CN121057751A - NK cell conjugates combining NKP80 and their applications - Google Patents

Abstract

This invention provides multispecific polypeptide constructs comprising: (a) one or more antigen-targeting domains binding to one or more tumor-associated antigens; and (b) one or more NK cell-targeting domains, wherein binding to NK cells can stimulate and/or inhibit the function of innate immune cells. Also disclosed are antigen-binding proteins or antigen-binding fragments thereof; nucleic acid sequences; vectors; host cells; methods for preparing multispecific polypeptide constructs or antibodies; methods for screening and/or identifying multispecific polypeptide constructs or antibodies as disclosed herein; pharmaceutical compositions; and methods for treating cancer.

Description

NK cell conjugates combining NKP80 and their applications

Technical Field

This disclosure relates to the field of multispecific peptide constructs engineered to conjugate NK cells and bind cell surface antigens, thereby inducing a desired immune response in various disease indications.

Background Technology

Natural killer (NK) cells are part of the innate immune system, making up 5–15% of circulating lymphocytes. NK cells perform innate immune surveillance against stress cells (such as tumor cells or virus-infected cells), triggering their lysis upon recognition. 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 inhibit NK cell activity by binding to killer cell immunoglobulin-like receptors (KIRs) on the NK cell surface. In contrast, ligands expressed by stress cells bind to activating receptors.

Both activating and inhibitory receptors are expressed on the surface of NK cells, contributing to the execution of NK cell functions. MHC-I (major histocompatibility complex class I) antigen-specific inhibitory receptors tightly 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, known as the "missing self," was initially proposed by Ljunggren and Karre. Such MHC-I-recognizing inhibitory receptors form three families of NK cell surface receptors: KIR (cytotoxic cell immunoglobulin-like receptors), LIR (leukocyte immunoglobulin-like receptors), and NKG2A (natural killer group 2A). KIR is a member of the immunoglobulin superfamily and recognizes type I transmembrane molecules (HLA class Ia) that recognize classic human leukocyte antigens A, B, and C. LIR, also known as ILT (immunoglobulin-like transcript), forms a second group of receptors and primarily recognizes non-classical HLA-G (class Ib) molecules other than HLA class Ia. LIRs belong to the same Ig superfamily as KIRs. NKG2A is a member of the NKG2 group of seven receptors (A, B, C, D, E, F, and H), which dimerizes with CD94 to form the NKG2A/CD94 receptor. It belongs to the C-type lectin family of receptors and recognizes non-classical HLA-E class I molecules as its ligands.

NK cell destruction requires not only the detection of MHC-I molecules on transformed cells via inhibitory receptors but also the activation of NK cells via activating receptors. Natural cytotoxic receptors (NCRs) represent a group of activating receptors on the surface of natural killer cells, including NKp46, NKp30, and NKp44. These receptors, along with NKG2D, DNAM-1 (DNAX helper molecule-1), and NKp80, recognize ligands expressed on the surface of virus-infected or malignantly transformed cells. CD16 (or FcγRIII) is also an activating receptor, primarily expressed by the CD56 _dim NK cell subset and essential for antibody-dependent cytotoxicity (ADCC) against IgG-coated target cells.

NK cells have recently gained attention as an important innate immune regulatory cell type, representing a promising alternative platform for cellular immunotherapy. NK cells can rapidly kill multiple adjacent cancer cells through non-MHC-restricted effects. Although tumors may develop various resistance mechanisms against endogenous NK cell attack, in vitro activation, expansion, and genetic modification of NK cells can significantly enhance their antitumor activity and endow them with the ability to overcome drug resistance. Some of these approaches have been translated into clinical applications, and to date, clinical trials infusing NK cells into patients with hematologic malignancies and solid tumors have yielded many encouraging clinical results. However, several challenges remain to be overcome, such as achieving clinical-grade in vitro expansion, limited in vivo persistence, limited invasion into solid tumors, and tumor editing to evade NK cell activity. Therefore, there is a need to provide alternative multispecific peptide constructs.

Summary of the Invention

In one aspect, a multispecific polypeptide construct is provided, comprising:

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

(b) One or more NK cell-targeting domains, wherein binding to NK cells can stimulate and/or inhibit the function of innate immune cells.

In some instances, one of the NK cell binding domains is the NKp80 targeting domain.

In some instances, the NKp80 targeting domain contains:

(1) A heavy chain variable domain (VH) comprising one, two, or three complementarity-determining regions (CDRs) selected from VHCDR1 of SEQ ID NO: 51-67, 250, VHCDR2 of SEQ ID NO: 68-85, 251, and/or VHCDR3 of SEQ ID NO: 86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein; and/or

(2) Light chain variable domain (VL) comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and/or VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein.

In some instances, the NKp80 targeting domain contains:

(1) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(2) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions thereon.

In some instances, the NKp80 targeting domain contains:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions thereon.

In some instances, such as the multispecific peptide constructs disclosed herein, a functional Fc domain is also included.

In some instances, the Fc struct is

(i) The natural/wild-type Fc domain (FcWT) or weakened Fc (FcX) domain of SEQ ID NO:224; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein;

(ii) The enhanced Fc domain (FcE) of SEQ ID NO:226; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein;

(iii) The silent Fc domain/inactivated mutant Fc domain (FcLALA) of SEQ ID NO:225; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(a) Targeting the first domain of NKp80;

(b) Targeting the second domain of CD16;

(c) Binding one or more antigen-targeting domains of one or more tumor-associated antigens.

In some instances, one or more antigen-targeting domains bind to members selected from HER-2, EGFR, and CD20.

In some instances, one or more antigen-targeting domains contain:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) VH (VH rituximab) of amino acid sequence SEQ ID NO:244, VL (VL rituximab) of amino acid sequence SEQ ID NO:243, CH (CH rituximab) of amino acid sequence SEQ ID NO:246 and/or CL (CL rituximab) of amino acid sequence SEQ ID NO:245; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(A) The NKp80 targeting domain includes:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three, or four VLFRs selected from FR1 of SEQ ID NO:105-118, 253, VLFR3 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255, and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and

(B) One or more antigen-targeting domains, comprising:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) VH (VH rituximab) of amino acid sequence SEQ ID NO:244, VL (VL rituximab) of amino acid sequence SEQ ID NO:243, CH (CH rituximab) of amino acid sequence SEQ ID NO:246 and/or CL (CL rituximab) of amino acid sequence SEQ ID NO:245; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(A) The NKp80 targeting domain includes:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three, or four VLFRs selected from FR1 of SEQ ID NO:105-118, 253, VLFR3 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255, and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and

(B) One or more antigen-targeting domains, comprising:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) The amino acid sequence SEQ ID NO:244 of VH (VH rituximab), the amino acid sequence SEQ ID NO:243 of VL (VL rituximab), the amino acid sequence SEQ ID NO:246 of CH (CH rituximab), and/or the amino acid sequence SEQ ID NO:245 of CL (CL rituximab); or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein; and

(C) Having an Fc domain with an amino acid sequence selected from SEQ ID:224-226; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein.

In some instances, the peptide construct is a trispecific antigen-binding construct, which comprises:

(a) Binding to the first target domain of NKp80;

(b) binding to the second targeting domain of CD16; and

(c) Binding to the third targeting domain of the target antigen,

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), secreted T-cell bispecific Ab (STAb), single-domain Ab (sdAb), single-domain CH antibody, single-domain CL antibody, VHH, variable domain of neoantigen receptor (VNAR), sdAb based on shark VNAR structure, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, anticalin, fibronectin, and binding sites, which are constructed into the constant region of the antibody.

In some instances, the peptide construct is a trispecific antigen-binding construct, which comprises:

(a) Binding to the first targeting domain of NKp80, wherein the targeting domain is 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 shark-based VNAR structure sdAb;

(b) Binding to the first targeting domain of CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO:224), FcX (SEQ ID NO:224), the silenced Fc/Fc inactivation mutant (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 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), secreted T-cell bispecific Ab (STAb), single-domain Ab (sdAb), single-domain CH antibody, single-domain CL antibody, VHH, variable domain of neoantigen receptor (VNAR), sdAb based on shark VNAR structure, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, anticalin, fibronectin, and binding sites constructed into the constant region of the antibody.

In some instances, the NKp80 targeting domain contains:

(1) VH, which contains an amino acid sequence selected from SEQ ID NO:203-222, 236; or has at least about 80% sequence identity with its amino acid sequence; or has 2 or 3 amino acid substitutions;

(2) VL, which contains an amino acid sequence selected from SEQ ID NO:183-202, 235;

VH and VL pairings 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 have at least about 80% sequence identity with their amino acid sequence; or have 2 or 3 amino acid substitutions.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

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

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

(iii) A first NK cell targeting domain, composed of an Fd or Fab fragment; a first [(G4S)n] linker; an antigen targeting domain, composed of scFv containing VH, a second [(G4S)n] linker, and VL; and a second NK cell targeting domain, composed 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; a first [(G4S)n] linker; a first NK cell-targeting domain consisting of a 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.

In some instances, the NKp80 targeting domain contains 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:138) 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:138) 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:138) 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:138) 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:138) 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:138) 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:139) 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:138) 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: 13) 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: 17) 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 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).

On the other hand, an antigen-binding protein, or an antigen-binding fragment thereof, is provided, comprising 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),

The CDR sequence has at least approximately 90% homology with the amino acid sequences selected from SEQ ID NO:1-104 and 247-252; and/or

The CDR sequences selected from SEQ ID NO:1-104, 247-252 contain 2 or 3 amino acid substitutions.

On the other hand, an antigen-binding protein, or an antigen-binding fragment thereof, is provided, comprising a CDR and FR sequence 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:138) 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:138) 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:138) 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:138) 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:138) 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:138) 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:139) 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:138) 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: 13) 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: 17) 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 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:256) 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),

The FR and CDR sequences share at least approximately 90% homology with amino acid sequences selected from SEQ ID NO:1-104 and 247-260; and/or

The FR and CDR sequences selected from SEQ ID NO:1-104, 247-260 contain 2 or 3 amino acid substitutions.

On the other hand, nucleic acid sequences encoding multispecific polypeptide constructs or antibodies as disclosed herein are provided.

On the other hand, vectors containing sequences for multispecific polypeptide constructs or antibodies as disclosed herein are provided.

On the other hand, host cells containing vectors as disclosed herein are provided.

On the other hand, methods for preparing multispecific polypeptide constructs or antibodies as disclosed herein are provided, comprising culturing host cells and optionally isolating the multispecific polypeptide constructs from said host cells and/or culture medium.

On the other hand, methods are provided for screening and/or identifying multispecific polypeptide constructs or antibodies as disclosed herein, wherein the NK cell targeting domain is anti-NKp80.

On the other hand, pharmaceutical compositions comprising multispecific polypeptide constructs or antibodies as disclosed herein are provided.

On the other hand, methods for treating cancer are provided, which include administering a pharmaceutical composition as disclosed herein to a subject in need of such treatment, wherein a multispecific polypeptide construct or antibody is administered in an effective amount for treating the subject's cancer.

In some instances, the subjects had cancer cells expressing HER2, CD20, and/or EGFR.

Brief description of the attached figures

To better understand the implementation of the various descriptions, reference should be made to the following detailed description together with the accompanying drawings, wherein the same reference numerals refer to the corresponding parts throughout the drawings.

Figure 1 shows a summary of the NKp80 activating binders identified from antibody discovery to functional characterization. FcX represents the Fc region with reduced ADCC function.

Figure 2 shows a representative plot of cytotoxicity (percentage of kill relative to untreated control) of NKp80-binding clones against the HER2-positive tumor cell line N87 at an appropriate effector cell:target cell ratio (ET ratio). Screening was performed in two non-overlapping batches. The NKp80 binders used in this experiment were in a trispecific format containing an Fc region with attenuated ADCC ability (anti-HER2-anti-NKp80-FcX). Activating clones were defined as clones that consistently showed greater cytotoxicity than the median cutoff value in at least two replicates. The 20 activating binders in this representative assay are highlighted using triangle symbols. FcX: Attenuated ADCC function. Triangle: Activating binder; Circle: Non-activating binder.

Figure 3A shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) for four selected NKp80 activation conjugates against HER2-positive OVCAR3 tumor cells. These four NKp80 conjugates are humanized and expressed in a trispecific format, containing fully functional Fc regions (anti-HER2-anti-NKp80-Fc and anti-HER2-Fc), while the trastuzumab control does not contain anti-NKp80 (and therefore anti-HER2-Fc). Figure 3B shows the cytotoxicity (percentage of kill relative to untreated control) of the four selected NKp80 activation conjugates against HER2-positive N87 cells. The NKp80 conjugates used in this experiment are in a trispecific format, containing Fc regions with attenuated ADCC ability (anti-HER2-anti-NKp80-FcX). This figure shows the mean cytotoxicity values calculated from three independent assays (n=3). These clones exhibited greater cytotoxicity than trastuzumab containing a weakened Fc region (anti-HER2-FcX), but weaker cytotoxicity than trastuzumab containing a fully functional Fc (anti-HER2-Fc), demonstrating that a functional Fc is essential for enhancing the performance shown in Figure 3A. This is likely due to the lower abundance of NKp80 on NK cells compared to CD16 (70,000 copies of CD16 per cell vs. 4,000 copies of NKp80 per cell), as reported in previous studies. FcX: Fc with weakened ADCC function.

Figure 4 shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) of the conjugate containing NKp80 clone 87-2 (trispecific, anti-HER2-anti-NKp80-Fc) relative to trastuzumab. Experiments were performed for each cell line using 9-dose series and at the same effector cell:target cell ratio using PBMCs from healthy donors. Data were normalized to the untreated control and plotted in Prism.

Figure 5 shows a representative flow cytometry analysis of CD25 and CD137 expression populations on NK and T cells in the presence of the indicated antibody (0.08 nM). Secreted IFN-γ levels were also measured in the presence of the indicated antibody (0.1 nM). Experiments were performed using the HER2-positive tumor cell line HCT116 as the target cells with an appropriate ET ratio (PBMC:HCT116 cells).

Figure 6 shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) of the NKp80 clone 87-2 conjugate (trispecific, anti-EGFR-anti-NKp80-Fc) against cetuximab tested in two cell lines (HCT116 and MDA-MB-231, top and middle). Anti-EGFR experiments were performed using PBMCs from healthy donors, with a 9-dose series and at the same effector cell:target cell ratio. Data were normalized to the untreated control and plotted in Prism. The bottom plot shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) of the NKp80 clone 87-2 conjugate (trispecific, anti-CD20-anti-NKp80-Fc) against rituximab tested in RAJI cells. Anti-CD20 experiments were performed using NK cells purified from PBMCs from healthy donors, with a 9-dose series and at an effector cell:target cell ratio of 2.5. The data were normalized to the unprocessed control and plotted in Prism.

Figure 7 shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated controls), demonstrating the safety and specificity of the conjugate containing NKp80 clone 87-2. The cytotoxicity of a fully functional trispecific conjugate (anti-HER2-anti-NKp80-Fc) containing an antibody against NKp80 clone 87-2 and trastuzumab against HER2-positive normal fetal lung fibroblasts (MRC-5 and WI-38, top and middle) was tested. An isotype control containing the conjugate containing NKp80 clone 87-2 (where anti-HER2 was replaced by non-targeting IgG) was added to HER2-positive colorectal cancer cells (HCT116, bottom) along with trastuzumab (positive control). Experiments were performed for each cell line using 9-dose series and at the same effector cell:target cell ratio using PBMCs from healthy donors. Data were normalized to untreated controls and plotted in Prism.

Figure 8 shows four clusters containing different NKp80 clones of the active binder. Clones 94-1 and 101-1 are far apart 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 profile values for each clone were constructed using the binding index input generated by tandem binning employing biological layer interferometry (BLI).

