and integrins / adhesion molecules. These signals are combined, including the integration of the NK cell's activation state influenced by other events such as cytokine priming and latent viral infection, to determine whether or not to respond to target cells. When properly triggered, NK cells respond by killing targets and producing cytokines including IFN-γ, TNF-α, GM-CSF, and MIP-1α.

[0163] In some examples, the multispecific polypeptide construct includes a second NK cell targeting domain capable of binding to NK cells. Exemplary second targeting domains are selected from sequences that bind to NKp80, CD16, NKp46, NKp30, NKp44, NKG2D, NKp65, DNAM, CD94, NKG2A, TIGIT, interleukin receptors, and the like. In some examples, the multispecific polypeptide constructs described herein include two NK targeting domains, where the first NK targeting domain is an NKp80 targeting domain and the second NK targeting domain is a CD16 targeting domain.

[0164] In some examples, the multispecific polypeptide construct further comprises a second NK cell targeting domain, which is selected from, but not limited to, domains targeting CD16, NKp46, NKp30, NKp44, NKG2D, NKp65, DNAM, CD94, NKG2A, TIGIT, and interleukin receptors.

[0165] I. NCR In humans, the NCRs NKp46, NKp80, and NKp30 are expressed in activated and quiescent NK cells, while NKp44 expression is elevated in some NK cells by interleukin-2 stimulation. Viral hemagglutinin has been reported as a ligand for NKp46 and NKp44. Since anti-NCR antibodies inactivate NK cell-mediated lysis in many tumor cell types, cellular ligands likely exist. Another ligand for NCRs is nuclear factor HLA-B-related transcript 3, which is released from tumor cells and binds to NKp30. Furthermore, NKp46 and NKp30 have been shown to bind to heparin sulfate proteoglycans, and NKp80 has been shown to bind to activation-inducible type C lectin (AICL). More recently, NKp30 has also been shown to bind to the B7-H6 tumor antigen. NCRs are 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)).

[0166] NKp80, an activated homodimeric C-type lectin-like receptor (CTLR), is expressed in virtually all human natural killer (NK) cells and stimulates cytotoxicity and cytokine release. The ligand for NKp80 is a bone marrow-specific CTLR activation-inducing C-type lectin (AICL), encoded by the natural killer gene complex (NKC) adjacent to NKp80. In some cases, NKp80 expressed in NK cells has accession number Q9NZS2. In some cases, the NKp80 receptor has a human sequence: MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLILLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY (huNKp80 sequence; sequence number: 223) Includes.

[0167] In some cases, the NKp80 receptor has a cynomolgus monkey sequence: VLLKCQKGSHSNTTEHEDIGDLKMNNGTRRNTSNKDLCVSRSADQTVLCQSEWLKYRGKCYWFSNEMKSWSDSYVYCLERKSHLLIIQDELEMAFIQKNLRQSNYVWMGLNFTSLKMTWTWVDGSPLDPKIFFIKGPAKENSCAAIKESKIYSETCSSVFKWICQY(cyNKp80 sequence; sequence number: 261) Includes.

[0168] In some cases, the NK targeting domain is a domain that targets NKp80.

[0169] In some examples, the multispecific polypeptide constructs described herein include a variable light chain amino acid sequence of an NKp80 targeting domain selected from the following: AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKLASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTEVVVK(VL sequence of anti-NKp80(13); Sequence ID: 183); DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVVVK (VL sequence of anti-NKp80(28)-FcX); Sequence ID: 184); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTTRSSSIYWPFGGGTEVVVK (VL sequence of anti-NKp80(36); Sequence ID: 185); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYTAYTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEVVVK (VL sequence of anti-NKp80(37); Sequence ID: 186); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTLESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEVVVK (VL sequence of anti-NKp80(45); Sequence ID: 187); AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEVVVK (VL sequence for anti-NKp80(50); SEQ ID NO: 188); ALVMTQTPSSVSAAVGGTVTIKCQASQSIGNDLAWYQQKPGQPPKLLIYAASNLESGVPSRFRGSGSGTKFTLTISDLECADAATYYCQGTYRGSSISWPFGGGTEVVVK(VL sequence for anti-NKp80(51); Sequence ID: 189); QIVVTQTPASVSAAVGGTVTISCQSSQNVYGNNELSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGYSGGMRSFGGGTEVVLV (VL sequence of anti-NKp80(71); Sequence ID: 190); QIVVTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLASTLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTEVVVK (VL sequence of anti-NKp80(74); Sequence ID: 191); AQVLTQTASSVSAAVGGTVTISCQSSQSVYNYNWLGWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSVDCNVFGGGTEVVVK (VL sequence for anti-NKp80(78); SEQ ID NO: 192); ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTKVVVK (VL sequence of anti-NKp80(79); Sequence ID: 193); AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLASGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEVVVK (VL sequence of anti-NKp80(81); Sequence ID: 194); DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSKCVFPFGGGTEVVVK(VL sequence for anti-NKp80(82); Sequence ID: 195); DIVMTQTPASVEAAVGGTVTIKCQASQSIGRDLAWYQQKPGQPPKLLIYGASILESGVPSRFKGNGSGTQFTLTISDLECADAATYYCQGADRSSTPSWPFGGGTEVVVK (VL sequence of anti-NKp80(87); Sequence ID: 196); AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKTSSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTEVVVK (VL sequence for anti-NKp80(94); Sequence ID: 197); AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTEVVVK (VL sequence for anti-NKp80(101); SEQ ID NO: 198); DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTEVVVK (VL sequence for anti-NKp80(102); Sequence ID: 199); DVVMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTLESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTEVVVK (VL sequence of anti-NKp80(106); Sequence ID: 200); DIVMTQTPASVSAAAGGTVTINCQASQSISNELSWYQQKSGQPPKLLIYGASNLESGVPSRFKGSGSGTDFTLTISDLECADGATYYCQSNYYDSSSPDFAFGGGTEVVVK(VL sequence for anti-NKp80(63); SEQ ID NO: 201); and / or DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSNCVFPFGGGTEVVVK (VL sequence for anti-NKp80(83); Sequence ID: 202).

[0170] In some examples, the multispecific polypeptide constructs described herein have sequences comprising a variable heavy chain domain of an amino acid sequence of an NKp80 targeting domain selected from the following: QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIYTGGGSADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGYGDATDIWGPGTLVTV(VH sequence of anti-NKp80(13); SEQ ID NO: 203); QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYAGSSGSTHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDYFKIWGPGTLVTV (VH sequence of anti-NKp80(28); Sequence ID: 204); QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRYNIWGPGTLVTV (VH sequence of anti-NKp80(36); Sequence ID: 205); QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRYNIWGPGTLVTV (VH sequence of anti-NKp80(37); Sequence ID: 206; QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWRYNIWGPGTLVTV(VH sequence of anti-NKp80(45); SEQ ID NO: 207); QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWRYNIWGPGTLVTV (VH sequence of anti-NKp80(50); Sequence ID: 208); QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYSDSGNTYYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRYNIWGPGTLVTV (VH sequence of anti-NKp80(51); Sequence ID: 209); QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSPGVAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWGQGTLVTV (VH sequence of anti-NKp80(71); Sequence ID: 210); QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSGGSAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWGQGTLVTV (VH sequence of anti-NKp80(74); Sequence ID: 211); QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNGGATYYASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLWGQGTLVTV (VH sequence for anti-NKp80(78); Sequence ID: 212); QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPSGSIWYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWGQGTLVTV (VH sequence of anti-NKp80(79); Sequence ID: 213); QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIADKIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQGTLVTV (VH sequence of anti-NKp80(81); Sequence ID: 214); QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYFASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLWGQGTLVTV (VH sequence of anti-NKp80(82); Sequence ID: 215); QSLEEYGGDVVQPEGSLTLTCTASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWKYNIWGPGTLVTV (VH sequence of anti-NKp80(87); Sequence ID: 216); QSVEESGGRLVTPGTPLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTGGSAYYANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWGQGTLVTV (VH sequence of anti-NKp80(94); Sequence ID: 217); QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISWGGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDPWGPGTLLTV (VH sequence of anti-NKp80(101); SEQ ID NO: 218); QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSGGDTSYATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLWGQGTLVTV (VH sequence of anti-NKp80(102); Sequence ID: 219); QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDGSAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWGQGTLVTV (VH sequence of anti-NKp80(106); Sequence ID: 220); QSLEESGGRLVKPDETLTITCTVSGIDLSSYIISWVRQAPGEGLEYIGFINTDGSAYYATWAKGRFTISRTSATVDLKMTSLTTEDTATYFCARDAGHRYLFYFKLWGQGTLVTV (VH sequence of anti-NKp80(63); Sequence ID: 221); and QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLWGQGTLVTV (VH sequence for anti-NKp80(83); Sequence ID: 222).

[0171] In some examples, the NKp80 targeting domain includes: (1) Sequence IDs: 203-222, 236; or sequences having at least approximately 80% sequence identity with those sequences; or VH sequences containing amino acid sequences selected from two or three of those amino acid substitutions; (2) Sequence ID: VL containing an amino acid sequence selected from 183-202, 235; Here, VH and VL pair up 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 having at least about 80% sequence identity with their amino acid sequences; or two or three amino acid substitutions thereof.

[0172] In some examples, the NKp80 targeting domain includes: (1) Sequence ID: VH containing an amino acid sequence selected from 203-222 and 236; (2) Sequence ID: VL containing an amino acid sequence selected from 183-202, 235; Here, VH and VL pair up 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.

[0173] In some cases, VH and / or 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 at least about 100% sequence identity with amino acid sequences selected from SEQ ID NOs: 203-222, 236, and / or amino acid sequences selected from SEQ ID NOs: 183-202, 235, respectively.

[0174] In some examples, VH and / or VL have amino acid sequences selected from SEQ ID NOs: 203-222, 236 and / or SEQ ID NOs: 183-202, 235, respectively, and include 1 or 2, 3 or 4, 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.

[0175] In some examples, the NKp80 targeting domain includes members selected from the following: (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 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 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 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 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 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 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); (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); (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).

[0176] In some examples, the multispecific polypeptide constructs described herein include antigen-binding fragment sequences comprising one, two, three, four, five, or six CDRs selected from SEQ ID NOs: 1-104, 247-252.

[0177] NKp46 is established as an important activating receptor because it is almost exclusively expressed by NK cells and is the only NCR with a mouse orthologue called Ncr1. Its ligand repertoire is diverse, ranging from viral ligands such as hemagglutinin (HA) and hemagglutinin-neuraminidase (HN) from influenza virus, Sendai virus, Newcastle disease virus, and poxvirus, to fungal ligands and unknown ligands found in tumors, adipocytes, human pancreatic β-cells, hepatic stellate cells, and bacteria such as Fusobacterium nucleatum. Recently, soluble NKp46 ligands have been identified. The identification of unknown membrane-bound ligands, particularly tumor ligands of NKp46, has been actively studied for over 20 years. In some cases, the NK-targeting domain is a domain that targets NKp46.

[0178] Like NKp80, NKp65 induces cytotoxicity in NK cells in redirected cell lysis assays. However, unlike NKp80, NKp65 is not expressed to a detectable level in human peripheral blood NK cells or T cells. NKp65 cDNA was originally cloned from IL-2 / IL-12 stimulated peripheral blood NK cells. To date, significant surface expression of NKp65 has only been noted in the NK cell line NK92 and its derivative NK92MI; therefore, the elucidation of cells that physiologically express NKp65 and the determinants of in vivo NKp65 expression is awaited. In some cases, the NK targeting domain is a domain that targets NKp65.

[0179] ii. Type C lectins NK cells recognize "stressed" cells via the activating receptor NKG2D, which is expressed in almost all mouse NK cells. This receptor has been shown to be important for several cancer control mechanisms mediated by NK cells. The NKG2D molecule recognizes several different ligands. This ability is thought to be due to a single binding site of the receptor with side chains that exhibit limited flexibility, resulting in a rigid interaction model of ligand binding. Ligands for NKG2D include MHC class I-related proteins, whose expression is regulated by both the DNA damage pathway and the heat shock response pathway. Given the immunostimulatory nature of NK cells, NKG2D-mediated recognition of tumor cells is essential for optimal immune responses against some tumors. In some cases, the NK targeting domain is a domain that targets NKG2D.

[0180] Another receptor in the C-type lectin family is the CD94-NKG2A / C / E heterodimer. These receptors are thought to respond to levels of non-classical MHC class I on the surface of potential target cells and are important in preventing inappropriate NK cell activation. The heterodimers CD94-NKG2C and CD94-NKG2E have been shown to associate with DAP-12 and are considered to be activating receptors. In humans, both the inhibitory, ITIM-containing CD94-NKG2A receptor and the activating, DAP-12-associating CD94-NKG2C receptor bind to HLA-E, a non-classical HLA class I molecule. The reason for the existence of both activating and inhibitory receptors specific to the same molecule remains unclear. Since the expression of this ligand does not necessarily lead to NK cell activation, this phenomenon may allow for more specific differentiation between normal tissue and damaged or infected tissue. In some examples, the NK targeting domain is a domain that targets CD94.

[0181] iii. Co-stimulatory receptors There are several other NK cell receptors that are considered co-stimulatory. These receptors, while insufficient on their own to trigger NK cell activation, provide further stimulation to the cells. Thus, these receptors not only provide an alternative activation mechanism but also prevent NK cells from being activated to respond to normal or healthy tissue. These receptors include DNAM-1, NKR-P1 receptor, and PILR receptor.

[0182] The DNAM-1 receptor (also known as CD226) is a member of the Ig superfamily and is constitutively expressed in approximately 50% of NK cells. The ligands for this costimulatory activating receptor are CD155 (also known as the poliovirus receptor, PVR, or Necl-5) and CD112 (Nectin-2), and the expression of these ligands may be upregulated in some tumor cells, suggesting that DNAM-1 is involved in some NK cell-mediated antitumor responses. In some cases, the NK targeting domain is a domain that targets DNAM-1.

[0183] iv. FcγR In humans, there are three classes of Fc receptors (FcγR) that bind to IgG: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). The expression of FcγR by leukocytes induces their activation (CD64, CD32A, CD32C, CD16A, and CD16B) or suppression (CD32B), thereby regulating the immune response and signal threshold.

[0184] CD16 is a prototype NK cell activating receptor because it itself triggers cytotoxic activity and the production of pro-inflammatory cytokines and chemokines, enough to unleash the antitumor function of NK cells. Human CD16 is also expressed in macrophages and some circulating monocytes and consists of two extracellular Ig domains, a short cytoplasmic tail, and a transmembrane domain that allows binding to CD3 and FcγRI chains in NK cells; these immunoreceptor tyrosine-based activation motif (ITAM)-containing subunits connect the receptor to intracellular signaling pathways, regulating the rearrangement of the actin and microtubule cytoskeleton and the activation of several transcription factors. In some examples, the NK targeting domain is a domain that targets CD16.

[0185] Furthermore, this disclosure also includes functional Fc domains having “effector functions” of the 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; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc domain to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using a variety of assays known in the art. In some examples, the Fc domain has functional antibody-dependent cytotoxicity (e.g., via CD16 binding), ADCC reduction (FcX) (e.g., via structural changes in the functional Fc domain), ADCC absence (Fc silent / inactivating mutant Fc domain (FcLALA)) (e.g., via specific Fc mutations), or ADCC enhancement (FcE).

[0186] In some examples, multispecific polypeptide constructs bind to a second modulator via an Fc domain or Fc component. In some examples, multispecific polypeptide constructs include, but are not limited to, an Fc domain, a native / wild-type Fc domain, an Fc-enhancing domain, an Fc-decreasing domain, an Fc-silent domain / Fc-inactivating domain, an Fc-mutant domain, or a heterodimer Fc domain. In some examples, the antigen-binding protein includes 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 includes a mutant Fc domain. In some examples, the mutant Fc domain is a de-functioning Fc domain. In some examples, the de-functioning Fc domain is represented as FcX. In some examples, the mutant Fc domain is an Fc-silent domain / inactivating mutant Fc domain (FcLALA). In some examples, the de-functioning Fc domain is constructed according to methods known in the art. In some examples, the mutant Fc domain is an enhancement Fc domain. In some examples, the enhanced Fc domain is represented as FcE.

[0187] In some examples, the multispecific polypeptide constructs disclosed herein further include a functional Fc domain.

[0188] In some cases, the Fc domain contains an amino acid sequence selected from sequence numbers 224-226.

[0189] In some examples, the multispecific polypeptide constructs include a native / wild-type Fc domain. In some examples, the native / wild-type Fc domain includes the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Native / wild-type Fc domain; Sequence ID: 224).

[0190] In some examples, the multispecific polypeptide constructs include native / wild-type Fc domain sequences configured to reduce ADCC function. In some examples, the reduced-function Fc domains include the following sequences: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Decreased Fc domain (FcX); Sequence ID: 224).

[0191] In some cases, the mutant Fc domain is a silent Fc domain. In some cases, the silent Fc domain contains the following sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Silent Fc domain / Inactivating mutant Fc domain (FcLALA); Sequence ID: 225).

[0192] In some cases, the mutant Fc domain is an enhanced Fc domain (FcE). In some cases, the enhanced Fc domain contains the following sequence: DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Enhanced Fc Domain (FcE); Sequence ID: 226).

[0193] In some cases, the Fc domain (i) Native / wild-type Fc domain (FcWT) or reduced-function Fc(FcX) domain of Sequence ID: 224; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (ii) Enhanced Fc domain (FcE) of Sequence ID No. 226; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (iii) Silent Fc domain / inactivated mutant Fc domain (FcLALA) of Sequence ID No. 225; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof. That is the case.

[0194] In some cases, the Fc domain (i) Native / wild-type Fc domain (FcWT) or degraded Fc(FcX) domain of sequence number: 224; (ii) Enhanced Fc domain (FcE) of sequence number: 226; (iii) This is the silent Fc domain / inactivating mutant Fc domain (FcLALA) of sequence number 225.

[0195] In some cases, the Fc domain shares at least approximately 80%, or at least approximately 85%, or at least approximately 90%, or at least approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99%, or at least approximately 100% sequence identity with an amino acid sequence selected from SEQ ID NOs: 224-226.

[0196] In some examples, the Fc domain has an amino acid sequence selected from SEQ ID NOs: 224-226, which includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid substitutions.

[0197] In some examples, the multispecific polypeptide constructs disclosed herein include: (i) an antigen-targeting domain consisting of an Fd fragment (containing VH and CH1) or a Fab fragment (containing VH, CH1, CL1, and VL); a first NK cell-targeting domain consisting of an Fc domain (containing CH2 and CH3); a first [(G4S)n] linker; and a second NK cell-targeting domain consisting of scFv containing VH, a second [(G4S)n] linker, and VL; (ii) A first NK cell targeting domain consisting of an Fd fragment (containing VH and CH1) or a Fab fragment (containing VH, CH1, CL1, and VL); a second NK cell targeting domain consisting of an Fc domain (containing CH2 and CH3); a first [(G4S)n] linker; and an antigen targeting domain consisting of scFv containing VH, a second [(G4S)n] linker, and VL; (iii) A first NK cell targeting domain consisting of an Fd fragment (containing VH and CH1) or a Fab fragment (containing VH, CH1, CL1 and VL); a first [(G4s)n] linker; an antigen targeting domain consisting of scFv containing VH, a second [(G4S)n] linker and VL; and a second NK cell targeting domain consisting of an Fc domain containing CH2 and CH3; or (iv) an antigen-targeting domain consisting of an Fd fragment (containing VH and CH1) or a Fab fragment (containing VH, CH1, CL1, and VL); a first [(G4S)n] linker; a first NK cell-targeting domain consisting of scFv containing VH, a second [(G4S)n] linker, and VL; and a second NK cell-targeting domain consisting of an Fc domain containing CH2 and CH3.

[0198] In some examples, the multispecific polypeptide constructs disclosed herein include: (a) A first domain targeting NKp80; (b) A second domain targeting CD16; (c) One or more antigen-targeting domains that bind to one or more tumor-associated antigens.

[0199] In some examples, one or more antigen-targeting domains include: (1) VH (VH of cetuximab) with amino acid sequence SEQ ID NO: 231, VL (VL of cetuximab) with amino acid sequence SEQ ID NO: 232, CH with amino acid sequence SEQ ID NO: 233, and / or CL with amino acid sequence SEQ ID NO: 234; or those having at least about 80% sequence identity with their amino acid sequences; or two or three amino acid substitutions thereof; (2) VH (VH of trastuzumab) with amino acid sequence SEQ ID NO: 227, VL (VL of trastuzumab) with amino acid sequence SEQ ID NO: 228, CH with amino acid sequence SEQ ID NO: 229, and / or CL with amino acid sequence SEQ ID NO: 230; or those having at least about 80% sequence identity with their amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH of rituximab) with amino acid sequence SEQ ID NO: 244, VL (VL of rituximab) with amino acid sequence SEQ ID NO: 243, CH (CH of rituximab) with amino acid sequence SEQ ID NO: 246, and / or CL (CL of rituximab) with amino acid sequence SEQ ID NO: 245; or those having at least about 80% sequence identity with their amino acid sequences; or two or three amino acid substitutions thereof.

[0200] In some examples, one or more antigen-binding domains include the following: (1) VH (VH of cetuximab) with amino acid sequence SEQ ID NO: 231, VL (VL of cetuximab) with amino acid sequence SEQ ID NO: 232, CH with amino acid sequence SEQ ID NO: 233, and / or CL with amino acid sequence SEQ ID NO: 234; (2) VH (VH of trastuzumab) with amino acid sequence SEQ ID NO: 227, VL (VL of trastuzumab) with amino acid sequence SEQ ID NO: 228, CH with amino acid sequence SEQ ID NO: 229, and / or CL with amino acid sequence SEQ ID NO: 230; and / or (3) VH (VH of rituximab) with amino acid sequence SEQ ID NO: 244, VL (VL of rituximab) with amino acid sequence SEQ ID NO: 243, CH (CH of rituximab) with amino acid sequence SEQ ID NO: 246, and / or CL (CL of rituximab) with amino acid sequence SEQ ID NO: 245.

[0201] In some examples, the antigen-targeting domains VH, VL, CH, and / or CL share an amino acid sequence selected from SEQ ID NOs: 227-238, 243-246 and 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.

[0202] In some examples, the antigen-targeting domains VH, VL, CH, and / or CL have an amino acid sequence selected from SEQ ID NOs: 227-238, 243-246 and contain 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.

[0203] In some examples, the multispecific polypeptide constructs disclosed herein include: (A) An NKp80-targeting domain comprising: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or those having at least about 80% sequence identity with their amino acid sequences; or VH containing 1, 2 or 3 CDRs selected from 2 or 3 amino acid substitutions thereof; (2) VLCDR1 of SEQ ID NOs: 1-16, 247; VLCDR2 of SEQ ID NOs: 17-31, 248 of 247; and VLCDR3 of SEQ ID NOs: 32-50, 249; or those having at least about 80% sequence identity with their amino acid sequences; or VL containing 1, 2 or 3 CDRs selected from 2 or 3 amino acid substitutions thereof; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with their amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of their amino acid substitutions, and / or (4) FR1 of SEQ ID NOs: 105-118, 253; a of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VL FRs selected from 2 or 3 of those amino acid substitutions; and (B) One or more antigen-targeting domains including the following: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or those having at least approximately 80% sequence identity with those amino acids; or two or three amino acid substitutions thereof.

[0204] In some examples, the multispecific polypeptide constructs disclosed herein include: (A) NKp80 targeting domains including the following: (1) VHCDR1 of sequence numbers 51-67, 250; VHCDR2 of sequence numbers 68-85, 251; and / or VHCDR3 of sequence numbers 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions therein; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions; and / or (4) FR1 of SEQ ID NOs: 105-118, 253; a of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VL FRs selected from 2 or 3 of those amino acid substitutions; and (B) One or more antigen-targeting domains including the following: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and (C) Sequence IDs: 224-226; or sequences having at least approximately 80% sequence identity with those sequences; or Fc domains having an amino acid sequence selected from two or three of those amino acid substitutions.

[0205] In some examples, the multispecific polypeptide constructs disclosed herein include: (A) NKp80 targeting domains including the following: (1) A VH containing one, two, or three CDRs selected from VHCDR1 with sequence numbers 51-67, 250; VHCDR2 with sequence numbers 68-85, 251; and / or VHCDR3 with sequence numbers 86-104, 252; (2) A VL containing one, two, or three CD-Rs selected from VLCDR1 with sequence numbers 1-16 and 247; VLCDR2 with sequence numbers 17-31 and 248; and VLCDR3 with sequence numbers 32-50 and 249; (3) One, two, three or four VH framework regions (FRs) selected from VHFR1 with sequence numbers 141-155, 257; VHFR2 with sequence numbers 156-162, 258; VHFR3 with sequence numbers 163-179, 259; and / or VHFR4 with sequence numbers 180-182, 260; and / or (4) FR1 of sequence numbers 105-118, 253; a of sequence numbers 119-121, 254; VLFR3 of sequence numbers 122-137, 255; and / or 1, 2, 3, or 4 VLFRs selected from sequence numbers 138-140, 256; and (B) One or more antigen-targeting domains including the following: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; and (C) Sequence ID: Fc domain having an amino acid sequence selected from 224-226.

[0206] In some cases, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and VLFR4 have sequence identity of 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% with amino acid sequences selected from SEQ ID NOs: 1-182, 247-260; Here, 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 with an amino acid sequence selected from SEQ ID NOs: 183-222, 235-236; and / or Here, the Fc domain has sequence identity of 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 at least about 100% with an amino acid sequence selected from sequence numbers 224-226.

[0207] In some examples, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and / or VLFR4 have an amino acid sequence selected from SEQ ID NOs: 1-182, 247-260, which includes two or three amino acid substitutions; Here, the VH and VL of the amino acid sequence selected from sequence numbers 183-222, 235-236 contain 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; and / or Here, Fc has an amino acid sequence selected from SEQ ID NOs: 224-226, which includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid substitutions.

[0208] In some examples, the multispecific polypeptide construct is a triplespecific antigen-binding construct that includes: (a) First targeting domain that binds to NKp80 (b) A second targeting domain that binds to CD16; and (c) A third targeting domain that binds to the target antigen, Here, the targeting domain is selected from Fab fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-domain Ab(dAb) fragments, isolated CDRs, single-chain Fv(scFv), disulfide-stabilized Fv(dsFv), single-chain Ab(scAb), secretory T cell bispecific Ab(STAb), single-domain Ab(sdAb), single-domain CH antibody, single-domain CL antibody, VHH, variable domains (VNARs) of novel antigen receptors, sdAbs based on shark VNAR structures, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, finomers, avimers, anticarin, fibronectin, and binding sites incorporated into the constant region of antibodies (e.g., f-star's Modular Antibody Technology™).