Figure 9 shows the cluster analysis of all 20 NKp80-activating clones. Clusters were generated by the sequence identity matrix of heavy chain CDR3. This sequence-based analysis shows that clones 87-2 and 45-2 are in the same cluster (cluster 2), while clones 101-1 and 94-2 are in separate clusters (clusters 5 and 3) and are far apart, consistent with the BLI binding shown in Figure 8. This analysis indicates that the 20 NKp80-activating clones are sequence-diverse.

Figure 10 shows the amino acid sequences of the complementarity-determining regions (CDRs) of the variable heavy chain (VH) and variable light chain (VL) of the NKp80-activated clone.

Figure 11 shows the amino acid sequences of the variable heavy chain (VH) and variable light chain (VL) framework regions (FR) of the NKp80-activated clone.

Figure 12 shows the amino acid sequences of the VH, VL, CH, and CL domains of the NKp80 NK cell receptor, wild-type Fc domain, silent Fc domain, and antigen-targeting domains that bind HER2 (trastuzumab), EGFR (cetuximab), and CD20 (rituximab).

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-targeting peptide constructs in which the NK cell conjugate and antigen-targeting domain are arranged in four different configurations. Figure 13E shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) for various configurations of a trispecific conjugate containing anti-EGFR (cetuximab), anti-NKp80, and wild-type Fc domains relative to cetuximab. MDA-MB-231 cells were used as target cells, PBMCs from healthy donors were used, experiments were conducted using a 9-dose series, and the experiment was performed at 28 E:T. Data taken at 48 hours were normalized to the untreated control and plotted in Prism.

Figure 14 shows the amino acid percentage (%) identity matrix of the variable heavy chain complementarity-determining region 3 (VHCDR3) for each of the 20 NKp80-binding polypeptide construct clones.

Figure 15 illustrates how the trispecific binder works: it binds to CD16 and NKp80 on innate immune cells, as well as the target antigen on target cells. This trispecific binding triggers antibody-dependent cytotoxicity (ADCC) of innate immune cells to kill target cells loaded with the target antigen of interest.

Figure 16 shows representative cytotoxicity dose-response curves (percentage of kill relative to untreated control) for the conjugate targeting anti-NKp80(87-2). The cytotoxicity of a trispecific conjugate containing an antibody against NKp80 clone 87-2 and a variant of the Fc region (anti-HER2-anti-NKp80-Fc(variant), where the Fc(variant) can be wild-type Fc (WT), inactivated Fc mutant (LALA), or enhanced (E)Fc) against HER2-positive breast cancer cell lines was tested. PBMCs from healthy donors were used, with a 9-dose series and experiments performed for each cell line at the same effector cell:target cell ratio. 24-hour data were normalized to the untreated control and plotted in Prism.

Detailed description

I. Introduction

Antibody-enhanced innate cell regulators (AIMs) are first-in-class, next-generation NK cell conjugate (NKCE)-based molecules with applications across a variety of indications, including cancer, infectious diseases, and autoimmune disorders. In cancer immunotherapy, treatment approaches face limitations; for example, monoclonal antibodies are limited to patients with high target expression. The benefits of checkpoint inhibitors are limited to a small patient population, and the high risk of cytokine storm release is associated with T-cell bispecific antibodies and CAR-T cell therapy.

Natural killer (NK) cells are an essential component of tumor immune surveillance, as evidenced by increased cancer susceptibility and metastasis associated with diminished NK activity in mouse models and clinical studies. Using a range of germline-encoded surface receptors, NK cells recognize and rapidly act on malignant cells without prior sensitization. Upon activation, NK cells release cytotoxic granules containing perforin and granzymes to directly lyse tumor cells, much like activated cytotoxic T cells. NK cells are also potent producers of chemokines and cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and are therefore essential in regulating adaptive immune responses. Due to their innate ability to eliminate tumor cells, NK cell-based immunotherapies for cancer have been investigated for decades. Early clinical trials have demonstrated the overall safety of NK cell infusions, even in allogeneic settings. The feasibility of utilizing allogeneic NK cells, the established safety profile, and the rapid onset of action of NK cells have largely contributed to the emerging effort to develop “off-the-shelf” NK cell-based cancer immunotherapies. However, there are many challenges to overcome, such as difficulty in achieving clinical-grade ex vivo expansion, limited in vivo persistence, limited infiltration into solid tumors, and tumor editing to evade NK cell activity.

In some instances, AIM NKCE enhances innate immune cell responses to improve anti-tumor efficacy and minimize side effects. This multispecific peptide construct can be used as a single agent or in combination with existing disease-targeting therapies, such as NK cell therapy, T-cell checkpoint inhibitors, or small molecules.

II. Definition

To make this disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly stated herein, the following terms shall have the following meanings. Other definitions are provided throughout the application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art related to this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, ^2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, ^3rd ed., 1999, Academia Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide general dictionaries for those skilled in the art of the many terms used in this disclosure.

Throughout this specification and in the appended claims, unless the context otherwise requires, the term "comprise" and its variations (such as "comprises" and "comprising") are to be understood as implying inclusion of the stated integers or steps or groups of integers or steps, but not excluding any other integers or steps or groups of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing" or "including," or sometimes by the term "having." It should be understood that whenever the term "comprising" is used in describing aspects herein, other similar aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

The terms “about” or “consistently composed of” refer to a value or composition within an acceptable range of error for a particular value or composition, as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, in some instances, according to convention in the art, “about” or “consistently composed of” can mean within one standard deviation or more than one standard deviation. Alternatively, “about” or “consistently composed of” can mean a range of up to 10% (i.e., +/- 10%).

When used herein, "consisting of" excludes any element, step, or component not specified in the elements of the claim. When used herein, "consisting substantially of" does not exclude materials or steps that do not significantly affect the essential and novel features of the claim.

The use of alternatives (such as "or") should be understood to mean any one, two, or any combination of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any written or enumerated elements.

Scope: Throughout this disclosure, various aspects of the disclosure are presented in scope format. The scope format is for convenience and brevity only and should not be construed as an immutable limitation on the scope of the disclosure. Therefore, in this document, a scope description is considered to specifically disclose all possible sub-ranges within that range, as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 is considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

In some instances, multispecific peptide constructs include domains that bind to one or more innate immune cell modulators. In some instances, these domains are referred to as “targeting domains” or “binding domains” associated with 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, when bound, modify cellular activity and induce changes in the overall immune response. In some instances, changes in the immune response triggered by modulators help the body fight cancer, infection, or other diseases.

In some instances, multispecific polypeptide constructs contain domains containing antibodies or fragments thereof. When used herein, the term "antibody" includes both the complete antibody and its binding fragment. The basic antibody structural unit is a tetramer of subunits. Each tetramer comprises 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 includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed as being linked to a cleavable signal peptide. Variable regions without a signal peptide are sometimes referred to as mature variable regions. Thus, for example, a light chain mature variable region refers to a light chain variable region without a light chain signal peptide. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Constant regions may include any one or all of the CH1, hinge, CH2, and CH3 regions. It should be understood that sequence modifications of the constant region domains may also be used. For example, one or more amino acids (such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) can be substituted, added, and/or deleted from the constant domain of an antibody without significantly altering its ability to bind to the target antigen.

In some instances, multispecific polypeptide constructs contain domains containing monoclonal antibodies or fragments thereof. As used herein, the term "monoclonal antibody" refers to an antibody with a substantially identical amino acid sequence or an antibody derived from the same genetic source. Monoclonal antibody compositions exhibit binding specificity and affinity for a specific epitope, or binding specificity and affinity for multiple specific epitopes.

In some instances, multispecific polypeptide constructs contain domains containing chimeric antibodies or fragments thereof. The term "chimeric antibody" (or its antigen-binding fragment) is an antibody molecule (or its antigen-binding fragment) in which (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and/or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged by a variable region with different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, chimeric antibodies can exhibit reduced antigenicity in humans while maintaining the specificity for recognizing antigens compared to the original mouse antibody.

In some instances, multispecific polypeptide constructs contain 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) with variable regions, where both the framework region and CDR region are derived from human-derived sequences. Antibodies or immunoglobulins are classified into categories such as IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of their heavy chain constant regions, and several of these can be further subdivided into subclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Therefore, when antibody molecules are used for therapeutic purposes and require antibody effector function, human IgG constant region domains, particularly those of the IgG1 and IgG3 isotypes, can be used. Alternatively, when antibody molecules are used for therapeutic purposes and do not require antibody effector function, IgG2 and IgG4 isotypes can be used. Furthermore, if the antibody contains a constant region, that constant region is also derived from these human sequences. Humanized antibodies (or their antigen-binding fragments) retain the reactivity of non-human antibodies but are less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR region and replacing the remainder of the antibody with its human counterpart (i.e., the frame portion of the constant region and the variable region). Additional frame region modifications can be made within the human frame sequence and within the CDR sequence of a germline derived from another mammalian species. Humanized antibodies of this disclosure may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo, or conserved substitutions to promote stabilization or manufacturing). The definition of a humanized antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using various techniques known in the art, including phage display libraries, and by administering antigens to transgenic animals that have been modified to produce such antibodies in response to an antigen challenge but whose endogenous loci have been inactivated, such as xenogeneic mice immunized via human B-cell hybridoma technology.

In some instances, multispecific polypeptide constructs contain domains containing recombinant humanized antibodies or fragments thereof. As used herein, the term "recombinant humanized antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from host cells transformed to express humanized antibodies, for example from transfected tumors, and antibodies prepared, expressed, created, or isolated by any other means involving splicing all or part of a human immunoglobulin gene or sequence with other DNA sequences.

In some instances, multispecific polypeptide constructs contain domains containing antibodies or fragments thereof. The term "isolated antibody" refers to an antibody that is substantially free of other cellular material and/or chemicals.

In some instances, multispecific peptide constructs contain binding domains. As used herein, the term "binding domain" refers to, but is not limited to, any of the following: a "Fab fragment," a monovalent fragment consisting of VL, VH, CL, and CH1 domains; an "F(ab)2 fragment," a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region; an "Fd fragment," consisting of VH and CH1 domains; an "Fv fragment," consisting of the VL and VH domains of an antibody single arm; a "single-domain antibody (dAb) fragment," consisting of a VH domain; a separated "complementarity-determining region" (CDR); a "single-chain Fv"; a "disulfide-bonded stable variable fragment (dsFv)"; a "single-chain antibody fragment (sca)." b)"; STAB, "single-domain antibody (sdAb or dAb)"; "single-domain heavy chain antibody (sdCH)"; "single-domain light chain antibody (sdCL)"; "nanobody" or "single variable domain on heavy chain (VHH)"; "variable novel antigen receptor (VNAR)" from sharks; single-domain antibodies based on VNAR structures; and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, antiicalin, fibronectin, and binding sites constructed into the constant region of the antibody (e.g., f-star technology (F-star's modular antibody technology™)).

In some instances, multispecific peptide constructs contain linkers. As used herein, the term "linker" refers to an intermediate peptide sequence that connects any two components within a multispecific peptide construct and contains major repeats of residues such as glycine (G) and serine (S). Such linkers are broadly classified as flexible linkers, rigid linkers, and cleavable linkers. A G4S linker is a polyglycine-serine linker having four glycine residues and one serine residue. A [(G4S)n] linker, as described herein, refers to a number of (n) consecutively repeating G4S blocks.

In some instances, multispecific peptide constructs include antigen-binding domains. As used herein, the term "antigen" refers to a structure on the surface of a target cell that is generally considered to be associated with a specific disease state and to which the peptide constructs of this disclosure bind. As used herein, the term "epitope" defines an antigenic determinant that an antibody, antibody fragment, or other binding domain specifically binds to. "Antigen" and "epitope" may be used interchangeably in the context of this disclosure and refer to target molecules on the surface of target cells.

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

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

In some instances, multispecific peptide constructs contain different regions or domains. As used herein, the terms “region” and “domain” are understood to describe the same component and are therefore used interchangeably.

In some instances, multispecific peptide constructs contain complementarity-determining regions (CDRs). The term “complementarity-determining region” (“CDR”) refers to the amino acid sequence whose boundaries are determined using any number of well-known schemes, including those described by Kabat (i.e., the “Kabat” numbering scheme); Al-Lazikani (“Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (“IMGT” numbering scheme), etc. For example, in the classical format, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (VHCDR1), 50-65 (VHCDR2), and 95-102 (VHCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (VLCDR1), 50-56 (VLCDR2), and 89-97 (VLCDR3). According to Chothia, the CDR amino acids in human VH are numbered 26-32 (VHCDR1), 52-56 (VHCDR2), and 95-102 (VHCDR3); and the amino acid residues in human VL are numbered 24-34 (VLCDR1), 50-56 (VLCDR2), and 89-97 (VLCDR3). Combining the CDR definitions of Kabat and Chothia, the CDR consists of amino acid residues 26-35 (VHCDR1), 50-65 (VHCDR2), and 95-102 (VHCDR3) in human VH and amino acid residues 24-34 (VLCDR1), 50-56 (LVCDR2), and 89-97 (VLCDR3) in human VL. According to IMGT, the CDR amino acid residues in VH are approximately numbered 26-35 (VHCDR1), 51-57 (VHCDR2), and 93-102 (VHCDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (VLCDR1), 50-52 (VLCDR2), and 89-97 (VLCDR3) (according to "Kabat" numbering). The CDRs of the antibody can be determined using the IMGT/DomainGap Align procedure based on IMGT.

In some instances, multispecific polypeptide constructs comprise light and heavy chains. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε. The heavy chains of antibodies define the antibody isotypes as IgG, IgM, IgA, IgD, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain also includes “D” regions of about 10 or more amino acids. As used herein, the terms “variable light chain CDR1,” “variable light chain CDR2,” “variable light chain CDR3,” “variable heavy chain CDR1,” “variable heavy chain CDR2,” and “variable heavy chain CDR3” refer to VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2, and VHCDR3, respectively.

In some instances, when the multispecific peptide construct is a multispecific antigen-binding peptide, the multispecific peptide construct as described herein comprises multiple binding domains. These domains bind to or recognize a group selected from NK regulators or target antigens. In some instances, each binding domain of the multispecific peptide construct comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs as described herein. In some instances, the multispecific peptide construct comprises a combination of one or more CDRs as described herein.

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

In some instances, the multispecific polypeptide construct contains an NKp80 conjugate. The term "NKp80 conjugate" refers to a molecule capable of binding NKp80, such as an antibody that binds to NKp80 expressed on NK cells. In some instances, the NKp80 conjugate is a human NKp80 conjugate (huNKp80 conjugate) and/or a cynomolgus monkey NKp80 conjugate (cyNKp80 conjugate).

In some instances, multispecific polypeptide constructs contain an Fc domain. As used herein, the term "Fc domain" refers to a dimer complex containing a C-terminal polypeptide sequence of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is available by papain digestion of the intact antibody. The Fc sequence of an immunoglobulin typically contains two constant regions, CH2 and CH3, and optionally includes a CH4 region. Also considered part of this disclosure are Fc polypeptides comprising the polypeptide constituting the Fc domain, such as monomeric Fc. Fc polypeptides are available from any suitable immunoglobulin, such as human IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. Fc polypeptides are available from humans or any other non-human mammal. The Fc domain contains the carboxyl-terminal portion of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc domain; this region is also the portion recognized by Fc receptors (FcRs) found on certain types of cells.

The multispecific peptide constructs disclosed herein also include functional Fc domains with “effective functions” having a native/wild-type Fc region (FcE). In some instances, the “effective function” is selected from CD16 binding; C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the Fc region to be combined with a binding domain (e.g., antibody variable domain) and can be evaluated using various assays known in the art. In some instances, the Fc domain includes functional antibody-dependent cytotoxicity (ADCC) (e.g., via CD16 binding), attenuated ADCC (FcX) (e.g., by Fc mutation to provide a attenuated Fc domain or by antibody conformation to achieve attenuated ADCC), Fc silencing domain/inactivation mutant Fc domain (FcLALA), which is achieved by Fc mutation, or enhanced ADCC (FcE) (e.g., by Fc mutation to provide enhanced activity of the Fc domain).

The portion considered to be part of this disclosure also includes multispecific polypeptide constructs comprising a natural/wild-type Fc domain and/or a variant Fc domain. The variant Fc domain (or Fc mutant domain) comprises an amino acid sequence that differs from the amino acid sequence of the natural/wild-type Fc domain due to at least one amino acid modification, preferably one or more amino acid substitutions. In some instances, the variant Fc domain has at least one amino acid substitution compared to the natural/wild-type Fc domain sequence or the Fc domain of the parent polypeptide. In some instances, the variant Fc region (or Fc mutant region) contains about 1 to about 10 amino acid substitutions in the natural/wild-type Fc region. In some instances, the variant Fc region has at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94% homology to the natural/wild-type Fc domain sequence, 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.

The exemplary multispecific peptide construct also includes an "Fc component", which may include a hinge domain, a CH2 domain, or a CH3 domain of the Fc domain.

In some instances, multispecific peptide constructs include framework regions. As used herein, the term "framework region (FR)" refers to each domain of the variable light or heavy chain that separates the CDR.

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” refer to VLFR1, VLFR2, VLFR3, VLFR4, VHFR1, VHFR2, VHFR3 and VHFR4, respectively.

In some instances, multispecific peptide constructs are bispecific antigen-binding peptide constructs. As used herein, the term "bispecific antigen-binding peptide construct" refers to a multispecific peptide construct containing two binding domains, such as an antibody domain, but other binding domains may also be used. When the binding domains contain an antibody domain, each domain contains at least three code-receiving domains (CDRs) and a frame; for example, VHH contains three CDRs, while Fab contains six CDRs.

In some instances, multispecific peptide constructs are trispecific antigen-binding peptide constructs. As used herein, the term "trispecific antigen-binding peptide construct" refers to a multispecific peptide construct that binds to three different epitopes or three different binding sites on three different targets.

In some instances, a multispecific peptide construct is a multispecific antigen-binding peptide construct. As used herein, the term "multispecific antigen-binding peptide construct" refers to a multispecific peptide construct having two or more binding domains that bind to two or more distinct epitopes on at least two or more distinct targets. The term "multispecific antigen-binding peptide construct" includes, but is not limited to, bispecific, trispecific, tetraspecific, pentaspecific, and hexaspecific constructs.

In some instances, the multispecific peptide constructs of this disclosure exhibit synergistic functions in their cytotoxicity. As used herein, the terms “synergistic function” or “synergistic biological function” refer to biological activity or the level of biological activity or the effect on biological function or activity that is: 1) not observed in the individual peptide components of the multispecific peptide construct; 2) observed when two (or more) binding domains are linked in a specific format; or 3) higher or lower activity compared to, for example, the activity observed when using the individual peptide components of the multispecific constructs of this disclosure alone, and enhanced activity observed only in bispecific peptide constructs.

Therefore, "synergy" includes new biological functions or new activities. Synergistic functions, as used in this article, generally do not involve simple targeting, i.e., based solely on binding, but rather usually involve some form of inhibition, activation, signal transduction, or similar function following binding.

In some instances, multispecific polypeptide constructs are fusion proteins. 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 chaperone X or Y (where appropriate). In some instances, fusion proteins are translated polypeptide constructs expressed by a genetic construct via recombinant technology. In some instances, fusion proteins are expressed in a host by a DNA construct. In the context of this disclosure, one of the key characteristics of fusion proteins is that they are expressed from the cell as a "single polypeptide".

In some instances, multispecific peptide constructs contain an antigen-binding domain, or a binding domain, or an antigen-binding fragment, or an antigen-targeting domain. As used herein, an "antigen-binding moiety," "binding domain," "antigen-binding fragment," or "antigen-targeting domain" of a multispecific peptide construct refers to one or more peptide sequences within the multispecific peptide construct that have the ability to specifically bind to a given antigen.

In some instances, multispecific peptide constructs are NK cell binders. As used herein, the terms "binding agent," "natural killer cell binder," "NK cell binder," or "NK binder" refer to synthetic peptides or multifunctional antibodies that enable tumor cells and NK cells to aggregate and trigger NK cells to destroy tumor cells. In some instances, multispecific peptide constructs are described as "binding agents" rather than binders. As used herein, the term "binding agent" includes both activating and inactivating binders, appearing in cases where some multispecific peptide constructs are found to be inactivating during the screening process.

In some instances, the binding affinity and/or specificity of multispecific peptide constructs are evaluated. As used herein, the terms “bind to” or “bind” refer to a measurable and reproducible interaction, such as the binding between a target and an antigen-binding peptide construct, which is determined by the presence or absence of a target in the presence of a heterogeneous molecular population including biomolecules. It refers to the ability of a single antibody to react with one antigenic determinant and not with different antigenic determinants.

In some instances, multispecific peptide constructs include an activation binding domain. As used herein, "activation binding" refers to a peptide construct that exhibits above-defined baseline cytotoxicity against target cells in a cytotoxicity assay (i.e., above the median cytotoxicity level of all screened clones) and is capable of mediating NK cell cytotoxicity to lyse target cells.

In some instances, multispecific peptide constructs contain a non-binding domain or a non-activating binding domain. As used herein, “non-binding” or “non-activating binding” refers to a peptide construct that does not exhibit significant cytotoxicity to target cells and shows below-defined median baseline activity in cytotoxicity assays. In some instances, the non-binding agent is used in conjunction with a target antigen antibody (such as an anti-HER2 antibody: anti-HER2-FcX) to determine baseline activity.

In some instances, multispecific peptide constructs are evaluated using half-maximal effective concentration (WMC) or EC50. As used herein, the term “half-maximal effective concentration” or “EC50” refers to the concentration of the antibody or a portion thereof that induces a response (whether measured in vivo or in vitro) that is 50% of the maximum response (i.e., half between the maximum response and baseline). The term “average EC50 fold change potency” refers to the fold change in EC50 of the multispecific peptide construct relative to a control drug. Trastuzumab is an exemplary control of exemplary HER2-specific multispecific peptide constructs as standard of care for the treatment of HER2-positive early and advanced breast cancer. Cetuximab is used as a control in the presentation data of EGFR-specific multispecific peptide constructs.

In some instances, multispecific polypeptide constructs are contacted with peripheral blood mononuclear cells (PBMCs). The term "peripheral blood mononuclear cells" or "PBMCs" refers to mononuclear blood cells harvested from healthy subjects and subsequently cultured for use in various bioassays, such as cytotoxicity assays.

In some instances, the cytotoxic potential of multispecific peptide constructs is evaluated in the presence of target cells and effector cells. As used herein, “effector cell” refers to a cell that performs a specific function in response to a stimulus, in this case, an NK cell. As used herein, “target cell” refers to a cell that expresses an antibody or a fragment thereof that specifically binds to a specific receptor and/or antigen and/or epitope. In some instances, cytotoxicity assays expose the multispecific peptide construct to a specific ratio of effector cells to target cells (referred to as “effector cell to target cell ratio” or “effector cell:target cell ratio”, “E:T ratio”, or “E/T ratio”).

In some instances, as used herein, the term "subject" includes both patients and non-patients. The term "patient" refers to an individual who has or is likely to have a medical condition, while "non-patient" refers to an individual who does not have and is unlikely to have said medical condition. "Non-patient" includes healthy individuals, individuals who are not ill, and/or individuals without said medical condition. The term "subject" includes both humans and animals. The terms "subject" and "patient" are used interchangeably herein.

In some instances, multispecific polypeptide constructs or formulations containing said constructs are used to administer treatment to subjects in need. The term “administration” means the physical introduction of a pharmaceutical agent into a subject using any of the various methods and delivery systems known in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means a method of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some instances, formulations are administered via non-parenteral routes, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal application, such as intranasal, vaginal, rectal, sublingual, or topical. Application may also be performed, for example, once, multiple times, and/or over one or more extended periods.

"Parenteral" administration of the compositions disclosed herein includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

In some instances, multispecific peptide constructs are used to treat diseases in subjects. As used herein, “treatment” or “curing” refers to a method of obtaining a beneficial or desired outcome, including and preferably a clinical outcome. Treatment can refer to the relief of symptoms of a disease or condition, or the delay of its progression. Treatment is generally effective by administering a therapeutically effective amount of the multispecific peptide construct to a subject in need.

In some instances, a multispecific polypeptide construct or a formulation containing the construct is administered to a subject in need at a therapeutically effective amount, or an effective dose, or a therapeutically effective dose. The "therapeutically effective amount," "effective dose," "effective quantity," or "therapeuticly effective dose" of the multispecific polypeptide construct refers to any quantity of the construct that, when used alone or in combination with other therapeutic agents, protects a subject from the onset of disease or promotes disease resolution, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of injury or disability due to disease involvement. The ability of a multispecific polypeptide construct to promote disease resolution can be assessed by various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting human efficacy, or by measuring the activity of the multispecific polypeptide construct in an in vitro assay.

In some instances, multispecific peptide constructs are used to treat cancer. "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also spread to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors.

In some instances, multispecific peptide constructs have exhibited antitumor effects. As used herein, "antitumor effect" refers to biological effects manifested as tumor volume reduction, tumor cell count reduction, tumor cell proliferation reduction, metastasis reduction, increased overall survival or progression-free survival, increased life expectancy, or improvement in various tumor-related physiological symptoms. Antitumor effect can also refer to the prevention of tumor development.

In some instances, treating subjects with multispecific peptide constructs has facilitated progression-free survival. As used herein, the term "progression-free survival," abbreviated as PFS, refers to the time from the date of treatment to the date of disease progression.

In some instances, treating subjects with multispecific peptide constructs has helped prevent or delay 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 specific disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignancies, tumor metastasis, and death.

As used in this article, “duration of response”, abbreviated as DOR, refers to the time between a subject’s first objective response and the date on which disease progression is confirmed or death occurs according to the revised IWG criteria for response to malignant lymphoma.

In some instances, treatment of subjects with multispecific peptide constructs has helped prevent and/or reduce the severity of symptoms. The terms “prevention” and/or “reduction of symptom severity” refer to delaying onset, reducing symptom severity, reducing and/or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and/or improving at least one sign or symptom of the disease.

In some instances, treatment of subjects with multispecific peptide constructs induces an immune response in the subjects. An “immune response” refers to the action of immune system cells (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and/or elimination from the vertebrate body of invading pathogens, infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation.

In some instances, multispecific polypeptide constructs are encoded by nucleic acids. As used herein, the term "nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. Furthermore, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In some instances, nucleic acid molecules can be modified. In the case of double-stranded polymers, "nucleic acid" can refer to either one or both strands of the molecule.

In some instances, multispecific polypeptide constructs are encoded by nucleotide sequences. For the purposes of nucleic acids, the term "nucleotide sequence" refers to a continuous series of nucleotides linked by covalent connections (such as phosphate linkages (e.g., phosphodiester, alkyl and aryl phosphonates, thiophosphates, phosphotriester bonds) and/or non-phosphate linkages (e.g., peptide and/or aminosulfonate bonds)). In some instances, the nucleotide sequences encoding target-binding molecules, such as those linked to localization domains, are heterologous sequences (e.g., genes originating from different species or cell types).

As used herein, the terms “sequence identity” or “homology” refer to the percentage of sequence identity determined by the largest alignment of the polypeptide sequences according to the Kabat numbering convention. After alignment, if a region of the subject polypeptide (e.g., the entire mature 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 subject and reference polypeptide regions is calculated by dividing the number of positions in both regions occupied by the same amino acid by the total number of aligned positions in both regions (excluding vacancies) and multiplying by 100 to convert to a percentage.

The best alignment of sequences for comparison can be achieved through, for example, local homology algorithms of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), homology alignment algorithms of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), search similarity methods of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.) or visual inspection (see generally Ausubel et al. 2000, Current Protocols in Molecular Biology). One example of an algorithm suitable for determining 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 publicly available from the National Center for Biotechnology Information (accessible via the National Institutes of Health NCBI internet server). Typically, default program parameters are used for sequence comparisons, but custom 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 as default parameters (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

In some instances, multispecific polypeptide constructs or any of their domains or segments contain substitutions. For example, “conservative substitutions” can be made based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or amphiphilicity of the amino acid residues involved. Twenty naturally occurring amino acids can be grouped 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitution” is defined as replacing an amino acid with another amino acid listed in the same group of the six standard amino acid groups mentioned above. For example, replacing Asp with Glu retains a negative charge in such a modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix.

As used herein, “non-conservative substitution” is defined as replacing an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1)-(6) above.

In some instances, substitutions also include non-classical amino acids. Illustrative non-classical amino acids generally include, but are not limited to, selenocysteine, pyrrolidone, 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, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfoalanine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs.

On the other hand, a host cell containing the vector of this disclosure is provided.

In some instances, such as those disclosed herein, the host cell contains a cloning vector or expression vector configured to express a multispecific polypeptide or multispecific antibody as disclosed herein.

In some instances, nucleic acids encoding multispecific polypeptide constructs are incorporated into vectors. A “vector” is any molecule or composition that can deliver a nucleic acid sequence into a suitable host cell, where, for example, the synthesis of the encoded polypeptide can occur. Typically and preferably, the vector is a nucleic acid engineered using recombinant DNA techniques known in the art to incorporate the desired nucleic acid sequence (e.g., the nucleic acid of this disclosure). Expression vectors typically contain one or more of the following components (if they are 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 containing donor and acceptor splicing sites, a leader sequence for secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting the nucleic acid encoding the polypeptide to be expressed, and optional labeling elements.

In some instances, the various domains of a multispecific polypeptide construct are operatively linked. As used herein, the term "operatively linked" can refer to the juxtaposition or arrangement of specified elements that allows them to work synergistically to produce an effect. For example, if a promoter controls the transcription of a coding sequence, the promoter can be operatively linked to the coding sequence.

In some instances, nucleic acids encoding multispecific polypeptide constructs are inserted into expression vectors. An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other expression elements are provided by a host cell or in vitro expression system. Expression vectors include all vectors known in the art, such as visceral particles, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus), which incorporate the recombinant polynucleotide.

In some instances, 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 from its natural state by human intervention. For example, a naturally occurring composition or substance is isolated if it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from the material coexisting in its natural state is isolated, as used herein.

In some instances, multispecific polypeptide constructs are encoded by nucleic acids. "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Thus, a gene encodes 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 sequence listings) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. In some instances, nucleotide sequences encoding proteins or RNA include introns to the extent that nucleotide sequences encoding the protein may contain one or more introns in some versions.

In some instances, vectors containing nucleic acids encoding multispecific polypeptide constructs include promoters. As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the cellular or introduced synthetic machinery and is required to initiate specific transcription of a polynucleotide sequence.

As used herein, the term "promoter/regulatory sequence" refers to the nucleic acid sequence required for the expression of a gene product operatively linked to a promoter/regulatory sequence. In some instances, this sequence is a core promoter sequence; in others, it may also include enhancer sequences and other regulatory elements required for the expression of the gene product. In some instances, the promoter/regulatory sequence expresses the gene product in a tissue-specific manner.

A "constitutive" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, enables the gene product to be produced in the cell under most or all physiological conditions.

An "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell essentially only if the inducer corresponding to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence that, when operatively linked to a polynucleotide that encodes or is specified by a gene, causes the gene product to be produced in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

As used in this article, "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

In some instances, multispecific polypeptide constructs comprise 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 a protein sequence or peptide sequence may contain. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers to short chains, such as those commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, which are commonly referred to in the art as proteins, among which many types exist. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

In some instances, the sequence encoding a multispecific polypeptide construct or any of its domains contains conserved sequence modifications. As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that may or may not significantly alter the binding characteristics of an antibody containing an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. In some instances, modifications are introduced into the sequences disclosed herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions are those in which amino acid residues are substituted with amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the sequence can be replaced with other amino acid residues from the same side chain family, and the ability of the altered antibody to bind to the antigen can be tested using recognized functional assays.