[0209] In some examples, a multispecific polypeptide construct is a triplespecific antigen-binding construct that includes the following: (a) A first targeting domain that binds to NKp80, wherein the targeting domain is selected from a Fab fragment, an Fv fragment; an sdAb fragment, an isolated CDR, a scFv, a dsFv, a scAb, a STAb, an sdAb, a single domain CH antibody, a single domain CL antibody, a VHH, a VNAR, and an sdAb based on a VNAR structure from shark; (b) A first targeting domain that binds to CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO: 224), FcX (SEQ ID NO: 224), a silent Fc / inactivated mutant Fc domain (FcLALA) (SEQ ID NO: 225), or FcE (SEQ ID NO: 226); and (c) A third targeting domain that binds to a tumor-associated antigen, optionally HER2, EGFR or CD20, wherein the targeting domain is selected from a Fab fragment, an F(ab)2 fragment, an Fd fragment, an Fv fragment, a single domain Ab (dsAb) fragment, an isolated CDR, a single-chain Fv (scFv), a disulfide-stabilized Fv (dsFv), a single-chain Ab (scAb), a secreted T cell bispecific Ab (STAb), a single domain Ab (sdAb), a single domain CH antibody, and a single domain CL antibody, a VHH, a variable domain of a novel antigen receptor (VNAR), an sdAb based on a VNAR structure from shark, and a binding domain based on an alternative scaffold including but not limited to ankyrin-based domains, fibronomers, avimers, anticalins, fibronectin and binding sites incorporated into the constant region of an antibody (e.g., f-star technology (F-star's Modular Antibody TechnologyTM)).

[0210] C. Antigen Target Exemplary polypeptides of the present disclosure include at least one antigen binder. The polypeptide is not limited by the identity of the antigen binder or its binding target. The polypeptide is exemplified with reference to binders to antigens of HER2, EGFR, and / or CD20, but is not limited to these antigen binders.

[0211] In some cases, one or more antigen-targeting domains bind to a member selected from HER-2, EGFR, and CD20.

[0212] In some examples, the multispecific polypeptide construct binds to one or more of these tumor antigens. In some examples, the multispecific antigen-binding polypeptide construct is a bispecific antigen-binding polypeptide (i.e., anti-NKp80-anti-HER2) that binds to NKp80 and HER2 as disclosed herein. In some examples, the bispecific antigen-binding polypeptide construct that binds to NKp80 and EGFR is anti-NKp80-anti-EGFR as disclosed herein. In some examples, the bispecific antigen-binding polypeptide construct that binds to NKp80 and CD20 is anti-NKp80-anti-CD20 as disclosed herein.

[0213] i. HER2 ADCC mediated by NK cells plays a crucial role in anti-HER2 therapy. However, the cytotoxicity of NK cells decreases along with changes in the activated receptor phenotype in breast cancer patients. Compared to healthy donors, breast cancer patients have lower expression levels of NKp30, NKp46, and NKG2D in their NK cells. Therefore, enhancing NK cells and their ADCC effects is an effective way to improve the efficacy and sensitivity of trastuzumab.

[0214] In some examples, the multispecific polypeptide constructs include a binding fragment known in the art to bind to HER2. In some examples, the multispecific polypeptide constructs that bind to HER2 include a heavy chain variable (VH) domain encoded by a sequence including: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Trastuzumab VH; Sequence ID: 227).

[0215] In some examples, HER2-binding multispecific polypeptide constructs include a light chain variable (VL) domain encoded by a sequence containing the following: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Trastuzumab VL; Sequence ID: 228).

[0216] In some examples, the HER2-binding multispecific polypeptide constructs include a constant heavy chain (CH) domain encoded by a sequence containing the following: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (Trastuzumab CH1; Sequence ID: 229).

[0217] In some examples, the HER2-binding multispecific polypeptide constructs include a constant light chain (CL) domain encoded by a sequence containing the following: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Trastuzumab CL; SEQ ID NO: 230).

[0218] In some examples, unless otherwise specified, the domain order expressions illustrated in this disclosure do not limit the general structure of the multispecific polypeptide constructs of this disclosure. Therefore, when a multispecific polypeptide construct is referred to as anti-NKp80-anti-HER2, this disclosure refers to an example where the anti-NKp80 arm of the multispecific polypeptide construct is either N-terminal or C-terminal, and anti-HER2 is at the other end of the anti-NKp80 arm (i.e., if anti-NKp80 is N-terminal, anti-HER2 is C-terminal, or if anti-NKp80 is C-terminal, anti-HER2 is N-terminal). These permutations are illustrated in Figure 13C with an EGFR-targeted multispecific polypeptide construct.

[0219] In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and HER2, in which the Fc domain is functional, is the anti-HER2-anti-NKp80-Fc disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and HER2, in which the Fc domain has reduced binding to CD16, is the anti-HER2-anti-NKp80-FcX disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and HER2, in which the Fc domain has enhanced binding to CD16, is the anti-HER2-anti-NKp80-FcE disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and HER2, in which the Fc domain is an inactivating variant / silent domain (FcLALA), is the anti-HER2-anti-NKp80-FcLALA disclosed herein.

[0220] In some cases, the humanized antibody clones anti-HER2-anti-NKp80(45-2)-Fc, anti-HER2-anti-NKp80(101-1)-Fc, anti-HER2-anti-NKp80(94-1)-Fc, and anti-HER2-anti-NKp80(87-2)-Fc are derived from the mother clones anti-HER2-anti-NKp80(45)-Fc, anti-HER2-anti-NKp80(101)-Fc, anti-HER2-anti-NKp80(94)-Fc, and anti-HER2-anti-NKp80(87)-Fc, respectively (Figure 3).

[0221] In some cases, the triple-specific antigen-binding polypeptide constructs (anti-HER2-anti-NKp80(94)-Fc, anti-HER2-anti-NKp80(101)-Fc, anti-HER2-anti-NKp80(45)-Fc, and / or anti-HER2-anti-NKp80(87)-Fc) engaged both NKp80 and CD16 in OVCAR3 with improved efficacy compared to trastuzumab (anti-HER2-anti-NKp80-Fc vs anti-HER2-Fc) (Figure 3A). Clonal anti-HER2-anti-NKp80(87)-Fc showed improved efficacy in four different cell lines tested (MKN1, OVCAR3, HCT116, MDA-MB-231) (Figure 4). In some cases, selected humanized clones of multispecific polypeptide constructs exhibit improved binding and / or cytotoxicity to cancer cells compared to the parent polypeptide constructs, while showing lower immunogenicity.

[0222] In the experimental data provided herein, MRC-5, a lung fibroblast cell line with low HER2 expression, was used as a control for tumor cell lines. The antigen-binding polypeptide construct, anti-HER2-Fc (trastuzumab), bound to MRC-5, suggesting that the anti-HER2 arm of the antigen-binding polypeptide construct described herein recognizes HER2 on MRC-5. However, the triply specific antigen-binding polypeptide construct (anti-HER2-anti-NKp80-Fc) disclosed herein showed cytotoxic specificity only in cancer cells, and MRC-5 was not killed. In some cases, multispecific polypeptide constructs specifically kill cancer cells but do not show cytotoxicity in non-cancer cells.

[0223] In some cases, co-inclusion of NKp80 and CD16 in the triple-specific antigen-binding polypeptide construct anti-HER2-anti-NKp80(87-2)-Fc enhanced NK cell function without affecting T cell activation (Figure 5). Therefore, the antigen-binding polypeptide constructs disclosed herein are specific to NK cells. In some cases, the antigen-binding polypeptide constructs disclosed herein do not induce T cell activation.

[0224] As shown in the experimental data of this disclosure, clone HER2-NKp80(87-2)-CD16 consistently demonstrated improved potency (mean EC50) compared to trastuzumab (Figure 4; Table 1, Column 4).

[0225] In some cases, the NKp80 engager contains clonal anti-HER2-anti-NKp80(87)-Fc. In some cases, the antigen-binding polypeptide construct has an average EC50 multiplier change of about 1 to about 1000. In some cases, the antigen-binding polypeptide construct has an average EC50 multiplier 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.

[0226] ii. EGFR In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and EGFR, in which the Fc domain is functional, is the anti-EGFR-anti-NKp80-Fc disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and EGFR, in which the Fc domain has reduced binding to CD16, is the anti-EGFR-anti-NKp80-FcX disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and EGFR, in which the Fc domain has enhanced binding to CD16, is the anti-EGFR-anti-NKp80-FcE disclosed herein. In some cases, a triplicate, multispecific polypeptide construct that binds to NKp80, CD16, and EGFR, in which the Fc domain is an inactivating variant / silent domain (FcLALA), is the anti-EGFR-anti-NKp80-FcLALA disclosed herein.

[0227] In some examples, the multispecific polypeptide constructs described herein include a variable light chain amino acid sequence selected from SEQ ID NOs: 183-202 as described above, where the trastuzumab VL sequence (SEQ ID NO: 228) is substituted with the following cetuximab VL sequence: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (Cetuximab VL; SEQ ID NO: 232).

[0228] In some examples, the EGFR-binding multispecific polypeptide constructs include a constant light chain (CL) domain encoded by a sequence containing the following: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAEC (Cetuximab CL; SEQ ID NO: 234).

[0229] In some examples, the multispecific polypeptide constructs include a binding fragment known in the art to bind to EGFR. In some examples, the multispecific polypeptide constructs that bind to EGFR include a heavy chain variable (VH) domain encoded by a sequence including: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (Cetuximab VH; Sequence ID: 231).

[0230] In some examples, the EGFR-binding multispecific polypeptide constructs include a constant heavy chain (CH) domain encoded by a sequence containing the following: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (Cetuximab CH1; Sequence ID: 233).

[0231] In the experimental data presented herein, isotypes were used as untargeted controls that do not recognize the target antigen.

[0232] As shown in the experimental data of this disclosure, clonal anti-EGFR-anti-NKp80(87)-Fc was more potent than cetuximab (anti-EGFR-Fc) in the cytotoxic killing assay (Figure 6). The control in the cytotoxic killing assay (isotype-anti-NKp80(87)-Fc) did not show NK cell cytotoxicity against HER2-positive cell lines, suggesting that cytotoxicity is antigen-dependent (Figure 7).

[0233] iii. CD20 In some examples, a triplicate, multispecific polypeptide construct (where the Fc domain is functional) that binds NKp80, CD16, and CD20 is the anti-CD20-anti-NKp80-Fc disclosed herein. In some examples, a triplicate, multispecific polypeptide construct that binds NKp80, CD16, and CD20, in which the Fc domain has reduced binding to CD16, is the anti-CD20-anti-NKp80-FcX disclosed herein. In some examples, a triplicate, multispecific polypeptide construct that binds NKp80, CD16, and CD20, in which the Fc domain has enhanced binding to CD16, is the anti-CD20-anti-NKp80-FcE disclosed herein. In some cases, triplicate and multispecific polypeptide constructs that bind to NKp80, CD16, and CD20, wherein the Fc domain is an inactivating mutant / silent domain (FcLALA), are the anti-CD20-anti-NKp80-FcLALA disclosed herein.

[0234] In some examples, the multispecific polypeptide constructs described herein include a variable light chain amino acid sequence selected from SEQ ID NOs: 183-202 as described above, where the trastuzumab VL sequence (SEQ ID NO: 228) is substituted with the following rituximab VL sequence: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (Rituximab VL; Sequence ID: 243).

[0235] In some examples, the multispecific polypeptide constructs that bind to CD20 include a constant light chain (CL) domain encoded by a sequence containing the following: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Rituximab CL; SEQ ID NO: 245).

[0236] In some examples, the multispecific polypeptide construct includes a binding fragment known in the art to bind to CD20. In some examples, the multispecific polypeptide construct that binds to CD20 includes a heavy chain variable (VH) domain encoded by a sequence including: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (Rituximab VH; Sequence ID: 244).

[0237] In some examples, the multispecific polypeptide constructs that bind to CD20 include a constant heavy chain (CH) domain encoded by a sequence containing the following: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC (Rituximab CH1; Sequence ID: 246).

[0238] D. Innate immune cell engagers In some examples, the multispecific polypeptide construct includes innate immune cell engagers, which include, but are not limited to, natural killer cells (NK cells), macrophages, dendritic cells, eosinophils, basophils, neutrophils, mast cells, and natural killer T cells (NKT cells). In some examples, when the multispecific polypeptide construct is a multispecific antigen-binding polypeptide, the multispecific polypeptide construct described herein includes multiple antigen-targeting domains. In some examples, each of these targeting domains binds to or recognizes an innate immune cell modulator or target antigen. In some examples, each targeting domain of the multispecific polypeptide construct includes 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 described herein. In some examples, the multispecific polypeptide construct includes a combination of one or more CDRs described herein.

[0239] In some examples, the antigen-binding polypeptide constructs described herein include multiple antigen-targeting domains. Each of these targeting domains binds to or recognizes an NK modulator or target antigen. Thus, each targeting domain of a multispecific polypeptide construct includes 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 described herein. In some examples, the multispecific polypeptide construct includes a combination of one or more CDRs described herein. In some examples, the multispecific polypeptide construct binds to one NK modulator, e.g., NKp80. In some examples, the multispecific polypeptide construct is an antigen-binding polypeptide containing 1 to 6 CDRs that bind to NKp80 as described herein.

[0240] In another aspect, a method for producing a multispecific polypeptide construct or antibody disclosed herein is provided, comprising culturing host cells and optionally isolating a multispecific polypeptide construct from the host cells and / or culture medium.