In some instances, exogenous nucleic acids encoding multispecific polypeptide constructs or any of their domains are used to transfect, transform, or transduce one or more host cells. As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary test cells and their progeny.

This disclosure is not limited to the specific methodologies, schemes, materials, reagents, and substances described herein, and therefore can be varied. The terminology used herein is for the purpose of describing particular examples only and is not intended to limit the scope of this disclosure, which is defined only by the claims.

In some instances, this disclosure includes one or more features as defined above herein.

III. Detailed Implementation

In some instances, the multispecific polypeptide construct has 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.

In some instances, the target cell antigen-binding domain of a multispecific peptide construct specifically binds to target cell antigens. In its most general form (and when references are not defined), the term "specific binding" refers to the ability of a multispecific peptide construct to distinguish between a target of interest and non-target molecules/parts, as determined, for example, by specificity assays known in the art. Such assays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradioassay (IRMA), surface plasmon resonance (SPR) assays, and peptide scanning.

In some instances, multispecific peptide constructs bind one or more innate immune cell modulators and/or target cell antigens with greater affinity, stronger affinity, and more readily and/or for longer duration than they bind to other antigens.

In some instances, the multispecific peptide construct includes one or more innate immune cell targeting domains and/or one or more antigen-targeting domains. In some instances, the multispecific peptide construct includes one innate immune cell targeting domain and one antigen-targeting domain. In some instances, the multispecific peptide construct includes two innate immune cell targeting domains and one antigen-targeting domain. In some instances, the multispecific peptide construct includes three innate immune cell targeting domains and one antigen-targeting domain. In some instances, the multispecific peptide construct includes four innate immune cell targeting domains and one antigen-targeting domain. In some instances, the multispecific peptide construct includes more than one antigen-targeting domain.

In some instances, the multispecific peptide construct is a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific antigen-binding peptide. In some instances, the multispecific peptide construct contains one or more NK cell-targeting domains and/or one or more antigen-targeting domains. In some instances, the multispecific peptide construct contains one NK cell-targeting domain and one antigen-targeting domain. In some instances, the multispecific peptide construct contains two NK cell-targeting domains and one antigen-targeting domain. In some instances, the multispecific peptide construct contains three NK cell-targeting domains and one antigen-targeting domain. In some instances, the multispecific peptide construct contains four NK cell-targeting domains and one antigen-targeting domain.

In one aspect, a multispecific polypeptide construct is provided, comprising:

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

(b) One or more NK cell targeting domains, wherein binding to NK cells can stimulate and/or inhibit the function of innate immune cells.

In some instances, the multispecific peptide construct further comprises one, two, three, four, five, six, or more antigen-targeting domains. In some instances, the multispecific peptide construct comprises one innate immune cell-targeting domain and two antigen-targeting domains. In some instances, the multispecific peptide construct comprises one innate immune cell-targeting domain and three antigen-targeting domains. In some instances, the multispecific peptide construct comprises two innate immune cell-targeting domains and two antigen-targeting domains. In some instances, the multispecific peptide 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.

In some instances, the multispecific peptide construct contains more than one antigen-targeting domain. In some instances, the multispecific peptide construct also contains one, two, three, four, five, six, or more target antigen-targeting domains.

In some instances, the multispecific peptide construct contains one NK cell targeting domain and two antigen-targeting domains. In some instances, the multispecific peptide construct contains one NK cell targeting domain and three antigen-targeting domains. In some instances, the multispecific peptide construct contains two NK cell targeting domains and two antigen-targeting domains. In some instances, the multispecific peptide construct contains 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 this disclosure.

In some instances, the NK cell targeting domain is the NKp80 targeting domain, or the anti-NKp80 domain. In some instances, 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 CDR, NKp80-binding single-chain Fv, NKp80-binding dsFv, NKp80-binding scab, NKp80-binding STAb, NKp80-binding single-domain heavy chain antibody, NKp80-binding single-domain light chain antibody, NKp80-binding VHH, NKp80-binding VNAR, and other NKp80-binding domains based on alternative scaffolds.

In some instances, one of the NK cell targeting domains is the NKp80 targeting domain.

In some instances, the NKp80 targeting domain contains:

(1) A heavy chain variable domain (VH) comprising one, two, or three complementarity-determining regions (CDRs) selected from VHCDR1 of SEQ ID NO: 51-67, 250, VHCDR2 of SEQ ID NO: 68-85, 251, and/or VHCDR3 of SEQ ID NO: 86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein; and/or

(2) Light chain variable domain (VL) comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and/or VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein.

In some instances, the NKp80 targeting domain contains:

(1) A heavy chain variable domain (VH) comprising one, two, or three complementarity-determining regions (CDRs) selected from VHCDR1 of SEQ ID NO: 51-67, 250, VHCDR2 of SEQ ID NO: 68-85, 251, and/or VHCDR3 of SEQ ID NO: 86-104, 252; and/or

(2) Light chain variable structural domain (VL) comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and/or VLCDR3 of SEQ ID NO:32-50, 249.

In some instances, the NKp80 targeting domain comprises VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3, which have 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 NO:1-104, 247-252.

In some instances, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and/or VLCDR3 have an amino acid sequence selected from SEQ ID NO:1-104, which contains 2 or 3 amino acid substitutions.

In some instances, the NKp80 targeting domain contains:

(1) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(2) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions thereon.

In some instances, the NKp80 targeting domain contains:

(1) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; and/or

(2) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256.

In some instances, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3 and/or VLFR4 have 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 NO:105-182, 253-260.

In some instances, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3 and/or VLFR4 have amino acid sequences selected from SEQ ID NO:105-182, 253-260, which contain 2 or 3 amino acid substitutions.

In some instances, the NKp80 targeting domain contains:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions thereon.

In some instances, the NKp80 targeting domain contains:

(1) VH, which includes one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252;

(2) VL, which includes one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; and/or

(4) One, two, three or four VLFRs selected from VLFR1 of SEQ ID NO:105-118, 253, VLFR2 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255 and/or VLFR4 of SEQ ID NO:138-140, 256.

In some instances, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3 and/or VLFR4 have 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 NO:1-182, 247-260.

In some instances, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and/or VLFR4 have amino acid sequences selected from SEQ ID NO:1-182, 247-260, which contain 2 or 3 amino acid substitutions.

Figure 1 summarizes exemplary NKp80 activating binders from antibody discovery to functional characterization. FcX represents the Fc region with reduced ADCC function (see Example 1).

This disclosure circumvents many of the difficulties associated with NK cell infusion and describes a novel family of NKp80 targeting domains capable of binding to and activating innate immune cells. In some instances, these domains are single innate immune cell targeting domains used in combinations comprising one, two, three, four, five, or six domains. In some instances, these domains are used in multispecific polypeptide modular constructs containing components targeting innate immune cells and/or specific antigens. In some instances, the multispecific polypeptide construct is a multispecific antigen-binding polypeptide construct. In some instances, the multispecific polypeptide construct is a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific multispecific multispecific polypeptide construct. In some instances, the multispecific polypeptide construct also binds to target cell antigens. In some instances, the multispecific polypeptide construct binds both an innate immune cell modulator and a target cell antigen. In some instances, the multispecific polypeptide construct specifically binds to one or more innate immune cell modulators and one or more target cell antigens.

A. Natural Killer (NK) Cells

Natural killer (NK) cells are specialized immune effector cells that play a crucial role in immune activation to target abnormal cells. Unlike the events required for T cell activation, NK cell activation is controlled by the interaction of NK receptors with target cells, independent of antigen processing and presentation. Due to the relatively uncomplicated cues of activation, NK cells have gained significant attention in the field of cancer immunotherapy. Numerous efforts from research in this field are emerging to develop and engineer NK cell-based cancer immunotherapies.

Various immunomodulatory molecules, including receptors, are involved in the loss and induction of self-recognition, affecting NK cell reactivity. Major activating receptors expressed on human NK cells include FcγRIIIa (CD16), NKG2D, DNAM-1, and native cytotoxic receptors containing NKp30, NKp44, NKp65, NKp80, and NKp46. Like NKG2D, NKp80, upon triggering with appropriate antibodies, stimulates NK cell cytotoxicity and induces calcium influx in human NK cells.

i.ADCC

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an effective cytotoxic mechanism primarily mediated by natural killer (NK) cells in humans. ADCC mediates the clinical benefits of several widely used cytolytic monoclonal antibodies (mAbs), and improving their efficacy will enhance cancer immunotherapy. CD16a is the receptor for the Fc portion of IgG and is responsible for triggering NK cell-mediated ADCC. Knowledge of the mechanism of action of CD16a has led to several strategies to improve ADCC by working with mAbs or NK cells.

In some instances, the multispecific peptide construct comprises an Fc domain variant. In some instances, the Fc domain variant exhibits attenuated activity or binding compared to the natural/wild-type Fc domain. In some instances, the attenuated Fc domain is referred to as FcX in this disclosure. In some instances, the attenuated Fc domain is constructed according to methods known in the art. In some instances, the components of the multispecific peptide construct are a natural/wild-type Fc domain and/or a variant Fc domain. Exemplary Fc domain variants (or Fc mutant domains) comprise an amino acid sequence that differs from the amino acid sequence of the natural/wild-type Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. In some instances, the variant Fc region has at least one amino acid substitution compared to the natural/wild-type sequence Fc region or the Fc region of the parent peptide. In some instances, the variant Fc region (or Fc mutant region) contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions in the natural/wild-type Fc region sequence. In some instances, multispecific constructs include variant Fc regions that have at least about 80% homology to the natural/wild-type sequence Fc region, 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%.

In some instances, the weakened Fc domain (FcX) confers weakened ADCC, which refers to a reduction in the measurable ADCC response of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% of the control. In some instances, the Fc silencing domain/Fc inactivation mutant (FcLALA) confers little or no measurable ADCC, which refers to a substantially complete silencing of the measurable ADCC response of at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the control, or substantially complete silencing of ADCC to the point that no measurable ADCC is detected. In some instances, the enhanced Fc domain imparts enhanced ADCC. The enhanced ADCC refers to an improvement, increase, or doubling of the measurable ADCC response, which is 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 control.

ii. Immunotherapy

The preclinical development of NK cell-based therapies is currently largely inspired by early clinical studies. With a better understanding of how NK cells are activated, the initial NK cell-based therapies were pioneered in the clinical setting of hematopoietic stem cell transplantation (HSCT), where NK cells were shown to exert graft-versus-leukemia effects. It is now believed that successful adoptive transfer requires creating a lymphopenic environment to provide a niche for donor cell survival and proliferation.

In some instances, this disclosure includes multispecific polypeptide constructs or compositions or pharmaceutical compositions or uses or methods as described herein, wherein the polypeptide or composition or pharmaceutical composition is administered to a subject via one or more routes of administration (including but not limited to local, intravascular, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intraperitoneal, intranasal, intradermal, intramuscular, etc.).

On the other hand, pharmaceutical compositions comprising multispecific polypeptide constructs or antibodies as disclosed herein are provided.

In some instances, pharmaceutical compositions for treating cancer are provided, wherein the multispecific polypeptide construct or the antibody is administered in an effective amount to a subject in need of treating cancer in the subject.

In some instances, the use of a pharmaceutical composition in the manufacture of a medicament for treating cancer is provided, wherein the multispecific polypeptide construct or the antibody is administered in an effective amount to a subject in need of treating cancer in the subject.

On the other hand, a method for treating cancer is provided, comprising administering a pharmaceutical composition as disclosed herein to a subject in need, wherein the multispecific polypeptide construct or the antibody is administered in an effective amount to treat cancer in the subject.

In some instances, the subjects had cancer cells expressing HER2, CD20, and/or EGFR.

In some instances, such as the methods disclosed herein, where:

(1) The cancer described is a solid tumor;

(2) The cancer is selected from breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and stomach cancer; and/or

(3) Cancers selected from lung adenocarcinoma, conventional glioblastoma multiforme, glioblastoma, colon adenocarcinoma and non-small cell carcinoma.

In some instances, such as the methods disclosed herein, the administration of a secondary therapeutic treatment may also be included, which may include chemotherapeutic agents, biologics, hormone therapy, radiation, or surgery.

B. NK cell receptors

In some instances, the multispecific peptide construct contains one, two, three, four, five, or six innate immune cell targeting domains, or more. In some instances, the multispecific peptide construct contains one or more innate immune cell targeting domains, wherein the innate immune cell targeting domain is an NK cell targeting domain (i.e., an NK targeting domain). In some instances, the NK cell targeting domain is selected from 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 CDR, NKp80-binding single-chain Fv, NKp80-binding dsFv, NKp80-binding scab, NKp80-binding STAb, NKp80-binding single-domain heavy chain antibody, NKp80-binding single-domain light chain antibody, NKp80-binding VHH, NKp80-binding VNAR, and other NKp80-binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avider, anticalin, fibronectin, and binding sites constructed into the constant region of the antibody. In some instances, the multispecific peptide construct also includes a second NK-targeting domain. In some instances, the second NK-targeting domain is an Fc domain.

In humans, NK cell receptors include cytotoxic cell immunoglobulin-like receptors (KIRs), C-type lectins (CD94/NKG2A/NKG2C, NKG2D), innate cytotoxic receptors (NCRs; NKp44, NKp30, NKp65, NKp80, and NKp46), CD16/FcγRIIIa, and integrins/adhesion molecules. These signals are combined when deciding whether to respond to target cells, including integrating the NK cell activation state influenced by cytokine initiation or other events (such as latent viral infection). Once properly triggered, NK cells respond by killing the target and producing cytokines including IFN-γ, TNF-α, GM-CSF, and MIP-1α.

In some instances, the multispecific peptide 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, etc. In some instances, the multispecific peptide construct described herein includes two NK targeting domains, wherein the first NK targeting domain is an NKp80 targeting domain and the second NK targeting domain is a CD16 targeting domain.

In some instances, the multispecific peptide construct also includes a second NK cell targeting domain, wherein the second targeting domain is selected from, but is not limited to, domains targeting CD16, NKp46, NKp30, NKp44, NKG2D, NKp65, DNAM, CD94, NKG2A, TIGIT, and interleukin receptors.

i.NCR

In humans, NCR NKp46, NKp80, and NKp30 are expressed on both activated and resting NK cells, but NKp44 is upregulated on some NK cells upon stimulation with interleukin-2. Reported ligands for NKp46 and NKp44 include viral hemagglutinin. Given that anti-NCR antibodies ablate NK cell-mediated lysis in many tumor cell types, cellular ligands may also be present. Other ligands for NCR include nuclear factor HLA-B-associated transcript 3, which can be released from tumor cells and bind to NKp30. NKp46 and NKp30 have also been shown to bind to heparan sulfate proteoglycans, and NKp80 binds to activation-induced C-type lectin (AICL). Recently, NKp30 has also been shown to bind to the B7-H6 tumor antigen. NCR has been suggested as one of the major 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)).

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

MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLILLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQ SEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY (huNKp80 sequence; SEQ ID NO:223).

In some instances, the NKp80 receptor contains cynomolgus monkey sequences:

VLLKCQKGSHSNTTEHEDIGDLKMNNGTRRNTSNKDLCVSRSADQTVLCQSEWLKYRGKCYWFSNEMKSWSDSYVYCLERKSHLLIIQDELEMAFIQKNLRQSNYVWMGLNFTSLKMTWTWVDGSPLDPKIFFIKGPAKENSCAAIKESKIYSETCSSVFKWICQY (cyNKp80 sequence; SEQ ID NO: 261).

In some instances, the NK targeting domain is the domain that targets NKp80.

In some instances, the multispecific peptide constructs described herein contain a variable light chain amino acid sequence selected from the following NKp80 targeting domains:

AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKLASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTEVVVVK (VL sequence against NKp80(13); SEQ ID NO: 183);

DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVVVK (VL sequence of anti-NKp80(28)-FcX; SEQ ID NO: 184);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTTRSSSIYWPFGGTEVVVVK (VL sequence against NKp80(36); SEQ ID NO: 185);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYTAYTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGTEVVVK (VL sequence against NKp80 (37); SEQ ID NO: 186);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTLESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGTEVVVK (VL sequence against NKp80 (45); SEQ ID NO: 187);

AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEVVVK (VL sequence of anti-NKp80 (50); SEQ ID NO: 188);

ALVMTQTPSSVSAAVGGTVTIKCQASQSIGNDLAWYQQKPGQPPKLLIYAASNLESGVPSRFRGSGSGTKFTLTISDLECADAATYYCQGTYRGSSISWPFGGTEVVVK (VL sequence of anti-NKp80 (51); SEQ ID NO: 189);

QIVVTQTPASVSAAVGGTVTISCQSSQNVYGNNELSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGYSGGMRSFGGGTEVVLV (VL sequence of anti-NKp80 (71); SEQ ID NO: 190);

QIVVTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLASTLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTEVVVK (VL sequence of anti-NKp80 (74); SEQ ID NO: 191);

AQVLTQTASSVSAAVGGTVTISCQSSQSVYNYNWLGWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSVDCNVFGGGTEVVVK (VL sequence of anti-NKp80 (78); SEQ ID NO: 192);

ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTKVVVK (VL sequence of anti-NKp80 (79); SEQ ID NO: 193);

AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLASGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEVVVVK (VL sequence of anti-NKp80 (81); SEQ ID NO: 194);

DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSKCVFPFGGGTEVVVK (VL sequence of anti-NKp80 (82); SEQ ID NO: 195);

DIVMTQTPASVEAAVGGTVTIKCQASQSIGRDLAWYQQKPGQPPKLLIYGASILESGVPSRFKGNGSGTQFTLTISDLECADAATYYCQGADRSSTPSWPFGGGTEVVVVK (VL sequence of anti-NKp80 (87); SEQ ID NO: 196);

AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKTSSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTEVVVK (VL sequence of anti-NKp80 (94); SEQ ID NO: 197);

AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTEVVVVK (VL sequence of anti-NKp80 (101); SEQ ID NO: 198);

DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTEVVVK (VL sequence of anti-NKp80 (102); SEQ ID NO: 199);

DVVMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTLESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTEVVVK (VL sequence of anti-NKp80 (106); SEQ ID NO: 200);

DIVMTQTPASVSAAAGGTVTINCQASQSISNELSWYQQKSGQPPKLLIYGASNLESGVPSRFKGSGSGTDFTLTISDLECADGATYYCQSNYYDSSSPDFAFGGGTEVVVK (VL sequence against NKp80 (63); SEQ ID NO: 201); and/or

DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSNCVFPFGGGTEVVVVK (VL sequence of anti-NKp80 (83); SEQ ID NO: 202).

In some instances, the multispecific peptide constructs described herein have sequences comprising a variable heavy chain domain containing a domain targeting NKp80 selected from the following amino acid sequences:

QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIYTGGGSADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGYGDATDIWGPGTLVTV (VH sequence of anti-NKp80 (13); SEQ ID NO: 203);

QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYAGSSGSTHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDYFKIWGPGTLVTV (VH sequence against NKp80 (28); SEQ IDNO: 204);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRYNIWGPGTLVTV (VH sequence against NKp80 (36); SEQ IDNO: 205);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRYNIWGPGTLVTV (VH sequence against NKp80 (37); SEQ IDNO: 206);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWRYNIWGPGTLVTV (VH sequence against NKp80 (45); SEQ IDNO: 207);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWRYNIWGPGTLVTV (VH sequence against NKp80 (50); SEQ IDNO: 208);

QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYSDSGNTYYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRYNIWGPGTLVTV (VH sequence against NKp80 (51); SEQ IDNO: 209);

QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSPGVAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWGQGTLVTV (VH sequence against NKp80(71); SEQ ID NO: 210);

QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSGGSAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWGQGTLVTV (VH sequence against NKp80(74); SEQ ID NO: 211);

QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNGGATYYASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLWGQGTLVTV (VH sequence against NKp80(78); SEQ IDNO: 212);

QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPSGSIWYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWGQGTLVTV (VH sequence against NKp80 (79); SEQ ID NO: 213);

QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIADKIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQGTLVTV (VH sequence against NKp80(81); SEQ ID NO: 214);

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYFASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLWGQGTLVTV (VH sequence against NKp80(82); SEQ IDNO: 215);

QSLEEYGGDVVQPEGSLTLTCTASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWKYNIWGPGTLVTV (VH sequence against NKp80(87); SEQ IDNO: 216);

QSVEESGGRLVTPGTPLLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTGGSAYYANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWGQGTLVTV (VH sequence against NKp80 (94); SEQ ID NO: 217);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISWGGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDPWGPGTLLTV (VH sequence against NKp80(101); SEQ IDNO: 218);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSGGDTSYATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLWGQGTLVTV (VH sequence against NKp80(102); SEQ IDNO: 219);

QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDGSAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWGQGTLVTV (VH sequence against NKp80(106); SEQ IDNO: 220);

and

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLWGQGTLVTV (VH sequence against NKp80 (83); SEQ ID NO: 222).

In some instances, the NKp80 targeting domain contains:

(1) VH, which contains an amino acid sequence selected from SEQ ID NO:203-222, 236; or has at least about 80% sequence identity with its amino acid sequence; or has 2 or 3 amino acid substitutions;

(2) VL, which contains an amino acid sequence selected from SEQ ID NO:183-202, 235;

VH and VL pairings 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 have at least about 80% sequence identity with their amino acid sequence; or have 2 or 3 amino acid substitutions.

In some instances, the NKp80 targeting domain contains:

(1) VH, which contains an amino acid sequence selected from SEQ ID NO:203-222, 236;

(2) VL, which contains an amino acid sequence selected from SEQ ID NO:183-202, 235;

VH and VL are paired to produce clones 13, 28, 36, 37, 45, 50, 51, 63, 71, 74, 78, 79, 81, 82, 83, 87, 94, 101, 102, 106 or humanized clone 87-2.

In some instances, VH and/or VL have 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 NO:203-222, 236 and/or selected from SEQ ID NO:183-202, 235, respectively.

In some instances, VH and/or VL have amino acid sequences selected from SEQ ID NO:203-222, 236 and/or selected from SEQ ID NO:183-202, 235, respectively, wherein the amino acid sequences contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more amino acid substitutions.

In some instances, the NKp80 targeting domain contains 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:138) 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:138) 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:138) 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:138) 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:138) 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:138) 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:139) 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:138) 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: 13) 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: 17) 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 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).

In some instances, the multispecific polypeptide constructs described herein include an antigen-binding fragment sequence comprising one, two, three, four, five, or six CDRs selected from SEQ ID NO:1-104, 247-252.

NKp46 has been established as a key activating receptor because it is expressed almost exclusively by NK cells and is the only NCR with a mouse ortholog (called Ncr1). Its ligand spectrum ranges from viral ligands (e.g., hemagglutinin (HA) and hemagglutinin-neuraminidase (HN) of influenza virus, Sendai virus, Newcastle disease virus, and poxvirus) to fungal ligands, and even unknown ligands found on tumors, adipocytes, human pancreatic β cells, hepatic stellate cells, and bacteria (e.g., Fusobacterium nucleatum). Recently, soluble NKp46 ligands have been identified. The identification of unknown membrane-bound ligands, particularly tumor ligands of NKp46, has been extensively studied for over two decades. In some instances, the NK-targeting domain is the domain that targets NKp46.

Like NKp80, NKp65 triggers NK cell cytotoxicity in redirected cell lysis assays. However, unlike NKp80, NKp65 is not detected on human peripheral blood NK cells or T cells, although NKp65 cDNA was initially cloned from IL-2/IL-12-stimulated peripheral blood NK cells. To date, significant NKp65 surface expression has only been observed in the NK cell line NK92 and its derivative NK92MI; therefore, physiologically expressive NKp65 cells and the determinants of NKp65 expression in vivo remain to be elucidated. In some instances, the NK-targeting domain is the domain that targets NKp65.

ii. C-type lectin

NK cells recognize “stressed” cells by activating the receptor NKG2D, which is expressed on almost all mouse NK cells. This receptor has been shown to be important for the control of some NK cell-mediated cancers. The NKG2D molecule recognizes several different ligands. This ability is thought to be due to a single binding site in the receptor with side chains exhibiting limited flexibility, resulting in a rigid-body interaction model for ligand binding. NKG2D ligands include MHC class I-related proteins, whose expression is regulated by DNA damage and heat shock response pathways typically activated in tumors. Given the immunostimulatory nature of NK cells, NKG2D-mediated recognition of tumor cells is necessary for optimal immune responses against some tumors. In some instances, the NK-targeting domain is the domain that targets NKG2D.

Another family of C-type lectin receptors is the CD94-NKG2A/C/E heterodimer. These receptors respond to levels of non-canonical MHC class I molecules on the surface of potential target cells and are considered important for preventing inappropriate NK cell activation. The heterodimers CD94-NKG2C and CD94-NKG2E have been shown to be associated with DAP-12 and are considered activating receptors. In humans, both the ITIM-containing CD94-NKG2A receptor and the DAP-12-associated CD94-NKG2C receptor bind to HLA-E, a non-canonical HLA class I molecule. The reason why the same molecule has one activating receptor and one inhibitory receptor is unclear. This phenomenon may allow for more specific differentiation between normal tissue and distressed or infected tissue, since expression of this ligand does not necessarily lead to NK cell activation. In some instances, the NK-targeting domain is the domain that targets CD94.

iii. Co-stimulatory receptors

Several other NK cell receptors are considered co-stimulatory. These receptors provide further stimulation to the cell, although they alone are insufficient to trigger NK cell activation. Therefore, they not only provide an alternative mechanism for activation but also ensure that NK cells are not activated in response to normal or healthy tissue. These receptors include DNAM-1, NKR-P1, and PILR receptors.

The DNAM-1 receptor (also known as CD226) is a member of the Ig superfamily and is constitutively expressed on approximately 50% of NK cells. The ligands for this co-stimulatory activation receptor are CD155 (also known as the poliovirus receptor (PVR) or Necl-5) and CD112 (Nectin-2), and these ligands can be upregulated on some tumor cells, suggesting that DNAM-1 is involved in some NK cell-mediated anti-tumor responses. In some instances, the NK-targeting domain is the domain that targets DNAM-1.

iv.FcγR

In humans, there are three classes of Fc receptors (FcγRs) that bind IgG: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγR expression in leukocytes induces activation (CD64, CD32A, CD32C, CD16A, and CD16B) or inhibition (CD32B) to regulate immune responses and signaling thresholds.

CD16 represents the prototypical NK cell activation receptor because its binding is sufficient to trigger cytotoxic activity and the production of pro-inflammatory cytokines and chemokines, thereby releasing the anti-tumor function of NK cells. Human CD16 is also expressed by macrophages and some circulating monocytes, and consists of two extracellular Ig domains, a short cytoplasmic tail, and a transmembrane domain. In NK cells, the transmembrane domain allows it to be associated with the CD3 and FcγRI chains; these subunits containing the immune receptor's tyrosine-based activation motif (ITAM) link the receptor to intracellular signal transduction pathways that coordinate the reorganization of the actin and microtubule cytoskeleton and the activation of several transcription factors. In some instances, the NK targeting domain is the domain that targets CD16.

This document also discloses functional Fc domains possessing “effective functions” of the natural/wild-type sequence Fc region. Examples of “effective functions” include, but are not limited to, CD16 binding; C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the combination of the Fc domain with a binding domain (e.g., antibody variable domain) and can be assessed using various assays known in the art. In some instances, the Fc domain exhibits functional antibody-dependent cytotoxicity (e.g., via CD16 binding), attenuated ADCC (FcX) (e.g., via conformational changes of the functional Fc domain), no ADCC (Fc silencing/inactivating mutant Fc domain (FcLALA)) (e.g., via specific Fc mutations), or enhanced ADCC (FcE).

In some instances, multispecific peptide constructs bind to a second regulator via an Fc domain or Fc component. In some instances, multispecific peptide constructs include, but are not limited to, Fc domains, native/wild-type Fc domains, Fc-enhancing domains, Fc-weakening domains, Fc-silencing/inactivating domains, Fc-mutant domains, heterodimeric Fc domains, etc. In some instances, antigen-binding proteins contain native/wild-type Fc domains. In some instances, native/wild-type Fc domains are denoted as FcWT. In some instances, antigen-binding proteins contain variant Fc domains. In some instances, variant Fc domains are weakened Fc domains. In some instances, weakened Fc domains are denoted as FcX. In some instances, variant Fc regions are Fc-silencing/inactivating mutant Fc domains (FcLALA). In some instances, weakened Fc domains are constructed according to methods known in the art. In some instances, variant Fc domains are enhanced Fc domains. In some instances, the enhanced Fc domain is denoted as FcE.

In some instances, such as the multispecific peptide constructs disclosed herein, a functional Fc domain is also included.

In some instances, the Fc domain contains an amino acid sequence selected from SEQ ID NO:224-226.

In some instances, the multispecific peptide construct contains a natural/wild-type Fc domain. In some instances, the natural/wild-type Fc domain contains the following sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

In some instances, the multispecific peptide construct includes a natural/wild-type Fc domain sequence configured to have attenuated ADCC function. In some instances, the attenuated Fc domain includes the following sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

In some instances, the variant Fc structure field is a silent Fc structure field. In some instances, the silent Fc structure field contains a sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Silenced Fc domain/Inactivated mutant Fc domain (FcLALA); SEQ ID NO:225).

In some instances, the variant Fc domain is an enhanced Fc domain (FcE). In some instances, the enhanced Fc domain contains a sequence:

DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Enhanced Fc domain (FcE); SEQ ID NO: 226).

In some instances, the Fc struct is

(i) The natural/wild-type Fc domain (FcWT) or weakened Fc (FcX) domain of SEQ ID NO:224; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein;

(ii) The enhanced Fc domain (FcE) of SEQ ID NO:226; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein;

(iii) The silent Fc domain/inactivated mutant Fc domain (FcLALA) of SEQ ID NO:225; or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein.

In some instances, the Fc struct is:

(i) The natural/wild-type Fc domain (FcWT) or weakened Fc (FcX) domain of SEQ ID NO:224;

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

(iii) Silenced Fc domain of SEQ ID NO:225 / inactivated Fc domain (FcLALA).

In some instances, the Fc domain has an amino acid sequence that is 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 identical to the amino acid sequence selected from SEQ ID NO:224-226.

In some instances, the Fc domain has an amino acid sequence selected from SEQ ID NO:224-226, comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more amino acid substitutions.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(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 a scFv containing VH, a second [(G4S)n] linker, and VL.

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

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(a) Targeting the first domain of NKp80;

(b) Targeting the second domain of CD16;

(c) Binding one or more antigen-targeting domains of one or more tumor-associated antigens.

In some instances, one or more antigen-targeting domains contain:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) VH (VH rituximab) of amino acid sequence SEQ ID NO:244, VL (VL rituximab) of amino acid sequence SEQ ID NO:243, CH (CH rituximab) of amino acid sequence SEQ ID NO:246 and/or CL (CL rituximab) of amino acid sequence SEQ ID NO:245; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein.

In some instances, one or more antigen-binding domains include:

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

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

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

In some instances, the antigen-targeting domains VH, VL, CH, and/or CL have 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 NO:227-238, 243-246.

In some instances, the antigen-targeting domains VH, VL, CH, and/or CL have an amino acid sequence selected from SEQ ID NO:227-238, 243-246, comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more amino acid substitutions.