[0241] In another context, a method is provided for screening and / or identifying multispecific polypeptide constructs or antibodies disclosed herein, in which the NK cell targeting domain is anti-NKp80.

[0242] In some examples, the present disclosure provides methods for screening and / or identifying NKp80 binders, non-binders, inactivated binders, activated binders, and / or engagers. In some examples, screening and / or identification of NKp80 engagers involves using biolayer interferometry (BLI) and the Xcelligence® cytotoxic killing assay.

[0243] In some examples, the multispecific polypeptide construct includes one or more CDRs selected from the group consisting of SEQ ID NOs: 1 to 50 (Figure 10), or fragments thereof, or a light chain variable region (VL) having sequences that 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% homology with them, and / or sequences having two or three amino acid substitutions. In some examples, the multispecific polypeptide constructs include one or more CDRs selected from the group consisting of SEQ ID NOs: 51-104 (Figure 10), or fragments thereof, or heavy chain variable regions (VHs) having sequences that share at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or sequences having two or three amino acid substitutions. In some examples, the multispecific polypeptide constructs include one or more framework regions (FRs) selected from the group consisting of SEQ ID NOs: 105-140 (Figure 11), or fragments thereof, or light chain variable regions (VLs) having sequences that share 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 with them, and / or sequences having two or three amino acid substitutions.In some examples, the multispecific polypeptide constructs include one or more framework regions (FRs) selected from the group consisting of SEQ ID NOs: 141-182 (Figure 11), or fragments thereof, or heavy chain variable regions (VHs) having sequences that share at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or sequences having two or three amino acid substitutions.

[0244] In some examples, the present disclosure provides a method for screening and / or identifying a multispecific polypeptide construct, the multispecific polypeptide construct comprising an NKp80 engager. In some examples, the screening and / or identification of the NKp80 engager comprises using biolayer interference (BLI) and the Xcelligence® cytotoxic killing assay. In some examples, the multispecific polypeptide construct binds to a single NK modulator, e.g., NKp80. In some examples, the multispecific polypeptide construct is an antigen-binding polypeptide comprising 1 to 6 CDRs that bind to NKp80 as described herein. In some examples, the multispecific polypeptide constructs include one or more CDRs selected from the group consisting of SEQ ID NOs: 1-50 (Figure 10), or fragments thereof, or sequences sharing at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or light chain variable regions having sequences with two or three amino acid substitutions. The heavy chain variable region (VH) is bound to one or more CDRs selected from the group consisting of SEQ ID NOs. 51 to 104 (Figure 10), or fragments thereof, or sequences that share at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or sequences having two or three amino acid substitutions.

[0245] In some examples, the multispecific polypeptide construct comprises a sequence that is at least 80% identical to any one of the sequences disclosed herein. In some examples, the multispecific polypeptide construct comprises a target binding site or CDR containing a sequence that 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%, 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 with any of the sequences disclosed herein. In some examples, the sequences 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.

[0246] In some examples, a multispecific 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, a multispecific polypeptide construct comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, one or more amino acid mutations are selected independently of substitutions, insertions, deletions, and shortenings. In some examples, the amino acid mutations are amino acid substitutions and include conserved substitutions and / or non-conserved substitutions.

[0247] In some examples, substitutions involve non-classical amino acids. In some examples, non-classical amino acids are selected from designer amino acids such as selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and amino acid analogs in general.

[0248] In some cases, the modification of the amino acid sequence is achieved using any known technique in the art, e.g., site-directed mutagenesis or PCR-based mutagenesis. In some cases, the mutation does not substantially reduce the ability of the antigen-binding polypeptide construct to specifically bind to the target. In some cases, the mutation does not substantially reduce the ability of the antigen-binding polypeptide construct to specifically bind to the target, and does not reduce the ability of the antigen-binding polypeptide construct without functionally modifying (e.g., partially or completely neutralizing) the target.

[0249] In some examples, the binding affinity of the multispecific polypeptide constructs of the present disclosure to the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or monomer and / or dimer and / or other naturally occurring or synthetic analogs, variants or variants (including monomer and / or dimer forms) of the multispecific polypeptide constructs is equal to the equilibrium dissociation constant (K d) is described by. In some examples, a multispecific polypeptide construct is described by the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or other naturally occurring or synthetic analogs, variants or variants (including monomeric and / or dimeric forms) of the multispecific polypeptide construct, and in concentrations of about 1 μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5 nM, or less than about 1 nM (K d They are joined together using ).

[0250] In one preferred embodiment, co-inclusion of NKp80 and CD16 in the triplicate multispecific polypeptide construct anti-HER2-anti-NKp80(87-2)-Fc enhanced NK cell function without affecting T cell activation (Figure 5). Therefore, the multispecific polypeptide constructs disclosed herein are specific to NK cells. In some examples, the multispecific polypeptide constructs disclosed herein do not induce T cell activation.

[0251] In some examples, triplicate and multispecific polypeptide constructs containing the NKp80 engager include clonal anti-HER2-anti-NKp80(13)-FcX, anti-HER2-anti-NKp80(28)-FcX, anti-HER2-anti-NKp80(36)-FcX, anti-HER2-anti-NKp80(37)-FcX, anti-HER2-anti-NKp80(45)-FcX, anti-HER2-anti-NKp80(50)-FcX, anti-HER2-anti-NKp80(51)-FcX, anti-HER2-anti-NKp80(63)-FcX, anti-HER2-anti-NKp80(71)-FcX, and anti HER2-anti-NKp80(74)-FcX, anti-HER2-anti-NKp80(78)-FcX, anti-HER2-anti-NKp80(79)-FcX, anti-HER2-anti-NKp80(81)-FcX, anti-HER2-anti-NKp80(82)-FcX, anti-HER2-anti-NKp80(83)- FcX, anti-HER2-anti-NKp80(87)-FcX, anti-HER2-anti-NKp80(94)-FcX, anti-HER2-anti-NKp80(101)-FcX, anti-HER2-anti-NKp80(102)-FcX, and / or anti-HER2-anti-NKp80(106)-FcX.

[0252] As demonstrated by the experimental data of this disclosure, the triplicate and multispecific polypeptide constructs (anti-HER2-anti-NKp80(94)-Fc, anti-HER2-anti-NKp80(101)-Fc, anti-HER2-anti-NKp80(45)-Fc, and / or anti-HER2-anti-NKp80(87)-Fc) co-retract both NKp80 and CD16 in OVCAR3 with improved efficacy compared to trastuzumab (anti-HER2-anti-NKp80-Fc vs anti-HER2-Fc) (Figure 3A). Clonal anti-HER2-anti-NKp80(87)-Fc shows improved efficacy in four different cell lines tested (MKN1, OVCAR3, HCT116, MDA-MB-231) (Figure 4) (see Example 3).

[0253] i. Immunoglobulins The primary effector function of IgG antibodies is antibody-dependent cytotoxicity (ADCC), where antibody-coated antigens activate effector cells such as NK cells and monocytes by binding to FcγR, thereby destroying the antibody-coated target. ADCC activity is highly dependent on the glycosylation of IgG and, further, on the net outcome of the binding of activated and inhibited FcγR.

[0254] In some examples, an Fc polypeptide contains an Fc domain, e.g., the polypeptide that makes up monomer Fc. In some examples, an Fc polypeptide is obtained from any suitable immunoglobulin, e.g., human IgG1, IgG2, IgG3, or IgG4 subtype, IgA, IgE, IgD, or IgM. In some examples, an Fc polypeptide is obtained from humans or any other non-human mammals. In some examples, the Fc domain contains the carboxyl-terminal portions of both H chains held together by a disulfide. In some examples, the effector function of the antibody is determined by the sequence of the Fc domain. This region is also the part recognized by an Fc receptor (FcR) found on certain cells.

[0255] In some cases, the native / wild-type Fc domain (FcX) results in a reduction in ADCC. This means that the measurable ADCC response is reduced by at least approximately 10%, or at least approximately 20%, or at least approximately 30%, or at least approximately 40%, or at least approximately 50%, or at least approximately 60%, or at least approximately 70%, or at least approximately 80%, or at least approximately 90%, or at least approximately 95%, or at least approximately 99% compared to the control. In some cases, Fc silent / inactivating mutant Fc domain (FcLALA) confers little to no measurable ADCC, which means substantially completely silencing 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 the measurable ADCC response, or substantially complete silencing of ADCC to the extent that no measurable ADCC is detected. In some cases, enhanced ADCC means an improvement, increase, or amplification 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 the measurable ADCC response compared to the control.

[0256] In some cases, multispecific polypeptide constructs bind to a second innate immune cell modulator via an Fc domain. In some cases, the second innate immune cell modulator-binding domain of the multispecific polypeptide construct is selected from native / wild-type Fc domains (FcWT), Fc-enhancing (FcE) domains, Fc-decreased (FcX) domains, Fc-silent domains / inactivating variants of Fc domains (FcLALA), Fc-mutant domains, heterodimer Fc domains, and the like. The terms “region” and “domain” as used herein are understood to represent the same component and can therefore be used interchangeably.

[0257] In some examples, the multispecific polypeptide constructs include an Fc domain. In some examples, the Fc domain includes a sequence containing the following: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Native / wild-type Fc domain; Sequence ID: 224).

[0258] In some examples, the multispecific polypeptide construct includes a mutant Fc domain. In some examples, the mutant Fc domain is a degraded Fc domain (FcX). In some examples, the degraded Fc domain is constructed according to methods known in the art. In some examples, the degraded Fc domain includes an amino acid sequence comprising: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Decreased Fc domain (FcX); Sequence ID: 224).

[0259] In some cases, the mutant Fc domain may be a silent Fc domain / inactivating variant. In some cases, the silent Fc domain / inactivating variant of the Fc domain includes a sequence containing the following: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Silent Fc domain / Inactivating mutant Fc domain (Fc LALA)); Sequence ID: 225).

[0260] In some cases, the mutant Fc domain may be an enhancing Fc domain. In some cases, the enhancing Fc domain contains a sequence that includes the following: DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Enhanced Fc Domain (FcE): Sequence ID: 226).

[0261] In some examples of this disclosure, the NK cell engager includes a native / wild-type Fc domain and / or a mutant Fc domain. The mutant Fc domain (or Fc mutant region) includes an amino acid sequence different from the native / wild-type sequence Fc domain by at least one amino acid modification, preferably one or more amino acid substitutions. In some examples, the mutant Fc domain has at least one amino acid substitution compared to the native / wild-type sequence Fc domain or the Fc domain of the parent polypeptide. For example, the mutant Fc domain (or Fc mutant domain) may include 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 the native / wild-type sequence Fc domain. The mutant Fc domains described 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% homology with the native / wild-type sequence Fc region.

[0262] In some examples, the "Fc domain" includes the hinge region, CH2 domain, or CH3 domain of the Fc region.

[0263] ii. CDR and FR Each domain of a naturally occurring antibody has a structure characterized by an “immunoglobulin fold” formed from two β-sheets (e.g., 3, 4, or 5-chain sheets) packed together in a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops known as “complementarity-determining regions” (CDR1, CDR2, CDR3) and four somewhat invariant “framework regions” (FR1, FR2, FR3, FR4). When a naturally occurring antibody folds, the FRs form the β-sheets that constitute the structural backbone of the domain, and the heavy and light chain CDR loop regions come together in three-dimensional space to form a single hypervariable antigen-binding site located at the tip of a Y structure. The Fc domains of naturally occurring antibodies bind to elements of the complement system and also to receptors on effector cells, such as effector cells that mediate cytotoxicity (U.S. Patent Application Publication 2022 / 0040231, which is incorporated herein by reference in its entirety).

[0264] 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,” and “Variable Heavy Chain CDR3” refer to VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3, respectively. In some examples, when the multispecific polypeptide construct is a multispecific antigen-binding polypeptide, the multispecific polypeptide construct described herein includes multiple antigen-targeting domains. Each of these targeting domains binds to or recognizes an innate immune cell modulator or target antigen. In some examples, each targeting domain of the multispecific polypeptide construct includes 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 multispecific polypeptide construct includes a combination of one or more CDRs as described herein.

[0265] In some examples, the multispecific polypeptide construct binds to a single modulator, such as NKp80. In some examples, the multispecific polypeptide construct is an antigen-binding polypeptide construct comprising 1 to 6 CDRs bound to NKp80, as described herein. In some examples, the multispecific polypeptide construct comprises one or more CDRs selected from the group consisting of sequences from Figure 10, or fragments thereof, or a light chain variable region (VL) having sequences that share at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or sequences having 2 or 3 amino acid substitutions.

[0266] In some examples, the multispecific polypeptide construct binds to a single modulator, e.g., NKp80. In some examples, the multispecific polypeptide construct is an antigen-binding polypeptide construct comprising 1 to 6 CDRs bound to NKp80, as described herein. In some examples, the multispecific polypeptide construct comprises one or more CDRs, or fragments thereof, selected from the group consisting of sequences from Figure 10, or sequences sharing at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with them, and / or sequences having 2 or 3 amino acid substitutions.