In some instances, such as the multispecific peptide constructs disclosed herein, the following are included:

(A) The NKp80 targeting domain includes:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three, or four VLFRs selected from FR1 of SEQ ID NO:105-118, 253, VLFR3 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255, and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and

(B) One or more antigen-targeting domains, comprising:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) VH (VH rituximab) of amino acid sequence SEQ ID NO:244, VL (VL rituximab) of amino acid sequence SEQ ID NO:243, CH (CH rituximab) of amino acid sequence SEQ ID NO:246 and/or CL (CL rituximab) of amino acid sequence SEQ ID NO:245; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein.

In some instances, such as the multispecific peptide constructs described herein, the following are included:

(A) The NKp80 targeting domain includes:

(1) VH, comprising one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(2) VL, comprising one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249; or having at least about 80% sequence identity with its amino acid sequence; or having two or three amino acid substitutions therein;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and/or

(4) One, two, three, or four VLFRs selected from FR1 of SEQ ID NO:105-118, 253, VLFR3 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255, and/or VLFR4 of SEQ ID NO:138-140, 256; or having at least about 80% sequence identity with their amino acid sequence; or having two or three amino acid substitutions therein; and

(B) One or more antigen-targeting domains, comprising:

(1) VH (VH cetuximab) of amino acid sequence SEQ ID NO:231, VL (VL cetuximab) of amino acid sequence SEQ ID NO:232, CH of amino acid sequence SEQ ID NO:233 and/or CL of amino acid sequence SEQ ID NO:234; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein;

(2) VH (VH trastuzumab) of amino acid sequence SEQ ID NO:227, VL (VL trastuzumab) of amino acid sequence SEQ ID NO:228, CH of amino acid sequence SEQ ID NO:229 and/or CL of amino acid sequence SEQ ID NO:230; or having at least about 80% sequence identity with their amino acid sequences; or having 2 or 3 amino acid substitutions therein; and/or

(3) The amino acid sequence SEQ ID NO:244 of VH (VH rituximab), the amino acid sequence SEQ ID NO:243 of VL (VL rituximab), the amino acid sequence SEQ ID NO:246 of CH (CH rituximab), and/or the amino acid sequence SEQ ID NO:245 of CL (CL rituximab); or having at least about 80% sequence identity with its amino acid sequence; or having 2 or 3 amino acid substitutions therein; and

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

In some instances, such as the multispecific peptide constructs described herein, the following are included:

(A) The NKp80 targeting domain includes:

(1) VH, which includes one, two or three CDRs selected from VHCDR1 of SEQ ID NO:51-67, 250, VHCDR2 of SEQ ID NO:68-85, 251 and/or VHCDR3 of SEQ ID NO:86-104, 252;

(2) VL, which includes one, two or three CDRs selected from VLCDR1 of SEQ ID NO:1-16, 247, VLCDR2 of SEQ ID NO:17-31, 248 and VLCDR3 of SEQ ID NO:32-50, 249;

(3) One, two, three, or four VH frame regions (FRs) selected from VHFR1 of SEQ ID NO:141-155, 257, VHFR2 of SEQ ID NO:156-162, 258, VHFR3 of SEQ ID NO:163-179, 259, and/or VHFR4 of SEQ ID NO:180-182, 260; and/or

(4) One, two, three, or four VLFRs selected from FR1 of SEQ ID NO:105-118, 253, VLFR3 of SEQ ID NO:119-121, 254, VLFR3 of SEQ ID NO:122-137, 255, and/or VLFR4 of SEQ ID NO:138-140, 256; and

(B) One or more antigen-targeting domains, comprising:

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

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

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

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

In some instances, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3, and VLFR4 have 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 NO:1-182, 247-260;

Wherein VH and VL have 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 the amino acid sequences selected from SEQ ID NO: 183-222, 235-236; and/or

The Fc domain has an amino acid sequence that is 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 identical to the amino acid sequence selected from SEQ ID NO:224-226.

In some instances, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, VHFR1, VHFR2, VHFR3, VHFR4, VLFR1, VLFR2, VLFR3 and/or VLFR4 have amino acid sequences selected from SEQ ID NO:1-182, 247-260, which contain 2 or 3 amino acid substitutions;

The VH and VL sequences selected from SEQ ID NO: 183-222, 235-236 contain one or two, three or four, five or six, seven or eight, nine or ten, eleven or twelve or thirteen or fourteen or fifteen or sixteen or seventeen or eighteen or nineteen or twenty or more amino acid substitutions; and/or

Fc has an amino acid sequence selected from SEQ ID NO:224-226, which contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more amino acid substitutions.

In some instances, multispecific peptide constructs are trispecific antigen-binding constructs, which include:

(a) Binding to the first target domain of NKp80;

(b) Combining with the second targeting domain of CD16; and

(c) Binding to the third targeting domain of the target antigen,

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), secreted T-cell bispecific Ab (STAb), single-domain Ab (sdAb), single-domain CH antibody and single-domain CL antibody, VHH, variable domain of neoantigen receptor (VNAR), sdAb based on shark VNAR structure, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, antiicalin, fibronectin, and binding sites are constructed into the constant region of the antibody (e.g., f-star technology (F-star's Modular Antibody Technology™)).

In some instances, multispecific peptide constructs are trispecific antigen-binding constructs, which include:

(a) Binding to the first targeting domain of NKp80, wherein the targeting domain is 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 shark-based VNAR structure sdAb;

(b) Binding to a first targeting domain of CD16, wherein the targeting domain is a functional Fc domain selected from FcWT (SEQ ID NO:224), FcX (SEQ ID NO:224), the silenced/inactivated Fc 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 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), secreted T-cell bispecific Ab (STAb), single-domain Ab (sdAb), single-domain CH antibodies and single-domain CL antibodies, VHH, variable domains of neoantigen receptors (VNAR), sdAbs based on shark VNAR structures, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, anticalin, fibronectin, and binding sites are constructed into the constant region of the antibody (e.g., f-star technology (F-star's Modular Antibody Technology™)).

C. Antigen Target

The exemplary peptides disclosed herein include at least one antigen binder. The peptides are not limited to the identity of the antigen binder or its binding target. The peptides are illustrated by reference to binders of HER2, EGFR, and/or CD20 antigens, but are not limited to these antigen binders.

In some instances, one or more antigen-targeting domains bind to members selected from HER-2, EGFR, and CD20.

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

i.HER2

NK cell-mediated ADCC plays a crucial role in anti-HER2 therapy. However, NK cell cytotoxicity decreases with altered activating receptor phenotypes in breast cancer patients. Compared to healthy donors, breast cancer patients express lower levels of NKp30, NKp46, and NKG2D on their NK cells. Therefore, enhancing NK cell activity and its ADCC effects is an effective way to improve the efficacy and sensitivity of trastuzumab.

In some instances, the multispecific peptide construct contains a HER2-binding fragment known in the art. In some instances, the HER2-binding multispecific peptide construct contains a heavy-chain variable (VH) domain encoded by a sequence comprising the following sequences:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Trastuzumab VH; SEQ ID NO: 227).

In some instances, multispecific peptide constructs that bind HER2 contain a light chain variable (VL) domain encoded by a sequence comprising the following sequences:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Trastuzumab VL; SEQ ID NO:228).

In some instances, multispecific peptide constructs that bind HER2 contain a constant heavy chain (CH) domain encoded by a sequence comprising the following sequences:

ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (Trastuzumab CH1; SEQ ID NO: 229).

In some instances, multispecific peptide constructs that bind HER2 contain a constant light chain (CL) domain encoded by a sequence comprising the following sequences:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Trastuzumab CL; SEQ ID NO: 230).

In some instances, unless otherwise stated, the representation of the order of domains as illustrated in this disclosure does not limit the general structure of the multispecific peptide constructs of this disclosure. Thus, in the case where a multispecific peptide construct is represented as anti-NKp80-anti-HER2, this disclosure refers to an instance where the anti-NKp80 arm of the multispecific peptide construct is located at either the N-terminus or the C-terminus, and the anti-HER2 arm is located at the other end of the anti-NKp80 arm (i.e., when anti-NKp80 is at the N-terminus, anti-HER2 is at the C-terminus, or when anti-NKp80 is at the C-terminus, anti-HER2 is at the N-terminus). These arrangements are illustrated in Figure 13C with an example of an EGFR-targeted multispecific peptide construct.

In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and HER2 (where the Fc domain is functional) is anti-HER2-anti-NKp80-Fc. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and HER2 (where the binding of the Fc domain to CD16 is weakened) is anti-HER2-anti-NKp80-FcX. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and HER2 (where the binding of the Fc domain to CD16 is enhanced) is anti-HER2-anti-NKp80-FcE. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and HER2 (where the Fc domain is an inactivating mutant/silenced domain (FcLALA)) is anti-HER2-anti-NKp80-FcLALA.

In some instances, the humanized antibody clones antiHER2-antiNKp80(45-2)-Fc, antiHER2-antiNKp80(101-1)-Fc, antiHER2-antiNKp80(94-1)-Fc, and antiHER2-antiNKp80(87-2)-Fc were derived from the parent clones of antiHER2-antiNKp80(45)-Fc, antiHER2-antiNKp80(101)-Fc, antiHER2-antiNKp80(94)-Fc, and antiHER2-antiNKp80(87)-Fc, respectively (Figure 3).

In some instances, trispecific antigen-binding peptide 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-binded both NKp80 and CD16 in OVCAR3 with improved potency compared to trastuzumab (anti-HER2-anti-NKp80-Fc vs. anti-HER2-Fc) (Fig. 3A). The clone anti-HER2-anti-NKp80(87)-Fc showed improved potency in four different cell lines tested (MKN1, OVCAR3, HCT116, MDA-MB-231) (Fig. 4). In some instances, selected humanized clones of multispecific peptide constructs showed improved binding and/or cytotoxic potential against cancer cells compared to parental peptide constructs, while exhibiting low immunogenicity.

In the experimental data provided in this disclosure, the lung fibroblast cell line MRC-5, with low HER2 expression, was used as a control against tumor cell lines. The antigen-binding peptide construct anti-HER2-Fc (trastuzumab) bound MRC-5, indicating that the anti-HER2 arm of the antigen-binding peptide construct as described herein recognizes HER2 on MRC-5. However, the trispecific antigen-binding peptide construct (anti-HER2-anti-NKp80-Fc) disclosed herein showed cytotoxic specificity only in cancer cells and did not kill MRC-5. In some instances, multispecific peptide constructs specifically killed cancer cells while showing no cytotoxicity to non-cancer cells.

In some instances, the co-conjugation of NKp80 and CD16 in the trispecific antigen-binding peptide construct antiHER2-antiNKp80(87-2)-Fc increased NK cell function without affecting T cell activation (Figure 5). Therefore, the antigen-binding peptide constructs disclosed herein are specific for NK cells. In some instances, the antigen-binding peptide constructs disclosed herein do not induce T cell activation.

As the experimental data disclosed herein show, compared with trastuzumab, clone HER2-NKp80(87-2)-CD16 consistently showed improved potency (mean EC50) (Figure 4; Table 1, column 4), with the fold change in potency increasing as the expression of the target antigen decreased in the target cells (Table 1, column 4).

In some instances, the NKp80 conjugate comprises a clonal anti-HER2-anti-NKp80(87)-Fc. In some instances, the antigen-binding polypeptide construct has an average EC50 fold change of about 1 to about 1000. In some instances, the antigen-binding polypeptide construct has an average EC50 fold change potency of about 1, about 5, about 10, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, and about 1000.

ii.EGFR

In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and EGFR (where the Fc domain is functional) is anti-EGFR-anti-NKp80-Fc. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and EGFR (where the binding of the Fc domain to CD16 is weakened) is anti-EGFR-anti-NKp80-FcX. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and EGFR (where the binding of the Fc domain to CD16 is enhanced) is anti-EGFR-anti-NKp80-FcE. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and EGFR (where the Fc domain is an inactivating mutant/silenced domain (FcLALA)) is anti-EGFR-anti-NKp80-FcLALA.

In some instances, the multispecific peptide constructs described herein comprise a variable light chain amino acid sequence selected from SEQ ID NO:183-202 as described above, wherein the trastuzumab VL sequence (SEQ ID NO:228) is replaced with the cetuximab VL sequence:

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (cetuximab VL; SEQ ID NO: 232).

In some instances, multispecific peptide constructs that bind to EGFR contain a constant light chain (CL) domain encoded by a sequence comprising the following sequences:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAEC (cetuximab CL; SEQ ID NO: 234).

In some instances, the multispecific peptide construct contains a binding fragment known in the art that binds to EGFR. In some instances, the EGFR-binding multispecific peptide construct contains a heavy-chain variable (VH) domain encoded by a sequence comprising the following sequences:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (cetuximab VH; SEQ ID NO: 231).

In some instances, multispecific peptide constructs that bind to EGFR contain a constant heavy chain (CH) domain encoded by a sequence comprising the following sequences:

ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (cetuximab CH1; SEQ ID NO: 233).

In the experimental data disclosed herein, isotypes were used as non-targeted controls that do not recognize the target antigen.

As the experimental data disclosed herein show, the clone anti-EGFR-anti-NKp80(87)-Fc was more effective than cetuximab (anti-EGFR-Fc) in the cytotoxicity assay (Figure 6). The control in the cytotoxicity assay (isotype-anti-NKp80(87)-Fc) showed no NK cell cytotoxicity in HER2-positive cell lines, indicating that the cytotoxicity was antigen-dependent (Figure 7).

iii.CD20

In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and CD20 (where the Fc domain is functional) is anti-CD20-anti-NKp80-Fc. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and CD20 (where the binding of the Fc domain to CD16 is weakened) is anti-CD20-anti-NKp80-FcX. In some instances, as disclosed herein, a trispecific multispecific peptide construct binding NKp80, CD16, and CD20 (where the binding of the Fc domain to CD16 is enhanced) is anti-CD20-anti-NKp80-FcE. In some instances, such as those disclosed herein, a trispecific multispecific peptide construct combining NKp80, CD16, and CD20 (where the Fc domain is an inactivating mutant/silenced domain (FcLALA)) is anti-CD20-anti-NKp80-FcLALA.

In some instances, the multispecific peptide constructs described herein comprise a variable light chain amino acid sequence selected from SEQ ID NO:183-202 as described above, wherein the trastuzumab VL sequence (SEQ ID NO:228) is replaced with the rituximab VL sequence:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (rituximab VL; SEQ ID NO: 243).

In some instances, multispecific peptide constructs that bind CD20 contain a constant light chain (CL) domain encoded by a sequence comprising the following sequences:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (rituximab CL; SEQ ID NO: 245).

In some instances, the multispecific peptide construct contains a CD20-binding fragment known in the art. In some instances, the CD20-binding multispecific peptide construct contains a heavy-chain variable (VH) domain encoded by a sequence comprising the following sequence:

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (rituximab VH; SEQ ID NO: 244).

In some instances, multispecific peptide constructs that bind CD20 contain a constant heavy chain (CH) domain encoded by a sequence comprising the following sequences:

ASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC (rituximab CH1; SEQ ID NO: 246).

D. Innate immune cell binding agent

In some instances, the multispecific peptide construct contains an innate immune cell binder, wherein the innate immune cells 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 instances, when the multispecific peptide construct is a multispecific antigen-binding peptide, the multispecific peptide construct as described herein contains multiple antigen-targeting domains. In some instances, each of these targeting domains binds to or recognizes an innate immune cell modulator or a target antigen. In some instances, each targeting domain of the multispecific peptide construct contains at least one, at least two, at least three, at least four, at least five, or all six CDRs as described herein. In some instances, the multispecific peptide construct contains a combination of one or more CDRs as described herein.

In some instances, antigen-binding peptide constructs, as described herein, comprise multiple antigen-targeting domains. Each of these targeting domains binds to or recognizes an NK regulator or a target antigen. Therefore, each targeting domain of a multispecific peptide construct comprises at least one, at least two, at least three, at least four, at least five, or all six CDRs as described herein. In some instances, multispecific peptide constructs comprise a combination of one or more CDRs as described herein. In some instances, multispecific peptide constructs bind an NK regulator, such as NKp80. In some instances, multispecific peptide constructs are antigen-binding peptides comprising one to six CDRs that bind NKp80 as described herein.