[0267] In another context, an antigen-binding protein, or an antigen-binding fragment thereof, comprising a CDR sequence selected from the following, is provided: (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 (sequence number: 6), VLCDR2 (sequence number: 18), VLCDR3 (sequence number: 38), VHCDR1 (sequence number: 57), VHCDR2 (sequence number: 73), and VHCDR3 (sequence number: 92) (clone 71); (9) VLCDR1 (sequence number: 7), VLCDR2 (sequence number: 23), VLCDR3 (sequence number: 39), VHCDR1 (sequence number: 57), VHCDR2 (sequence number: 74), and VHCDR3 (sequence number: 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 (sequence number: 9), VLCDR2 (sequence number: 25), VLCDR3 (sequence number: 41), VHCDR1 (sequence number: 59), VHCDR2 (sequence number: 76), and VHCDR3 (sequence number: 95) (clone 79); (12) VLCDR1 (SEQ ID NO: 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 NO: 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), Here, the CDR sequence shares at least approximately 90% homology with an amino acid sequence selected from SEQ ID NOs: 1-104, 247-252, and / or Here, the CDR sequence selected from sequence numbers 1-104 and 247-252 contains two or three amino acid substitutions.

[0268] In some examples, an antigen-binding protein or its antigen-binding fragment is provided, containing a CDR sequence selected from the following: (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 (sequence number: 6), VLCDR2 (sequence number: 18), VLCDR3 (sequence number: 38), VHCDR1 (sequence number: 57), VHCDR2 (sequence number: 73), and VHCDR3 (sequence number: 92) (clone 71); (9) VLCDR1 (sequence number: 7), VLCDR2 (sequence number: 23), VLCDR3 (sequence number: 39), VHCDR1 (sequence number: 57), VHCDR2 (sequence number: 74), and VHCDR3 (sequence number: 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 (sequence number: 9), VLCDR2 (sequence number: 25), VLCDR3 (sequence number: 41), VHCDR1 (sequence number: 59), VHCDR2 (sequence number: 76), and VHCDR3 (sequence number: 95) (clone 79); (12) VLCDR1 (SEQ ID NO: 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 NO: 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), Here, the CDR sequence shares 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 with an amino acid sequence selected from SEQ ID NOs: 1-104, 247-252, and / or Here, the CDR sequence selected from sequence numbers 1-104 and 247-252 contains two or three amino acid substitutions.

[0269] In another context, an antigen-binding protein, or an antigen-binding fragment thereof, comprising CDR and FR sequences selected from the following, is provided: (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 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 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 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 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 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 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); (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); (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), Here, the FR and CDR sequences share at least approximately 90% homology with amino acid sequences selected from SEQ ID NOs: 1-104, 247-260, and / or Here, the FR and CDR sequences selected from sequence numbers 1-104 and 247-260 contain two or three amino acid substitutions.

[0270] In some examples, an antigen-binding protein or its antigen-binding fragment is provided, comprising CDR and FR sequences selected from the following: (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 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 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 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 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 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 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); (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); (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), Here, 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 with an amino acid sequence selected from SEQ ID NOs: 1-104, 247-260, and / or Here, the FR and CDR sequences selected from sequence numbers 1-104 and 247-260 contain two or three amino acid substitutions.

[0271] iii. Light chains and heavy chains The maturation variable region of each light / heavy chain pair forms the antibody binding site. Therefore, an intact antibody has two binding sites. Except for multispecific antibodies, the two binding sites are identical. All chains exhibit the same general structure: a relatively conserved framework region (FR) linked by three hypervariable regions, also called complementarity-determining regions (CDRs). The CDRs of the two chains in each pair are aligned by the framework region, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chains consist of domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the Kabat definition, Sequences of Polypeptide constructs of Immunological Interest. Kabat also provides a widely used numbering rule (Kabat numbering) where corresponding residues are assigned the same number between different heavy chains or between different light chains. In contrast, in multispecific antigen-binding polypeptide constructs, the binding sites of each multispecific polypeptide construct are distinct. In other words, in a dual-functional or dual-specificity multispecific polypeptide construct, the multispecific polypeptide construct has two different binding sites, etc.

[0272] The binding fragment is selected from a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), an F(ab)2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge in 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, an scAb, an 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 sharks, and binding domains based on alternative scaffolds including but not limited to ankyrin-based domains, finomers, avimers, anticalins, fibronectin, and binding sites incorporated into the constant regions of antibodies (e.g., F-star's Modular Antibody TechnologyTM). A single-domain antibody in which one chain is separated from its natural partner may sometimes be known as Dabs. Whether or not a constant region or a part of a constant region is included in a single-domain antibody is not a concern. In some examples, the antigen-targeting domain is selected from a Fab fragment, an F(ab)2 fragment, an Fd fragment, an Fv fragment, a dAb, an isolated CDR, an scFv, a dsFv, an scAb, an STAb, an sdAb, a CH domain, a CL domain, a VHH, a VNAR, an sdAb derived from VNAR, an ankyrin-based domain, a finomer, an avimer, a fibronectin domain, and an F-star's Modular Antibody TechnologyTM domain.

[0273] The polypeptide constructs disclosed herein typically have at least 10 6 、10 7 、10 8 、10< 9 、or 10 10The antibody binds to its designated target with an association constant of M. Such binding is specific in that it is detectable and distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding occurs as a result of bond formation between specific functional groups or specific spatial fit (e.g., lock-and-key type), while nonspecific binding usually arises from van der Waals forces. Specific binding does not necessarily mean that the antibody binds to only one target. In some examples, multispecific polypeptide constructs bind specifically to one or more antigens.

[0274] In some examples, the antigen-binding polypeptide constructs described herein include an NKp80-binding VL having a sequence selected from the group consisting of SEQ ID NOs: 183-202 (Figure 13A). AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKLASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTEVVVK (VL sequence of anti-NKp80(13); Sequence ID: 183); DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVVVK (VL sequence of anti-NKp80(28); Sequence ID: 184); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTTRSSSIYWPFGGGTEVVVK (VL sequence of anti-NKp80(36); Sequence ID: 185); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYTAYTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEVVVK (VL sequence of anti-NKp80(37); Sequence ID: 186); DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTLESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGGTEVVVK (VL sequence of anti-NKp80(45); Sequence ID: 187); AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEVVVK (VL sequence for anti-NKp80(50); SEQ ID NO: 188); ALVMTQTPSSVSAAVGGTVTIKCQASQSIGNDLAWYQQKPGQPPKLLIYAASNLESGVPSRFRGSGSGTKFTLTISDLECADAATYYCQGTYRGSSISWPFGGGTEVVVK (VL sequence of anti-NKp80(51); Sequence ID: 189); QIVVTQTPASVSAAVGGTVTISCQSSQNVYGNNELSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGYSGGMRSFGGGTEVVLV (VL sequence of anti-NKp80(71); Sequence ID: 190); QIVVTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLASTLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTEVVVK (VL sequence of anti-NKp80(74); Sequence ID: 191); AQVLTQTASSVSAAVGGTVTISCQSSQSVYNYNWLGWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSVDCNVFGGGTEVVVK (VL sequence for anti-NKp80(78); SEQ ID NO: 192); ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTKVVVK (VL sequence of anti-NKp80(79); Sequence ID: 193); AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLASGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEVVVK (VL sequence of anti-NKp80(81); Sequence ID: 194); DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSKCVFPFGGGTEVVVK (VL sequence for anti-NKp80(82); SEQ ID NO: 195); DIVMTQTPASVEAAVGGTVTIKCQASQSIGRDLAWYQQKPGQPPKLLIYGASILESGVPSRFKGNGSGTQFTLTISDLECADAATYYCQGADRSSTPSWPFGGGTEVVVK (VL sequence of anti-NKp80(87); Sequence ID: 196); AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKTSSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTEVVVK (VL sequence for anti-NKp80(94); Sequence ID: 197); AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTEVVVK (VL sequence for anti-NKp80(101); SEQ ID NO: 198); DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTEVVVK(VL sequence of anti-NKp80(102); Sequence ID: 199); DVVMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTLESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTEVVVK (VL sequence of anti-NKp80(106); Sequence ID: 200); DIVMTQTPASVSAAAGGTVTINCQASQSISNELSWYQQKSGQPPKLLIYGASNLESGVPSRFKGSGSGTDFTLTISDLECADGATYYCQSNYYDSSSPDFAFGGGTEVVVK(VL sequence of anti-NKp80(63); SEQ ID NO: 201); and DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSNCVFPFGGGTEVVVK (VL sequence for anti-NKp80(83); Sequence ID: 202).

[0275] In some examples, the antigen-binding polypeptide constructs described herein include NKp80-binding VH having a sequence selected from the group consisting of (SEQ ID NOs: 203-222) (Figure 13B). QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIYTGGGSADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGYGDATDIWGPGTLVTV(VH sequence of anti-NKp80(13); SEQ ID NO: 203); QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYAGSSGSTHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDYFKIWGPGTLVTV (VH sequence of anti-NKp80(28); Sequence ID: 204); QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRYNIWGPGTLVTV (VH sequence of anti-NKp80(36); Sequence ID: 205); QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRYNIWGPGTLVTV (VH sequence of anti-NKp80(37); Sequence ID: 206); QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWRYNIWGPGTLVTV (VH sequence of anti-NKp80(45); Sequence ID: 207); QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWRYNIWGPGTLVTV (VH sequence of anti-NKp80(50); Sequence ID: 208); QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYSDSGNTYYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRYNIWGPGTLVTV (VH sequence of anti-NKp80(51); Sequence ID: 209); QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSPGVAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWGQGTLVTV (VH sequence of anti-NKp80(71); Sequence ID: 210); QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSGGSAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWGQGTLVTV (VH sequence of anti-NKp80(74); Sequence ID: 211); QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNGGATYYASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLWGQGTLVTV (VH sequence for anti-NKp80(78); Sequence ID: 212); QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPSGSIWYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWGQGTLVTV (VH sequence of anti-NKp80(79); Sequence ID: 213); QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIADKIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQGTLVTV (VH sequence of anti-NKp80(81); Sequence ID: 214); QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYFASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLWGQGTLVTV (VH sequence of anti-NKp80(82); Sequence ID: 215); QSLEEYGGDVVQPEGSLTLTCTASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWKYNIWGPGTLVTV (VH sequence of anti-NKp80(87); Sequence ID: 216); QSVEESGGRLVTPGTPLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTGGSAYYANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWGQGTLVTV (VH sequence of anti-NKp80(94); Sequence ID: 217); QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISWGGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDPWGPGTLLTV (VH sequence for anti-NKp80(101); (Sequence ID: 218); QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSGGDTSYATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLWGQGTLVTV (VH sequence for anti-NKp80(102); SEQ ID NO: 219); QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDGSAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWGQGTLVTV (VH sequence of anti-NKp80(106); Sequence ID: 220); QSLEESGGRLVKPDETLTITCTVSGIDLSSYIISWVRQAPGEGLEYIGFINTDGSAYYATWAKGRFTISRTSATVDLKMTSLTTEDTATYFCARDAGHRYLFYFKLWGQGTLVTV (VH sequence of anti-NKp80(63); Sequence ID: 221); and QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLWGQGTLVTV (VH sequence for anti-NKp80(83); Sequence ID: 222).

[0276] With respect to each of the sequences described herein above, in some examples, one or more of the sequences described 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 with any of the sequences disclosed herein. In some examples, the sequences disclosed herein have one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty, or more amino acid substitutions.

[0277] E. Diseases In some cases, a multispecific polypeptide construct binds to a target antigen, which is a disease-related antigen. In some cases, the target antigen is a disease-related molecule. In some cases, the target antigen molecule is selected from extracellular molecules, intracellular molecules, and transmembrane molecules. In some cases, the molecule is selected from polypeptides, polynucleotides, carbohydrates, etc. In some cases, the disease is a proliferative disorder selected from proliferative disorders (tumors / cancer, inflammatory diseases, etc.), infectious diseases, autoimmune diseases, autoimmune disorders, etc. In some cases, the disease is a tumor / cancer.

[0278] In some cases, the disease is selected from proliferative disorders (such as cancer), infectious diseases, autoimmune diseases, and autoimmune disorders. In some cases, the disease is a tumor.

[0279] In some examples, the targets include tumor antigens selected from, but not limited to, HER2 and EGFR. In some examples, the multispecific polypeptide construct binds to one or more of these tumor antigens. In some examples, the multispecific polypeptide construct binds to target cells, which include, but are not limited to, tumor cells and cancer cells. In some examples, tumor cells or cancer cells express HER2 and / or EGFR.

[0280] In some cases, tumor cells, or cancer cells, include bladder cancer cells, breast cancer cells, cervical cancer cells, bile duct cancer cells (extrahepatic or intrahepatic), colorectal cancer cells, esophageal cancer cells or gastroesophageal junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck cancer cells, hepatocellular carcinoma cells, intestinal (minor) malignant tumor cells, lung cancer cells (non-small cell), 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, cancer cells of unknown primary origin, or uterine cancer cells.

[0281] In some cases, multispecific polypeptide constructs bind to tumor cells, including bladder cancer cells, breast cancer cells, cervical cancer cells, cholangiocarcinoma cells (extrahepatic or intrahepatic), colorectal cancer cells, esophageal cancer cells or gastroesophageal junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck cancer cells, hepatocellular carcinoma cells, intestinal (small) malignant tumor cells, lung cancer cells (non-small cell), 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, cancer cells of unknown primary origin, or uterine cancer cells. In some cases, multispecific polypeptide constructs bind to cells such as immortalized cell lines and primitive cells, but are not limited to these. In some cases, multispecific polypeptide constructs bind to cancer cell lines such as immortalized cell lines. In some examples, the multispecific polypeptide constructs bind to cancer cell lines such as MKN1, OVCAR3, HCT116, MDA-MB-231, N87, and RAJI, but are not limited to these.

[0282] In some cases, a disease is an infectious disease. In some cases, an infectious disease is caused by a bacterial pathogen and / or a viral pathogen. In some cases, a multispecific polypeptide construct binds to one or more bacterial and / or viral antigens.