On the other hand, methods for producing multispecific polypeptide constructs or antibodies as disclosed herein are provided, comprising culturing host cells and optionally isolating the multispecific polypeptide constructs from said host cells and/or culture medium.

On the other hand, methods are provided for screening and/or identifying multispecific polypeptide constructs or antibodies as disclosed herein, wherein the NK cell targeting domain is anti-NKp80.

In some instances, this disclosure is a method for screening and/or identifying NKp80 binders, non-binding agents, non-activating binders, activated binders, and/or binding agents. In some instances, screening and/or identifying NKp80 binders includes using biological layer interferometry (BLI) and the Xcelligence® cytotoxicity assay.

In some instances, the multispecific polypeptide construct includes a light chain variable region (VL) having one or more CDRs selected from the following: SEQ ID NO:1-50 (Figure 10) or fragments thereof, or having 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, and/or having a sequence with 2 or 3 amino acid substitutions. In some instances, the multispecific polypeptide construct includes a heavy chain variable region (VH) having one or more CDRs selected from the following: SEQ ID NO:51-104 (Figure 10) or fragments thereof, or having 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, and/or having a sequence with 2 or 3 amino acid substitutions. In some instances, the multispecific polypeptide construct includes a light chain variable region (VL) having one or more frame regions (FRs) selected from: SEQ ID NO:105-140 (Figure 11) or fragments thereof, or having 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 therewith, and/or having a sequence with 2 or 3 amino acid substitutions. In some instances, the multispecific polypeptide construct includes a heavy chain variable region (VH) having one or more frame regions (FRs) selected from: SEQ ID NO:141-182 (Figure 11) or fragments thereof, or having 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 therewith, and/or having 2 or 3 amino acid substitutions.

In some instances, this disclosure is a method for screening and/or identifying multispecific peptide constructs, wherein the multispecific peptide construct includes an NKp80 conjugate. In some instances, screening and/or identifying the NKp80 conjugate includes using biological layer interferometry (BLI) and the ^{Xcelligence®} cytotoxicity assay. In some instances, the multispecific peptide construct binds to an NK modulator, such as NKp80. In some instances, the multispecific peptide construct is an antigen-binding peptide containing one to six CDRs that bind NKp80 as described herein. In some instances, the multispecific polypeptide construct comprises a light chain variable region (VL) having one or more CDRs selected from: SEQ ID NO:1-50 (Figure 10) or fragments thereof, or having 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 therewith, and/or a sequence having 2 or 3 amino acid substitutions, which binds to a heavy chain variable region (VH) having one or more CDRs selected from: SEQ ID NO:1-50 (Figure 10) NO:51-104 (Figure 10) or a fragment thereof, or a sequence having 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, and/or having 2 or 3 amino acid substitutions.

In some instances, the multispecific polypeptide construct contains at least 80% sequence identity with any sequence disclosed herein. In some instances, the multispecific polypeptide construct contains a target binding site or CDR that contains a sequence having 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 sequence disclosed herein. In some instances, sequences such as those disclosed herein have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or twenty or more amino acid substitutions.

In some instances, the multispecific polypeptide construct comprises an amino acid sequence having one or more amino acid mutations relative to any sequence disclosed herein. In some instances, the multispecific polypeptide construct comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty amino acid mutations relative to any sequence disclosed herein. In some instances, the one or more amino acid mutations are independently selected from substitution, insertion, deletion, and truncation. In some instances, the amino acid mutation is an amino acid substitution, and includes both conserved and/or non-conserved substitutions.

In some instances, substitutions include non-classical amino acids. In some instances, non-classical amino acids are generally selected from selenocysteine, pyrrolidone, 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, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfoalanine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs.

In some instances, the modification of the amino acid sequence is achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. In some instances, the mutation does not significantly reduce the ability of the antigen-binding peptide construct to specifically bind to the target. In some instances, the mutation does not significantly reduce the ability of the antigen-binding peptide construct to specifically bind to the target and does not functionally modulate (e.g., partially or completely neutralize) the target.

In some instances, the binding affinity of the multispecific polypeptide constructs of this disclosure to the full-length and/or mature and/or isoform and/or splice variants and/or fragments and/or monomeric and/or dimeric forms and/or any other naturally occurring or synthetic analogs, variants or mutants (including monomeric and/or dimeric forms) of the multispecific polypeptide constructs is described by an equilibrium dissociation constant ( _Kd ). In some instances, the multispecific peptide construct is bound to the full-length form and/or mature form and/or isoform and/or splice variant and/or fragment and/or any other naturally occurring or synthetic analog, variant or mutant (including monomeric and/or dimer forms ₎ of the multispecific peptide construct at a concentration of less than 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, or about 5 nM, or about 1 nM.

In a preferred embodiment, the co-binding of NKp80 and CD16 in the trispecific multispecific peptide construct antiHER2-antiNKp80(87-2)-Fc increases NK cell function without affecting T cell activation (Figure 5). Therefore, the multispecific peptide constructs disclosed herein are specific for NK cells. In some instances, the multispecific peptide constructs disclosed herein do not induce T cell activation.

In some instances, trispecific and multispecific peptide constructs containing NKp80 binders include clones of antiHER2-antiNKp80(13)-FcX, antiHER2-antiNKp80(28)-FcX, antiHER2-antiNKp80(36)-FcX, antiHER2-antiNKp80(37)-FcX, antiHER2-antiNKp80(45)-FcX, antiHER2-antiNKp80(50)-FcX, antiHER2-antiNKp80(51)-FcX, antiHER2-antiNKp80(63)-FcX, antiHER2-antiNKp80(71)-FcX, and antiHER2- 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.

As the experimental data disclosed herein show, the trispecific multispecific peptide 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-conjugated both NKp80 and CD16 in OVCAR3 with improved potency compared to trastuzumab (anti-HER2-anti-NKp80-Fc vs anti-HER2-Fc) (Figure 3A). The clone anti-HER2-anti-NKp80(87)-Fc showed improved potency in four different cell lines tested (MKN1, OVCAR3, HCT116, MDA-MB-231) (Figure 4) (see Example 3).

i. Immunoglobulins

The key effector function of IgG antibodies is antibody-dependent cytotoxicity (ADCC), in which antibody-coated antigens activate effector cells (e.g., NK cells or monocytes) to destroy antibody-coated targets via the binding of a complex to FcγR. ADCC activity is significantly dependent on the glycan composition of IgG and, moreover, on the net result of activating and inhibiting FcγR binding.

In some instances, Fc polypeptides comprise polypeptides that constitute the Fc domain, such as monomeric Fc. In some instances, Fc polypeptides are derived from any suitable immunoglobulin, such as human IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. In some instances, Fc polypeptides are derived from humans or any other non-human mammal. In some instances, the Fc domain comprises the carboxyl-terminal portion of two H chains held together by disulfide bonds. In some instances, the effector function of the antibody is determined by the sequence within the Fc domain; this region is also the part recognized by Fc receptors (FcRs) found on certain types of cells.

In some instances, the natural/wild-type Fc domain (FcX) confers attenuated ADCC, which refers to a measurable reduction in the ADCC response of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% of the control. In some instances, the Fc silencing/inactivating mutant Fc domain (FcLALA) confers little or no measurable ADCC. This refers to substantially complete silencing of the measurable ADCC response, which is 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 control, or substantially complete silencing of ADCC to the point that no measurable ADCC is detected. In some instances, enhanced ADCC refers to an improvement, increase, or doubling of the measurable ADCC response, which is 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 control.

In some instances, the multispecific peptide construct binds to a second innate immune cell modulator via its Fc domain. In some instances, the second innate immune cell modulator-binding domain of the multispecific peptide construct is selected from wild-type Fc domains (FcWT), Fc-enhancing domains (FcE), Fc-depressing domains (FcX), Fc-silencing domains/inactivating mutants of Fc domains (FcLALA), Fc mutant domains, heterodimeric Fc domains, etc. As used herein, the terms "region" and "domain" are understood to describe the same component and are therefore used interchangeably.

In some instances, the multispecific peptide construct includes an Fc domain. In some instances, the Fc domain includes a sequence comprising:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

In some instances, the multispecific peptide construct includes a variant Fc domain. In some instances, the variant Fc domain is a weakened Fc domain (FcX). In some instances, the weakened Fc domain is constructed according to methods known in the art. In some instances, the weakened Fc domain comprises an amino acid sequence comprising:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

In some instances, the variant Fc domain can be a silenced Fc domain/inactivation mutant. In some instances, the silenced Fc domain/inactivation mutant of the Fc domain contains a sequence comprising:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Silenced Fc domain/Inactivated mutant Fc domain (Fc LALA); SEQ ID NO:225)

In some instances, the variant Fc domain can be an enhanced Fc domain. In some instances, the enhanced Fc domain contains a sequence comprising:

DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Enhanced Fc domain (FcE): SEQ ID NO: 226).

In some examples of this disclosure, the NK cell conjugate contains a natural/wild-type Fc domain and/or a variant Fc domain. The variant Fc domain (or Fc mutant region) comprises an amino acid sequence that differs from the amino acid sequence of the natural/wild-type Fc domain due to at least one amino acid modification, preferably one or more amino acid substitutions. In some examples, the variant Fc domain has at least one amino acid substitution compared to the natural/wild-type Fc domain or the Fc domain of the parental polypeptide. For example, the variant Fc domain (or Fc mutant domain) may contain 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 natural/wild-type Fc domain. The variant Fc domain in this paper has 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 Fc region of the natural/wild-type sequence.

In some instances, the “Fc domain” includes the hinge region of the Fc region, the CH2 domain, or the CH3 domain.

ii.CDR and FR

Each domain in a natural antibody has a characteristic structure of an "immunoglobulin fold" formed by two β-sheets (e.g., 3-, 4-, or 5-chain sheets) stacked together to form a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops, called "complementation-determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework regions" (FR1, FR2, FR3, and FR4). When the natural antibody folds, the FRs form the β-sheets that provide the structural framework for the domain, and the CDR loop regions from both the heavy and light chains are aggregated together in three-dimensional space to create a single hypervariable antigen-binding site located at the top of the Y-structure. The Fc domain of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity (U.S. Patent Application No. 20220040231, incorporated herein by reference in its entirety).

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 instances, when the multispecific peptide construct is a multispecific antigen-binding peptide, the multispecific peptide construct as described herein contains multiple antigen-targeting domains. Each of these targeting domains binds to or recognizes an innate immune cell modulator or a target antigen. In some instances, each targeting domain of the multispecific peptide construct contains 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 instances, the multispecific peptide construct contains a combination of one or more CDRs as described herein.

In some instances, the multispecific polypeptide construct binds a modulator, such as NKp80. In some instances, the multispecific polypeptide construct is an antigen-binding polypeptide construct comprising one to six CDRs that bind NKp80 as described herein. In some instances, the multispecific polypeptide construct comprises a light chain variable region (VL) having one or more CDRs selected from the sequence of Figure 10 or a fragment thereof, or having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having sequences with 2 or 3 amino acid substitutions.

In some instances, the multispecific polypeptide construct binds a modulator, such as NKp80. In some instances, the multispecific polypeptide construct is an antigen-binding polypeptide construct comprising one to six CDRs that bind NKp80 as described herein. In some instances, the multispecific polypeptide construct comprises a heavy chain variable region (VH) having one or more CDRs selected from the sequence of Figure 10 or a fragment thereof, or having at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% homology thereto, and/or having sequences with 2 or 3 amino acid substitutions.

On the other hand, an antigen-binding protein or an antigen-binding fragment thereof is provided, comprising 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),

The CDR sequence thereon has at least about 90% homology with the amino acid sequences selected from SEQ ID NO:1-104, 247-252; and/or

The CDR sequences selected from SEQ ID NO:1-104, 247-252 contain 2 or 3 amino acid substitutions.

In some instances, an antigen-binding protein, or an antigen-binding fragment thereof, is provided, comprising 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),

The CDR sequence thereon has 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 the amino acid sequences selected from SEQ ID NO: 1-104, 247-252; and/or

The CDR sequences selected from SEQ ID NO:1-104, 247-252 contain 2 or 3 amino acid substitutions.

On the other hand, an antigen-binding protein, or an antigen-binding fragment thereof, is provided, comprising a CDR and FR sequence 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:138) 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:138) 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:138) 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:138) 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:138) 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:138) 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:139) 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:138) 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: 13) 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: 17) 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 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:256) 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),

The FR and CDR sequences thereon have at least about 90% homology with amino acid sequences selected from SEQ ID NO:1-104, 247-260; and/or

The FR and CDR sequences selected from SEQ ID NO:1-104, 247-260 contain 2 or 3 amino acid substitutions.

In some instances, an antigen-binding protein, or an antigen-binding fragment thereof, is provided, comprising a CDR and FR sequence 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:138) 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:138) 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:138) 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:138) 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:138) 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:138) 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:139) 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:138) 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: 13) 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: 17) 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 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:256) 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),

The FR and CDR sequences thereon have 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 the amino acid sequences selected from SEQ ID NO:1-104, 247-260; and/or

The FR and CDR sequences selected from SEQ ID NO:1-104, 247-260 contain 2 or 3 amino acid substitutions.

iii. Light chains and heavy chains

The mature variable region of each light/heavy chain pair forms the antibody binding site. Therefore, a complete antibody has two binding sites. These two binding sites are identical except in multispecific or bispecific antibodies. All chains exhibit the same general structure of relatively conserved frame regions (FRs) linked by three hypervariable regions also known as complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the frame regions, enabling them to bind to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are assigned to each domain according to the definition in Kabat, Sequences of Polypeptide Constructs of Immunological Interest. Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. On the other hand, in multispecific antigen-binding polypeptide constructs, each binding site of the multispecific polypeptide construct is different. That is, in bifunctional or bispecific multispecific polypeptide constructs, the multispecific polypeptide construct has two distinct binding sites, etc.

The binding fragments are selected from Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains), F(ab)2 fragments (bivalent fragments containing two Fab fragments linked by disulfide bonds at the hinge region), Fd fragments (consisting of VH and CH1 domains), Fv fragments (consisting of VL and VH domains of the antibody arm), single-domain antibody (dAb) fragments (consisting of VH domain), separate complementarity-determining regions (CDRs), single-chain Fv (scFv), dsFv, scAb, STAb, single-domain antibodies (sdAb or dAb), single-domain heavy chain antibodies, and single-domain light chain antibodies, VHH, VNAR, single-domain antibodies based on shark VNAR structures, and binding domains based on alternative scaffolds, including but not limited to ankyrin-based domains, fynomer, avimer, anticalin, fibronectin, and binding sites constructed into the constant region of the antibody (e.g., f-star's Modular Antibody Technology™). A single-domain antibody in which one chain is separated from its natural chaperone is sometimes referred to as a Dab. A constant region, or a portion thereof, may or may not be included in a single-domain antibody. In some instances, the antigen-targeting domain is selected from the Fab fragment, F(ab)2 fragment, Fd fragment, Fv fragment, dAb, isolated CDR, scFv, dsFv, scAb, STAb, sdAb, CH domain, CL domain, VHH, VNAR, sdAb derived from VNAR, ankyrin-based domains, fynomer, avimer, fibronectin domains, and F-star’s Modular Antibody Technology™ domain.

The peptide constructs disclosed herein typically bind to their designated targets with an association constant of at least ^10⁶ , ^10⁷ , ^10⁸ , ^10⁹ , or ^10¹⁰ M. This binding is specific, meaning it is detectably larger in size than and distinguishable from nonspecific binding to at least one unrelated target. Specific binding results from bond formation between specific functional groups or between specific spatial matches (e.g., lock and key types), while nonspecific binding is typically a result of van der Waals forces. Specific binding does not necessarily mean that the antibody binds to one and only one target. In some instances, multispecific peptide constructs specifically bind to one or more antigens.

In some instances, such as the antigen-binding polypeptide constructs described herein, the VL that binds to NKp80 has a sequence selected from SEQ ID NO:183-202 (Figure 13A).