[0283] In some cases, the disease is an autoimmune disease or autoimmune disorder. In some cases, an autoimmune disease / disorder includes any disorder, condition, or disease in which the immune system reacts against its own cells or tissues, resulting from a breakdown in its ability to distinguish between self and non-self, among other reasons.

[0284] i. Diagnostic drugs In some examples, the present disclosure includes a method for detecting a disease in a subject where such detection is required, the method comprising contacting a sample obtained from the subject with a multispecific polypeptide construct or composition described herein.

[0285] In some examples, the sample is a biological sample obtained from a biological subject, including a sample of biological tissue or body 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 biopsy. In another embodiment, the fluid biological sample or liquid biological sample is selected from blood, serum, plasma, sputum, lavage fluid (such as peritoneal lavage), cerebrospinal fluid, urine, semen, sweat, tears, saliva, etc. As used herein, the terms “blood,” “plasma,” and “serum” include their fractions or processed portions. Similarly, if the sample is taken from a biopsy, swab, smear, etc., “sample” includes the processed fraction or portion derived from the biopsy, swab, smear, etc.

[0286] ii. Pharmaceutical compositions In some examples, this disclosure includes compositions comprising multispecific polypeptide constructs described herein. In some examples, this disclosure includes pharmaceutical compositions comprising multispecific polypeptide constructs described herein and suitable pharmaceutical compositions thereof. In some examples, this disclosure includes compositions or pharmaceutical compositions, said compositions being preventive and / or therapeutic compositions.

[0287] In some examples, pharmaceutically acceptable agents for use in the pharmaceutical composition are selected from carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonics, cosolvents, wetting agents, complexing agents, buffers, antimicrobial agents, and surfactants.

[0288] In some cases, the compositions described herein are used for therapeutic / pharmaceutical purposes. In some cases, the compositions described herein further comprise excipients and / or stabilizers. In some cases, the compositions described herein are used as monotherapies and / or multispecific polypeptide constructs in combination with disease-targeted therapies such as NK cell therapy, T cell checkpoint inhibitor therapy, and small molecule therapies that can stimulate NK cells to enhance the antitumor response, but are not limited to these uses.

[0289] In some examples, the present disclosure provides a method for preventing and / or treating a disease in a subject where such treatment is needed, the method comprising administering one of the multispecific polypeptide constructs or compositions of the preceding examples to a subject. In some examples, the present disclosure provides for the use of the multispecific polypeptide constructs described herein in the manufacture of a pharmaceutical product for preventing and / or treating a disease.

[0290] F. Antibody / Engager Production Monoclonal antibodies (mAbs) are often selected from antigen-specific single B cells derived from different hosts. The development of several new techniques and protocols has made the isolation and recovery of antibody-coding sequences from antigen-specific B cells easier, also by utilizing smaller reaction volumes. Alternatively, mAbs can be produced independently of antigen-specific B cells, including display technologies and, more recently, artificial intelligence-based algorithms. As a result, a wide variety of techniques are being used, and better integration is needed.

[0291] In some cases, NKp80 engagers are prepared by methods known in the art, such as rabbit single B cell cloning or phage libraries. In some cases, nucleic acids (such as DNA) encoding the sequence of the NKp80 engager clone are isolated, and the NKp80 engager is identified by methods known in the art, such as ELISA screening.

[0292] i. Vector Vectors are typically selected to be functional in the host cell in which they are used (the vector is compatible with the host cell's machinery so that gene amplification and / or gene expression can occur). The vectors described herein are expression vectors and / or cloning vectors.

[0293] In some examples, the vector is selected from the group consisting of plasmids, viral particles, phages, baculoviruses, yeast plasmids, lipid-based vehicles, polymer microspheres, liposomes, and cell-based vehicles, and is derived from colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral vehicles, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, foam viruses, cytomegaloviruses, Semryki forest viruses, poxviruses, pseudorabies viruses, RNA viral vectors, DNA viral vectors, and vectors derived from combinations of plasmids and phage DNA, further optionally, the polynucleotides are operably linked to expression control sequences to direct peptide synthesis, and further optionally, the vector includes one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0294] ii. Host cells In some examples, the present disclosure is a host cell comprising a vector comprising a nucleic acid sequence encoding any one of the multispecific polypeptide constructs described herein. In some examples, the host cell comprises a cloning or expression vector as described above, and / or a nucleic acid sequence encoding a multispecific polypeptide construct, an antibody and its binding fragment as described above. In some examples, the exemplary host cell comprises a cloning or expression vector configured to express a multispecific polypeptide construct disclosed herein.

[0295] In another context, the Specified Multispecific polypeptide constructs or nucleic acids encoding antibodies are provided. In some examples, the host cell is any type of cell that can be transformed or transfected with a nucleic acid or vector to produce a multispecific polypeptide construct or its binding fragment / polypeptide construct encoded thereby. In some embodiments, the host cell containing the nucleic acid or vector is used to produce the multispecific polypeptide construct or its binding fragment / polypeptide construct, or a portion thereof (e.g., a heavy chain sequence or 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 the expression of the encoded sequence. In some examples, the antibody, multispecific polypeptide construct, or fragment, or a portion of the antibody, is then isolated from the cell.

[0296] In some examples, the host cell is a prokaryotic host cell (e.g., Escherichia coli) or a eukaryotic host cell (e.g., yeast cell, insect cell, vertebrate cell). In some examples, the host cell, when cultured under appropriate conditions, expresses the antibody or its conjugated fragment, which is then recovered from the culture medium (if the host cell secretes it into the culture medium) or directly from the host cell producing it (if it is not secreted). In some examples, the selection of an appropriate host cell depends on the desired expression level, polypeptide modifications desirable or required for activity such as glycosylation or phosphorylation, ease of folding into a biologically active molecule, or other factors routinely considered in the art. In some examples, the selection of the host cell depends in part on whether the antibody or its conjugated fragment is post-transcriptionally modified (e.g., glycosylation and / or phosphorylation). In one embodiment, the host cell includes bacterial cells, yeast cells, animal cells, e.g., mammalian cells and / or plant cells.

[0297] In some cases, suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells. Many host cell lines are available from the American Type Culture Collection (ATCC), Manassas, Va. Some examples include mammalian cells such as Chinese hamster ovary cells (CHO) (ATCC number CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC number CRL1573), 3T3 cells (ATCC number CCL92), or PER.C6 cells. Other cell types that can be used to express antibodies include lymphocyte cell lines, such as NSO myeloma cells, SP2 cells, and COS cells.

[0298] iii. Clone selection Developing and engineering antibodies for various purposes, such as diagnostic and therapeutic agents, requires comprehensive characterization to determine affinity, specificity, and mechanism of action. Biolayer Interferometry (BLI) is widely used to analyze interactions between two biomolecules, allowing for relatively simple and rapid antibody characterization. In BLI, binding of a ligand immobilized on a biosensor chip to the analyte in solution increases the optical thickness of the biosensor chip, resulting in a wavelength shift proportional to the degree of binding. The sensor chip collects measurements in real time while remaining immersed in the analyte solution ("dip and read"). Therefore, this system enables the measurement of various antibody-antigen interactions using a variety of sensors suitable for label-free molecules and widely used tags.

[0299] The xCELLigence platform utilizes gold microelectrodes embedded in the bottom of microtiter wells to monitor the state of adherent and suspended cells anchored to the plate bottom. The basic measurement principle is based on impedance measurement across the surface of the gold electrodes, where adherent cells act as insulators, hindering the flow of microampere alternating current between the electrodes. This impedance signal is automatically measured at a user-defined frequency (e.g., every 10 seconds, once per hour), allowing for highly sensitive readings of cell number, cell size, and cell-substrate adhesion strength. Unlike cancer cell targets adhered to the surface, immune effector cells are non-adherent and therefore do not directly affect the impedance signal. This characteristic allows for real-time, selective monitoring of cytotoxic activity of NK cells, T cells, CART, oncolytic viruses, checkpoint inhibitors, bispecific antibodies, BiTE, and others.

[0300] In some cases, screening and / or identification of NKp80 engagers involves using biolayer interference (BLI) and the Xcelligence® cytotoxic killing assay. In some cases, the selected engagers exhibit the highest binding affinity determined by BLI. In some cases, the selected NK engagers exhibit the highest cytotoxic profile determined by xCELLigence.

[0301] iv. Humanization Antibodies and antigen-targeting domains have emerged as effective tools for the treatment and diagnosis of various human diseases. Non-human antibodies and antigen-targeting domains have been shown to induce a human immune response, resulting in neutralization of administered antibodies and limiting their application in the treatment of human diseases. To overcome this problem, antibody humanization technology has been developed. Antibody humanization is an efficient approach to eliminate or reduce the immunogenicity of these antibodies and antigen-targeting domains. Researchers have devised various methods to humanize non-human antibodies and antigen-targeting domains, improving their affinity, specificity, and other properties. Each of these methods has its own advantages and disadvantages.

[0302] A common method for humanizing non-human antibodies and antigen-targeting domains is complementary determinant region (CDR) transplantation, which involves transplanting the CDR of the non-human antibody or antigen-targeting domain into a human framework region. Typically, the human framework region with the highest homology to the framework region of the non-human antibody or antigen-targeting domain is selected as the acceptor for the CDR graft. While directly transplanting the CDR loop of a mouse antibody or antigen-targeting domain into a human framework may not affect the affinity of the antibody or antigen-targeting domain, it often significantly reduces affinity. Several mouse residues in the framework region, known as vernier zone residues, have been shown to affect the conformation of the CDR loop and the affinity of the antibody or antigen-targeting domain. These residues are localized in the β-sheet framework region immediately beneath the CDR. Therefore, after selecting the desired human framework region, these residues are retained in the humanized antibody or antigen-targeting domain.

[0303] Human germline genes can be used as an alternative source of framework regions for the humanization of mouse antibodies and antigen-targeting domains. Compared to IgG-derived framework regions, germline genes exhibit less intraclonal somatic hypermutation. Therefore, humanized antibodies and antigen-targeting domains with germline framework regions are expected to be less immunogenic than those with IgG framework regions. These characteristics have encouraged research into applying these sequences to the humanization of antibodies and antigen-targeting domains.

[0304] To improve the affinity of humanized antibodies and antigen-targeting domains, researchers employ several approaches, which involve modifying specific residues in the framework or CDR regions of the designed antibodies or antigen-targeting domains. Of all CDRs, modifications to heavy chain CDR3 (VHCDR3) have been the most frequently used to enhance the affinity and specificity of antibodies and antigen-targeting domains. This CDR is the most variable, and its mutations arise from somatic mutations and recombinations of the variability (V), diversity (D), and binding (J) fragment coding sequence.

[0305] Antibody re-emergence is another method for humanizing non-human antibodies and antigen-targeting domains. This method replaces potential antigenic surface skeleton residues with the most common human residues at those locations. The basis of this method is that the response of human anti-mouse antibodies (HAMA) to variable regions is triggered solely by surface residues. Antibodies and antigen-targeting domains humanized by this method typically show little change in stability or affinity.

[0306] Humanization based on CDR homology is based on the idea that mouse antibodies with similar CDRs and the framework regions of human antibodies or antigen-targeting domains can support each other's CDR structures while maintaining high affinity. In this method, framework region homology is not considered in order to select human framework regions, and mouse key residues (vernier zone residues) are not restored in humanized antibodies or antigen-targeting domains. This method reduces the formation of motifs that may be recognized as contaminants. Antibodies or antigen-targeting domains produced by this method have been found to maintain relatively better affinity than those produced by framework-homology-based humanization methods.

[0307] Fully human antibodies or antigen-targeting domains obtained from transgenic animals account for the increase in novel therapeutic agents. After immunization, a diverse range of high-affinity human monoclonal antibodies or antigen-targeting domains can be obtained from transgenic rodents, while larger animals such as transchromosomal cattle produce substantial amounts of specific human immunoglobulin (Ig) in their serum. In some cases, selected multispecific polypeptide construct clones include humanized variable regions, humanized CDRs, and / or humanized framework regions.

[0308] In some cases, the NKp80 targeting domain is humanized. In some cases, the humanized NKp80 targeting domain includes the VL domain sequence (Figure 13C): DIQMTQSPSSVSASVGDRVTITCQASQSIGRDLAWYQQKPGKAPKLLIYGASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGADRSSTPSWPFGQGTKVEIK (VL sequence of the humanized anti-NKp80 domain; SEQ ID NO: 235)

[0309] In some cases, the humanized NKp80 targeting domain contains a humanized VH sequence: QVQLVESGGGVVQPGGSLRLSCAASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKDLSKNTVYLQMNSLRAEDTAVYYCARDTGSGYWKYNIWGRGTLVTVSS (VH sequence of the humanized anti-NKp80 domain; SEQ ID NO: 236)

[0310] In some cases, the humanized NKp80 targeting domain includes a CL domain sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL sequence of the anti-NKp80 domain; SEQ ID NO: 237)

[0311] In some cases, the humanized NKp80 targeting domain includes a CH domain sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (CH sequence of the anti-NKp80 domain; SEQ ID NO: 238)

[0312] In some examples, the humanized NKp80 targeting includes 1, 2, 3, 4, 5, or 6 CDRs selected from SEQ ID NOs: 247-252. In some examples, the humanized NKp80 targeting domain includes 1, 2, 3, 4, 5, 6, 7, or 8FRs selected from SEQ ID NOs: 253-260.

[0313] G. Gene editing methods In some cases, the modification of the amino acid sequence is achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

[0314] i. Site-directed mutagenesis Mutagenesis is typically used to understand the relationship between the structure and function of regulatory regions of genes or polypeptide constructs. Depending on the number of sites being mutated, site-directed mutagenesis is divided into two types: simple mutation and multiple mutation. For single mutations, this method is based on amplifying double-stranded DNA from a plasmid using a complementary oligonucleotide containing the desired mutation. Due to its simplicity, short time, and high efficiency, this is one of the most common strategies for introducing mutations into DNA fragments. For multiple mutations, this method either incorporates the desired mutations simultaneously in the same reaction or obtains them after several mutations. In some examples, nucleic acid sequences, domains, or fragments encoding multiple specific polypeptide constructs are modified using site-directed mutagenesis.

[0315] ii. PCR-based mutagenesis PCR-based mutagenesis is fundamental to molecular biology and protein engineering research. This specification describes a rapid and highly efficient mutagenesis method using IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor primers and mutagenic primers. The mutant sequence is located near the recognition site of the IIs restriction enzyme. After digestion of the two fragments with the IIs enzyme, complementary exposed sticky ends are ligated to generate the mutant gene. Major strategies for PCR-based mutagenesis include base substitution, deletion, insertion, chimeric gene generation, multisite mutagenesis, and single-site or multi-site random mutagenesis. Numerous PCR-based methods have been developed commercially and non-commercially. Among these methods, the overlap extension method, megaprimer method, Quick Change method (Stratagene, La Jolla, CA), and their modifications are currently common. In some cases, the nucleic acid sequence of a multispecific polypeptide construct, or its domain or fragment, is modified using PCR-based mutagenesis. [Examples]

[0316] Example 1: Screening and identification of novel NKp80 engagers New Zealand white (NZW) rabbits were used for immunization via biological DNA delivery containing the NKp80 gene of interest. Titer was monitored during the immunization period. Peripheral whole blood was collected for B cell isolation. Briefly, B cells positive for anti-rabbit IgG staining were selected by flow cytometry and cultured. Using the B cell culture supernatant, positive NKp80 binders were identified by ELISA screening, followed by mRNA isolation and cDNA synthesis. The congeneral gene segments of VH and VL were amplified by PCR and validated by sequencing. The DNA coding sequences of VH and VL were cloned into their respective expression vectors for the expression of recombinant multispecific polypeptide constructs. The multispecific polypeptide construct containing the HER2 target domain, ADCC dysfunction in the Fc constant region, and a specific NKp80 engager is hereafter referred to as anti-HER2-anti-NKp80-FcX (FcX: ADCC dysfunction).

[0317] Multiple specific polypeptide construct clones were recombinantly expressed in mammalian systems via transient transfection. Candidate multiple specific polypeptide constructs were purified using a polypeptide construct A column and evaluated for purity of 95% or higher by SDS-PAGE under non-reducing conditions. Different formats of the candidate multiple specific polypeptide constructs exemplified in this study, including a triple-specific multiple specific polypeptide construct containing a HER2 targeting domain, an Fc constant region with ADCC function, and a humanized NKp80 targeting domain (hereinafter referred to as anti-HER2-anti-NKp80-Fc), were also recombinantly expressed and purified using a similar approach.

[0318] Next, the DNA coding sequences of NKp80 binder clones were isolated, cloned into rabbit IgG format, and validated using ELISA screening. A total of 108 NKp80 binder DNA coding sequences were inserted into mammalian expression vectors and expressed in a mammalian system in a triple-specific format (anti-HER2-anti-NKp80-FcX) (Figure 1, point 1). The Fc constant region of the bispecific polypeptide construct (denoted here as FcX) showed reduced ADCC function. The inclusion of a reduced-function Fc domain was important in the validation process: it demonstrated that the detected cytotoxicity was due to the involvement of NKp80.

[0319] Example 2: NKp80 Engager Clone: ​​Binding and Cytotoxic Killing Screening Assay

[0320] Potential NKp80-binding clones were identified using the ForteBio Octet Bio-Layer Interferometry (BLI) system. Streptavidin biosensors were loaded with different biotinylated antigens containing both human and cynomolgus monkey NKp80, and the binding kinetics of these candidates were determined.

[0321] Of the 108 clones with reduced ADCC function (triple-specific, anti-HER2-anti-NKp80-FcX), binding between human and cynomolgus monkey NKp80 was confirmed in 78 and 67 clones, respectively, using bio-layer interferometry (BLI) (Figure 1, point 2a).

[0322] The ability of the NKp80 binder to redirect NK cell cytotoxicity against the HER2-positive target cell line N87 was determined by the Xcelligence cytotoxic killing assay, which tracks cell death in real time. A single dose concentration of the target clone was incubated with primary PBMCs (from a healthy donor) in an effector-to-target cell ratio. The effector was primary PBMC isolated from a healthy donor.

[0323] A total of 92 clones were assayed for cytotoxicity using this method (70 with NKp80 binder, 22 without binder). The non-binder clones were included to measure baseline activity along with an anti-HER2 antibody (anti-HER2-FcX).

[0324] Activated binders were defined as clones that showed consistent killing activity above the median in two independent assays using two different PBMC donors. A total of 20 NKp80 clones were identified as activated binders (out of 70 binders) (Figure 2 shows the killing activity of all 20 activated binders). Importantly, 22 non-binders did not induce significantly higher cytotoxicity than baseline (data not shown). Taken together, these data reveal that a unique subset of NKp80 binders is involved in and enhances the cytotoxicity of NK cells.

[0325] Example 3: Enhancement of cytotoxicity by binding of NKp80 and CD16 Xcelligence real-time assays were performed to evaluate the cytotoxic performance of various multispecific polypeptide construct candidates. Briefly, target cells were seeded into Xcelligence microtiter plates on day 0. On day 1, PBMCs from healthy donors were added along with the NKp80-binding candidate or engager of study at a predetermined effector:target (ET) ratio. Cell lysis of target cells was tracked over time based on electrical impedance detected by the Xcelligence instrument. The output, the cell index, was a measure of the detected electrical impedance and was proportional to the number of attached target cells.

[0326] For hit screening, single concentrations of potential multispecific NKp80-binding polypeptide candidate constructs in a triple-specific format (anti-HER2-anti-NKp80-FcX) were used to identify NKp80 activation binders in the presence of N87 cancer cells.

[0327] In some cases, cytotoxic assays are used to investigate the innate immune cytotoxicity of multispecific polypeptide constructs, although these assays are not limited to the Xcelligence® cytotoxicity assay.

[0328] While we do not wish to be constrained by theory, the real-time Xcelligence® cytotoxic killing assay investigates the ability of therapeutics to redirect the cytotoxicity of innate immune cells against target antigen-positive target cells. For example, an NKp80 engager containing an anti-HER2 arm can be evaluated for its ability to redirect the cytotoxicity of NK cells against HER2-positive target cells such as N87.

[0329] As shown in the experimental data of this disclosure, the NKp80 activating binder exhibited above-median cytotoxicity in cytotoxicity assays compared to other populations. Of 108 clones (triple-specific, anti-HER2-anti-NKp80-FcX) containing Fc domains with reduced ADCC function, 78 and 67 clones were identified as binders for human and cynomolgus monkey NKp80, respectively, using bio-layer interferometry (BLI) (Figure 1, point 2a). A total of 92 triple-specific clones (70 NKp80 binders and 22 non-binders) were subjected to cytotoxicity assays, and a total of 20 NKp80 activating binder clones were identified. Of the 20 activated binders, 13 were humanized and constructed into a triplicate engager with a fully functional Fc domain (triplicately specific anti-HER2-anti-NKp80-Fc). All clones were reconfirmed to be human NKp80 binders by single-point measurements using BLI, with dissociation constants (KD) ranging from low nM to sub-pM.

[0330] To validate the synergistic effect of NKp80 and CD16 co-engagement, anti-NKp80 clones identified as activating binders were humanized and formatted as triplicate engagers (anti-HER2-anti-NKp80-Fc) containing a fully functional ADCC Fc region. These clonal engagers were again bonded, and four clones were selected for further study. For these four clones, dose-dependent cytotoxicity was measured using OVCAR3 cells as target cells. All clones showed improved cytotoxicity compared to trastuzumab controls (Figure 3A). In the absence of Fc function, these four clones were able to induce cytotoxicity, but at a lower level than that induced by trastuzumab (Figure 3B). This suggests that co-engagement of NKp80 and CD16 is necessary to enhance the synergistic cytotoxicity of NK cells. Dose-response curves and EC50 values ​​were plotted using Prism-normalized cell indices (PBMCs and engager-added points). One of these four clones was selected and further evaluated in four different cancer-derived cell lines using 3-4 independent PBMC donors for each cell line. Dose-response curves and EC50 values ​​were calculated similarly as described above.

[0331] Example 4: Efficacy of anti-NKp80 clone 87-2 in multiple target cell lines and PBMC donors Of the four engagers, including anti-NKp80 clones, that showed improved efficacy with the NKp80 and CD16 combination, the engager containing clone 87-2 was selected for further demonstration (triply specific anti-HER2-anti-NKp80(87-2)-Fc). This engager was tested for cytotoxicity using the Xcelligence assay in four different cell lines of different cancer origins, using 3-4 independent PBMC lots obtained from healthy donors (Table 1, column 3). Trastuzumab was used as a benchmark in these experiments. The selected cell lines and their tumor antigen copy number / cells are shown. The selected cell lines and their target copy number / cells are shown (Table 1, columns 1 and 2).

[0332] In these experiments, EC50 values ​​were used as an indicator of potency. Next, the percentage change in EC50 values ​​(compared to trastuzumab) was calculated for each experiment and averaged across independent experiments to obtain an overall EC50 percentage change value. Engagers containing anti-NKp80 clone 87-2 consistently showed improved potency (mean EC50) compared to trastuzumab in different cell lines (Table 1, column 4; Figure 3). Interestingly, potency increased as the copy number expression of the target antigen decreased (Table 1, column 4). This is likely because the potency of trastuzumab weakens with decreasing target antigen copy number (MKN1, mean EC50 0.71 nM vs. MDA-MB-231, mean EC50 6.85 nM) (Table 1, column 5).

[0333] The increased cytotoxicity is thought to be due to the promotion of NK cell activation through the synergistic action of NKp80 and CD16 (Figure 5). (Figure 5). Engagers containing anti-NKp80 clone 87-2 increased the expression of NK activation markers and cytokine secretion as measured by flow cytometry (vs. trastuzumab). Importantly, this engager did not induce T cell activation.

[0334] Next, anti-NKp80 clone 87-2 was cloned into a trispecific format in which anti-HER2 was replaced with anti-EGFR. This version of the trispecific engager targeted EGFR instead of HER2, as seen in all previous experiments. In the Xcelligence cytotoxicity assay, this engager was confirmed to induce cytotoxicity in EGFR+ cell lines with higher potency than cetuximab (anti-EGFR-Fc) (Figure 6). As shown in the experimental data, anti-EGFR-anti-NKp80(87-2)-Fc was more potent than cetuximab (anti-EGFR-Fc) in the cytotoxic killing assay. The control in the cytotoxic killing assay (isotype-anti-NKp80(87-2)-Fc) did not show NK cytotoxic killing against HER2-positive cell lines, suggesting that cytotoxicity is antigen-dependent.

[0335] [Table 1]

[0336] Example 5: Extratarget activation of NK cells in antigen-negative cell lines To evaluate the potential on-target, off-termer effect, a trispecific engager (anti-HER2-anti-NKp80-Fc) containing clone 87-2 was tested against the normal, healthy fibroblast cell line MRC-5. No cell killing was observed, suggesting no "on-target-off-tumor" effect (Figure 7). Furthermore, when the HER2-targeted antibody arm was removed (isotype-anti-NKp80-Fc, where HER2 is replaced by an isotype control), no cell killing was observed compared to the positive control trastuzumab, indicating that NK cell cytotoxicity is target antigen-dependent (Figure 7).

[0337] Example 6: Binding site and sequence similarity of identified NKp80-binding clones To analyze the epitope binding patterns on NKp80, BLI epitope binning experiments were performed using four anti-NKp80 clones selected for cytotoxicity studies, namely humanized clones 45-2, 87-2, 94-1, and 101-1 (Table 2). In these experiments, the target antigen was first immobilized on a biosensor, and (potentially competing) antibodies were added sequentially. If the second antibody generated a signal after the addition of the first antibody, it meant that the second antibody bound to a different epitope than the first antibody. Clones 45-2 and 87-2 bound to similar epitopes, while clones 94-1 and 101-1 bound to different epitopes.

[0338] [Table 2]

[0339] To assess the overall diversity of the 20 activated binders, including the four clones analyzed in Figure 8 (Figure 1, point 2b), a sequence identity matrix was created using the sequences of the heavy chain CDR3 regions of the 20 NKp80 binders (Figure 14). Clustering analysis of the output revealed five distinct clusters (Figure 9), confirming the diversity of the 20 multispecific polypeptide constructs. Consistent with the epitope binning data in Figure 8, clones 45 and 87 belonged to the same cluster (C2), clone 94 belonged to cluster 3 (C3), and clone 101 belonged to cluster 5 (C5), suggesting that the similar basal sequences of clones 45 and 87 explain the binding of NKp80 to similar epitopes.

[0340] Example 7: Permutations and combinations of domains in a multispecific polypeptide construct Figure 13D shows different possible permutations of the multispecific polypeptide construct domains. The selected exemplary constructs are as follows: 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 (Sequence ID: 241) 4) Cetuximab (Fd) / anti-NKp80(87-2)(scFv) / -FcWT (Sequence ID: 242) As shown in Figure 13E, various permutations of triple-specific engagers, including anti-EGFR, wild-type Fc, and anti-NKp80(87-2), consistently yielded superior cytotoxicity against breast cancer MDA-MB-231 cells compared to cetuximab, suggesting that the superior activity conferred by the additional anti-NKp80 targeting domain is not limited to a specific form. Dose-response curves and EC50 values ​​were plotted using Prism-normalized cellular indices (PBMCs and up to the time of engager addition).

[0341] Example 8: Further demonstration of the flexibility of antigen target domains using CD20 To further illustrate the flexibility of NKp80 targeting, CD20 was used as another target antigen. Anti-CD20 (the Fab portion of rituximab) was cloned in a triplicate format by directly substituting the trastuzumab Fab in the respective pcDNA-based VL and VH anti-HER2-anti-NKp80-Fc expression plasmids, while all other components remained identical.

[0342] These plasmids are recombinantly expressed in the EXPI-CHO mammalian cell line. Expression vectors containing different fragments of each multispecific polypeptide construct (one containing VH and one containing VL) are co-introduced into EXPI-CHO cells according to the manufacturer's manual, eluted with 0.2 M Tris-glycine pH 2.7, and purified using Protein A column chromatography neutralized with 1 M Tris pH 8.0. Protein purity is assessed by SDS-PAGE under non-reducing conditions (ideally 95% purity or higher). Constructs are buffer-exchanged with 1x PBS in an ultracentrifuge tube, and the concentration is measured using Nanodrop.

[0343] To assess the potential for cytotoxicity, the triple-specific engager anti-CD20-anti-NKp80-Fc is tested alongside a rituximab control in cytotoxicity assays such as the Xcelligence® assay or a Calcein AM-based staining assay. The processing is the same as that used with anti-HER2-anti-NKp80-Fc.

Claims

1. Multiple specific polypeptide constructs including the following: (a) One or more antigen-targeting domains that bind to one or more cancer-related antigens; and (b) One or more NK cell targeting domains that can stimulate and / or suppress innate immune cell function by binding to NK cells.

2. The multispecific polypeptide construct according to claim 1, wherein one of the NK cell targeting domains is an NKp80 targeting domain.

3. A multispecific polypeptide construct according to claims 1 to 2, comprising the following NKp80 targeting domain: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or heavy chain variable domains (VH) containing one, two, or three complementarity-determining regions (CDRs) selected from two or three amino acid substitutions therein, and / or (2) VLCDR1 of SEQ ID NOs: 1-16, 247; VLCDR2 of SEQ ID NOs: 17-31, 248; and / or VLCDR3 of SEQ ID NOs: 32-50, 249; or having at least about 80% sequence identity with their amino acid sequences; or light chain variable domains (VLs) containing one, two, or three CDRs selected from two or three amino acid substitutions of those.

4. A multispecific polypeptide construct according to claims 1 to 3, comprising the following NKp80 targeting domain: (1) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (2) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from the 2 or 3 amino acid substitutions thereof.

5. A multispecific polypeptide construct according to claims 1 to 4, comprising an NKp80 targeting domain: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions; and / or (4) VLFR1 of SEQ ID NOs: 105-118, 253; VLFR2 of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VLFRs selected from 2 or 3 amino acid substitutions thereof.

6. A multispecific polypeptide construct according to claims 1 to 5, further comprising a functional Fc domain.

7. The multispecific polypeptide construct according to claim 6, wherein the Fc domain is as follows: (i) The native / wild-type Fc domain (FcWT) or reduced-function Fc (FcX) domain of Sequence ID No. 224; or one having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (ii) Enhanced Fc domain (FcE) of Sequence ID No. 226; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof; (iii) Silent Fc domain / inactivating mutant Fc domain (FcLALA) of Sequence ID No. 225; or having at least approximately 80% sequence identity with its amino acid sequence; or two or three amino acid substitutions thereof.

8. The multispecific polypeptide construct according to claims 6 to 7, comprising the following: (a) The first domain targeting NKp80 (b) Second domain targeting CD16 (c) One or more antigen-targeting domains that bind to one or more tumor-associated antigens.

9. A multispecific polypeptide construct according to claims 1 to 8, wherein one or more antigen-targeting domains bind to a member selected from HER-2, EGFR, and CD20.

10. A multispecific polypeptide construct according to claims 1 to 9, comprising one or more antigen-targeting domains: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or any amino acid sequence having at least approximately 80% sequence identity with those; or any two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or any amino acid sequence having at least approximately 80% sequence identity with those; or any two or three amino acid substitutions thereof.

11. A multispecific polypeptide construct according to claims 1 to 10, comprising the following: (A) NKp80 targeting domains including the following: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions, and / or (4) FR1 of SEQ ID NOs: 105-118, 253; a of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VL FRs selected from the 2 or 3 amino acid substitutions thereof; and, (B) One or more antigen-targeting domains including the following: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or any amino acid sequence having at least approximately 80% sequence identity with those; or any two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or any amino acid sequence having at least approximately 80% sequence identity with those; or any two or three amino acid substitutions thereof.

12. A multispecific polypeptide construct according to claims 6 to 11, comprising the following: (A) NKp80 targeting domains including the following: (1) VHCDR1 of SEQ ID NOs: 51-67, 250; VHCDR2 of SEQ ID NOs: 68-85, 251; and / or VHCDR3 of SEQ ID NOs: 86-104, 252; or having at least approximately 80% sequence identity with those amino acid sequences; or VH containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (2) VLCDR1 of sequence numbers 1-16, 247; VLCDR2 of sequence numbers 17-31, 248; and VLCDR3 of sequence numbers 32-50, 249; or having at least approximately 80% sequence identity with their amino acid sequences; or VL containing one, two, or three CDRs selected from two or three amino acid substitutions of those; (3) VHFR1 of SEQ ID NOs: 141-155, 257; VHFR2 of SEQ ID NOs: 156-162, 258; VHFR3 of SEQ ID NOs: 163-179, 259; and / or VHFR4 of SEQ ID NOs: 180-182, 260; or having at least approximately 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VH framework regions (FRs) selected from 2 or 3 of those amino acid substitutions; and / or (4) FR1 of SEQ ID NOs: 105-118, 253; a of SEQ ID NOs: 119-121, 254; VLFR3 of SEQ ID NOs: 122-137, 255; and / or VLFR4 of SEQ ID NOs: 138-140, 256; or having at least about 80% sequence identity with those amino acid sequences; or 1, 2, 3, or 4 VL FRs selected from 2 or 3 of those amino acid substitutions; and (B) One or more antigen-targeting domains including the following: (1) VH (VH cetuximab) of amino acid sequence number 231, VL (VL cetuximab) of amino acid sequence number 232, CH of amino acid sequence number 233, and / or CL of amino acid sequence number 234; or any amino acid sequence having at least approximately 80% sequence identity with those; or any two or three amino acid substitutions thereof; (2) VH (VH trastuzumab) of amino acid sequence number 227, VL (VL trastuzumab) of amino acid sequence number 228, CH of amino acid sequence number 229, and / or CL of amino acid sequence number 230; or those having at least approximately 80% sequence identity with those amino acid sequences; or two or three amino acid substitutions thereof; and / or (3) VH (VH rituximab) of amino acid sequence number 244, VL (VL rituximab) of amino acid sequence number 243, CH (CH rituximab) of amino acid sequence number 246, and / or CL (CL rituximab) of amino acid sequence number 245; or those having at least approximately 80% sequence identity with those amino acids; or two or three amino acid substitutions thereof; and (C) Sequence IDs: 224-226; or sequences having at least approximately 80% sequence identity with those sequences; or Fc domains having an amino acid sequence selected from two or three of those amino acid substitutions.

13. A multispecific polypeptide construct according to claims 7 to 12, wherein the polypeptide construct is a triple-specific antigen-binding construct comprising the following: (a) A first targeting domain that binds to NKp80; (b) A second targeting domain that binds to CD16; and (c) A third targeting domain that binds to the target antigen, Here, the targeting domain is selected from Fab fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-domain Ab(dAb) fragments, isolated CDRs, single-chain Fv(scFv), disulfide-stabilized Fv(dsFv), single-chain Ab(scAb), secretory T cell bispecific Ab(STAb), single-domain Ab(sdAb), single-domain CH antibody, single-domain CL antibody, VHH, variable domains (VNARs) of novel antigen receptors, sdAb based on shark-derived VNAR structures, and binding domains based on alternative scaffolds including, but not limited to, ankyrin-based domains, finomers, avimers, anticarin, fibronectin, and binding sites incorporated into the constant region of antibodies.

14. A multispecific polypeptide construct according to claims 7 to 13, wherein the polypeptide construct is a triplespecific antigen-binding construct comprising the following: (a) A first targeting domain that binds to NKp80, the targeting domain being selected from Fab fragment, Fv fragment; sdAb fragment, isolated CDR, scFv, dsFv, scAb, STAb, sdAb, single-domain CH antibody, single-domain CL antibody, VHH, VNAR, and sdAb based on the VNAR structure from shark; (b) A first targeting domain that binds to CD16, the targeting domain being a functional Fc domain selected from FcWT (SEQ ID NO: 224), FcX (SEQ ID NO: 224), silent Fc / Fc inactivation variant (FcLALA) (SEQ ID NO: 225), or FcE (SEQ ID NO: 226); and (c) A third targeting domain that binds to a tumor-associated antigen, optionally to HER2, EGFR, or CD20, the targeting domain being selected from Fab fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-domain Ab (dsAb) fragments, isolated CDRs, single-chain Fv (scFv), disulfide-stabilized Fv (dsFv), single-chain Ab (scAb), secretory T cell bispecific Ab (STAb), single-domain Ab (sdAb), single-domain CH antibody, and single-domain CL antibody, VHH, variable domain (VNAR) of a novel antigen receptor, sdAb based on a shark-derived VNAR structure, and binding domains based on alternative scaffolds including, but not limited to, ankyrin-based domains, finomers, avimers, antikalin, fibronectin, and binding sites incorporated into the constant region of an antibody.

15. A multispecific polypeptide construct according to claims 7 to 14, comprising the following NKp80 targeting domain: (1) Sequence IDs: 203-222, 236; or sequences having at least approximately 80% sequence identity with those sequences; or VH containing an amino acid sequence selected from two or three amino acid substitutions therewith; (2) Sequence ID: VL containing an amino acid sequence selected from 183-202, 235; Here, VH and VL pair up 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 having at least about 80% sequence identity with their amino acid sequences; or producing two or three amino acid substitutions thereof.

16. A multispecific polypeptide construct according to claims 7 to 15, comprising the following: (i) an antigen targeting domain consisting of an Fd fragment or a Fab fragment; a first NK cell targeting domain consisting of an Fc domain; a first [(G4S)n] linker; and a second NK cell targeting domain consisting of scFv including VH, a second [(G4S)n] linker and VL; (ii) A first NK cell targeting domain consisting of an Fd fragment or a Fab fragment; a second NK cell targeting domain consisting of an Fc domain; a first [(G4S)n] linker; and an antigen targeting domain consisting of scFv including VH, a second [(G4S)n] linker and VL; (iii) A first NK cell targeting domain consisting of an Fd fragment or a Fab fragment; a first [(G4s)n] linker; an antigen targeting domain consisting of scFv including VH, a second [(G4S)n] linker and VL; and a second NK cell targeting domain consisting of an Fc domain including CH2 and CH3; or (iv) an antigen-targeting domain comprising an Fd fragment (containing VH and CH1) or a Fab fragment; a first [(G4S)n] linker; a first NK cell-targeting domain comprising scFv containing VH, a second [(G4S)n] linker and VL; and a second NK cell-targeting domain comprising an Fc domain containing CH2 and CH3.

17. A multispecific polypeptide construct according to claims 1 to 16, wherein the NKp80 targeting domain includes a member selected from the following: (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 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 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 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 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 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 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); (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); (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).

18. An antigen-binding protein or its antigen-binding fragment containing a CDR sequence selected from the following: (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 NO: 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 NO: 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), Here, the CDR sequence shares at least approximately 90% homology with an amino acid sequence selected from SEQ ID NOs: 1–104, 247–252, and / or Here, the CDR sequence selected from sequence numbers 1-104 and 247-252 contains two or three amino acid substitutions.

19. An antigen-binding protein or its antigen-binding fragment containing CDR and FR sequences selected from the following: (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 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 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 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 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 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 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); (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); (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), Here, the FR and CDR sequences share at least approximately 90% homology with amino acid sequences selected from SEQ ID NOs: 1–104, 247–260, and / or Here, the FR and CDR sequences selected from sequence numbers 1-104 and 247-260 contain two or three amino acid substitutions.

20. A nucleic acid sequence encoding a multispecific polypeptide construct or antibody according to any one of claims 1 to 19.

21. A vector comprising a sequence of a multispecific polypeptide construct or antibody according to any one of claims 1 to 19.

22. A host cell comprising the vector according to claim 21.

23. A method for producing a multispecific polypeptide construct or antibody according to claims 1 to 19, comprising culturing host cells and optionally isolating a multispecific polypeptide construct from the host cells and / or culture medium.

24. A method for screening and / or identifying multispecific polypeptide constructs or antibodies according to claims 1 to 19, wherein the NK cell targeting domain is anti-NKp80.

25. A pharmaceutical composition comprising a multispecific polypeptide construct or antibody according to claims 1 to 19.

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

27. The method according to claim 26, wherein the subject has cancer cells expressing HER2, CD20, and / or EGFR.

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