AYDMTQTPASVEVAVGGTVTINCQASQSISSYLAWYQQKPGQRPKLLIYDASKLASGVPSRFSGSGSGTQFTLTISGVECADAATYYCQQAYSRSNVDNSFGGGTEVVVVK (VL sequence against NKp80(13); SEQ ID NO: 183);

DIVMTQTPASVEAAVGGTVTIKCQASQSIYSWLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTDFTLTISDLECDDAATYYCQGNSWGAFGGGTEVVVK (VL sequence against NKp80 (28); SEQ ID NO: 184);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTTRSSSIYWPFGGTEVVVVK (VL sequence against NKp80(36); SEQ ID NO: 185);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLSWYQQKPGQPPKLLIYTAYTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGTEVVVK (VL sequence against NKp80 (37); SEQ ID NO: 186);

DVVMTQTPASVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYTASTLESGVPSRFRGSGSGTEFTLTISDLECADAATYYCQGTYRSSSISWPFGGTEVVVK (VL sequence against NKp80 (45); SEQ ID NO: 187);

AFELTQTPSSVEAAVGGTVTIKCQASQSIGSDLAWYQQKPGQPPKLLIYGASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQGTDRSSAPTWPFGGGTEVVVK (VL sequence against NKp80 (50); SEQ ID NO: 188);

ALVMTQTPSSVSAAVGGTVTIKCQASQSIGNDLAWYQQKPGQPPKLLIYAASNLESGVPSRFRGSGSGTKFTLTISDLECADAATYYCQGTYRGSSISWPFGGTEVVVK (VL sequence against NKp80 (51); SEQ ID NO: 189);

QIVVTQTPASVSAAVGGTVTISCQSSQNVYGNNELSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGGYSGGMRSFGGGTEVVLV (VL sequence of anti-NKp80 (71); SEQ ID NO: 190);

QIVVTQTPASVSAAVGGTVTISCQSSQNLYGNKELSWYQQKPGQPPKLLIYLASTLSSGVPSRFKGSGSGTQFTLTISDLECDDAAAYYCAGGYSGGMRAFGGGTEVVVK (VL sequence of anti-NKp80 (74); SEQ ID NO: 191);

AQVLTQTASSVSAAVGGTVTISCQSSQSVYNYNWLGWYQQKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSVDCNVFGGGTEVVVK (VL sequence of anti-NKp80(78); SEQ ID NO: 192);

ASDMTQIPASVSAVVGGTVTIDCQASEDIESYLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTQFTLTITGVECADAAVYYCQQGHGYAHVDNAFGGGTKVVVK (VL sequence against NKp80(79); SEQ ID NO: 193);

AFELTQTPVPVEAAVGGTVTIKCQASQSISIYLAWYQQKPGQPPKLLIYSASTLASGVSSRFKGIGSGTDFTLTISDLECADAATYYCQSYYGTSDTDWNTFGGGTEVVVVK (VL sequence against NKp80(81); SEQ ID NO: 194);

DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSKCVFPFGGGTEVVVK (VL sequence against NKp80(82); SEQ ID NO: 195);

DIVMTQTPASVEAAVGGTVTIKCQASQSIGRDLAWYQQKPGQPPKLLIYGASILESGVPSRFKGNGSGTQFTLTISDLECADAATYYCQGADRSSTPSWPFGGGTEVVVK (VL sequence against NKp80(87); SEQ ID NO: 196);

AQVLTQTASSVSAAVGGTVTINCQSSQSVYGNNWLPWYQQKPGQPPKLLIYKTSSLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGGYSGAIRAFGGGTEVVVK (VL sequence of anti-NKp80 (94); SEQ ID NO: 197);

AFELTQTPSSVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYYGTDSTGFFAFGGGTEVVVVK (VL sequence of anti-NKp80 (101); SEQ ID NO: 198);

DYDMTQTPASVEVAVGGTVTINCQASQSINSWLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGKQFTLTISGVECADAATYYCQQGYSDSDVENLFGGGTEVVVVK (VL sequence of anti-NKp80(102); SEQ ID NO: 199);

DVVMTQTPASVSEPVGGTVTIKCQASQSIGRNLAWYQQKPGQPPKLLIYSASTLESGVSSRFKGSGSGTEFTLTISGVQCADAATYYCQCTDYGSSGLFFAFGGGTEVVVK (VL sequence of anti-NKp80 (106); SEQ ID NO: 200);

and

DVVMTQTPSSASEPVGGTVTIKCQASESISSDLAWYQQKPGQPPKLLIYGASTLESGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSTYYSWYSSNCVFPFGGGTEVVVVK (VL sequence against NKp80(83); SEQ ID NO: 202).

In some instances, such as the antigen-binding polypeptide constructs described herein, the VH that binds to NKp80 has a sequence selected from (SEQ ID NO:203-222) (Figure 13B).

QEQLEESGGGLVKPEGSLTLPCKASGFSFSSSYYMCWVRQAPGKGLELIACIYTGGGSADYASWVNGRFTISRSTSLNTVDLKMTSMTAADTATYFCARFGISVGYGDATDIWGPGTLVTV (VH sequence of anti-NKp80(13); SEQID NO: 203);

QSLEESGGDLVKPGASLTLTCTASGFSFSSGYYMCWVRQAPGKGLEWIACIYAGSSGSTHYASWAKGRFTISKTSSTTVTLQMTSLTAADTATHFCARDDGNSGDYFKIWGPGTLVTV (VH sequence against NKp80 (28); SEQ IDNO: 204);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYCMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCTRDAGTTYWRYNIWGPGTLVTV (VH sequence against NKp80(36); SEQ IDNO: 205);

QSLEESGGDLVQPEGSLTLTCTASGFFFSSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTSSTTVTLQMTSLTAADTATYFCARDAGTTYWRYNIWGPGTLVTV (VH sequence against NKp80 (37); SEQ IDNO: 206);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYASWAKGRFTITKTLSTTVTLQMTSLTAADTATYFCARDTGSTYWRYNIWGPGTLVTV (VH sequence against NKp80 (45); SEQ IDNO: 207);

QSLEESGGDLVQPEGSLTLTCTASGFSFSGSYYMCWVRQAPGKGLEWIGCIYTGSSGSTYYTSWAKGRFTITKTSSTTVTLQMTGLTAADTATYFCARDTGTTNWRYNIWGPGTLVTV (VH sequence against NKp80(50); SEQ IDNO: 208);

QSLEESGGDLVQPEGSLTLTCTASGFSFSSSYCICWVRQAPGKGLEWIGCIYSDSGNTYYASWAKGRFTISKASSTTVTLQMTTLTAADTATYFCARDSGTTSWRYNIWGPGTLVTV (VH sequence against NKp80 (51); SEQ IDNO: 209);

QSLEESGGRLVTPGGSLTLTCTVSGIDLSSAYMNWVRQAPGKGLEWIGAINSPGVAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAATTSANNLWGQGTLVTV (VH sequence against NKp80(71); SEQ ID NO: 210);

QSLEESGGRLVTPGTPLTLTCTASGFSLFSAYMNWVRQSPGKGLEWIGAINSGGSAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCAREAADTSANNLWGQGTLVTV (VH sequence against NKp80(74); SEQ ID NO: 211);

QSLEESGGRLVTPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEWIGIIDNGGATYYASWAKGRFTISKTSTTVDLKISSPTTEDTATYFCARENPTTHSLVWGLWGQGTLVTV (VH sequence against NKp80(78); SEQ IDNO: 212);

QSLEESGGRLVTPGTPLTLTCTASGLTVGSSYMSWVRQAPGKGLEWIGVIVPSGSIWYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDGASSGFYFDLWGQGTLVTV (VH sequence against NKp80 (79); SEQ ID NO: 213);

QSLEESGGRLVTPGTPLTLTCTASRFSLGSNAMSWVRQAPGEGLEWIGYISIADKIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAGYRIDTHFNLWGQGTLVTV (VH sequence against NKp80(81); SEQ ID NO: 214);

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYFASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNDHFVFSLWGQGTLVTV (VH sequence against NKp80(82); SEQ IDNO: 215);

QSLEEYGGDVVQPEGSLTLTCTASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKTLSTTVTLQMTSLTATDTATYFCARDTGSGYWKYNIWGPGTLVTV (VH sequence against NKp80(87); SEQ IDNO: 216);

QSVEESGGRLVTPGTPLLTLTCKVSGFSLSSYDMIWVRQAPGEGLEWIGFINTGGSAYYANWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARDPDGLPYCNVWGQGTLVTV (VH sequence against NKp80 (94); SEQ ID NO: 217);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYGMNWVRQAPGKGLEWIGSISWGGNTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARTRSSNFDAPFDPWGPGTLLTV (VH sequence against NKp80(101); (SEQ IDNO: 218);

QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYWMSWVRQAPGKGLEYIGIISSGGDTSYATWAKGRFTISKTSTTVDLEITSPTTEDTATYFCARDRNSNSWGSFYLWGQGTLVTV (VH sequence against NKp80(102); SEQ IDNO: 219);

QSVEESGGRLVTPGTPLTLTCTVSGIDLSSCAMIWVRQAPGEGLEYIGLINTDGSAYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRDGGTDDHFYFNLWGQGTLVTV (VH sequence against NKp80(106); SEQ IDNO: 220);

and

QSLEESGGRLVTPGTPLTLTCTVSGFSLSNNGMIWVRQAPGEGLEYIGIMNTDGSAYYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARDAGSNEHFVFNLWGQGTLVTV (VH sequence against NKp80 (83); SEQ ID NO: 222).

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

E. Disease

In some instances, the multispecific polypeptide construct binds to a target antigen that is a disease-associated antigen. In some instances, the target antigen is a disease-associated molecule. In some instances, the target antigen is a disease-associated antigen. In some instances, the target antigen molecule is selected from extracellular molecules, intracellular molecules, and transmembrane molecules. In some instances, the molecule is selected from polypeptides, polynucleotides, carbohydrates, etc. In some instances, the disease is a proliferative disease selected from proliferative diseases (e.g., tumors/cancer, inflammatory diseases, etc.), infectious diseases, autoimmune diseases, autoimmune disorders, etc. In some instances, the disease can be a tumor/cancer.

In some instances, the disease is selected from proliferative diseases (such as cancer), infectious diseases, autoimmune diseases, autoimmune disorders, etc. In some instances, the disease is a tumor.

In some instances, the targets include tumor antigens selected from, but not limited to, HER2 and EGFR. In some instances, the multispecific peptide construct binds to one or more of these tumor antigens. In some instances, the multispecific peptide construct binds to target cells, wherein the target cells include, but are not limited to, tumor cells, cancer cells, etc. In some instances, the tumor cells or cancer cells express HER2 and/or EGFR.

In some instances, tumor cells or cancer cells include, but are not limited to, bladder cancer cells, breast cancer cells, cervical cancer cells, bile duct cancer cells (extrahepatic or intrahepatic), colorectal cancer cells, esophageal or esophagogastric junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck cancer cells, hepatocellular carcinoma cells, intestinal (small) malignant 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, unknown primary cancer cells, or uterine cancer cells.

In some instances, the multispecific polypeptide construct binds to tumor cells, including bladder cancer cells, breast cancer cells, cervical cancer cells, cholangiocarcinoma cells (extrahepatic or intrahepatic), colorectal cancer cells, esophageal or esophagogastric junction cancer cells, endometrial cancer cells, gallbladder cancer cells, gastric adenocarcinoma cells, head and neck cancer cells, hepatocellular carcinoma cells, small intestinal malignant 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, unknown primary cancer cells, or uterine cancer cells. In some instances, the multispecific polypeptide construct binds to cells, including, but not limited to, immortalized cell lines and primary cells. In some instances, the multispecific polypeptide construct binds to cancer cell lines, such as immortalized cell lines. In some instances, multispecific peptide constructs are bound to cancer cell lines, such as, but not limited to, MKN1, OVCAR3, HCT116, MDA-MB-231, N87, RAJI, etc.

In some instances, the disease is infectious. In some instances, infectious diseases are caused by bacterial pathogens and/or viral pathogens. In some instances, multispecific polypeptide constructs bind to one or more bacterial antigens and/or viral antigens.

In some instances, the disease is an autoimmune disease or autoimmune condition. In some instances, an autoimmune disease/condition includes any condition, illness, or disease in which the immune system reacts against its own cells or tissues due to a breakdown in its ability to distinguish between self and non-self, or for other reasons.

i. Diagnosis

In some instances, this disclosure includes methods for detecting diseases in subjects of need, the methods comprising contacting the multispecific polypeptide constructs or compositions described herein with a sample obtained from the subject.

In some instances, the sample is a biological sample obtained from a biological subject, including samples of biological tissues or fluids obtained in vivo or in vitro. In some instances, the biological sample is a solid biological sample or a liquid biological sample. In some instances, a solid biological sample includes a tissue specimen or biopsy. In another exemplary embodiment, the fluid biological sample or liquid biological sample is selected from blood, serum, plasma, sputum, lavage fluid (e.g., peritoneal lavage fluid), cerebrospinal fluid, urine, semen, sweat, tears, saliva, etc. As used herein, the terms “blood,” “plasma,” and “serum” include its fraction or processed portion. Similarly, when the sample is taken from a biopsy, swab, smear, etc., “sample” includes the processed fraction or portion derived from the biopsy, swab, smear, etc.

ii. Pharmaceutical composition

In some instances, this disclosure includes compositions comprising the multispecific polypeptide constructs described herein. In some instances, this disclosure includes pharmaceutical compositions comprising the multispecific polypeptide constructs described herein and suitable pharmaceutical compositions thereof. In some instances, this disclosure includes compositions or pharmaceutical compositions wherein the compositions are prophylactic and/or therapeutic compositions.

In some instances, the pharmaceutically acceptable agents used in the pharmaceutical compositions herein are selected from carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluents, emulsifiers, suspending agents, solvents, fillers, swelling agents, buffers, delivery media, tensioning agents, cosolvents, wetting agents, complexing agents, buffers, antimicrobial agents, and surfactants.

In some instances, the compositions described herein are used in therapeutic/medical applications. In some instances, the compositions described herein also contain excipients and/or stabilizers. In some instances, the compositions described herein are used as a single agent and/or in combination with multispecific peptide constructs and disease-targeting therapies (e.g., but not limited to NK cell therapy, T cell checkpoint inhibitor therapy, small molecule therapies capable of stimulating NK cells to enhance anti-tumor responses, etc.).

In some instances, methods for preventing and/or treating a disease in a subject of need include administering to the subject a multispecific polypeptide construct or composition of any of the foregoing examples. In some instances, this disclosure includes the use of the multispecific polypeptide constructs described herein in the manufacture of medicaments for the prevention and/or treatment of diseases.

F. Antibody/Conjugate Production

Monoclonal antibodies (mAbs) are typically derived from antigen-specific single B cells from different hosts, which are significantly short-lived under in vitro culture conditions, making them difficult to study. The development of several new techniques and protocols has facilitated the isolation and recovery of antibody-coding sequences from antigen-specific B cells by also utilizing miniaturization of reaction volumes. Alternatively, mAbs can be generated independently of antigen-specific B cells, including demonstration techniques and, more recently, AI-driven algorithms. Therefore, a considerable variety of techniques are employed, increasing the need for better consolidation.

In some instances, NKp80 conjugates are generated using methods known in the art (e.g., rabbit single B cell clones, phage libraries, etc.). In some instances, the nucleic acid (e.g., DNA) coding sequence of the NKp80 conjugate clone is isolated and the NKp80 conjugate is identified using methods known in the art (e.g., ELISA screening).

i. Carrier

Vectors are typically selected to be functional in the host cell where they will be used (the vector is compatible with the host cell machinery, enabling gene amplification and/or gene expression to occur). The vectors described herein are expression vectors and/or cloning vectors.

In some instances, the vector is selected from plasmids, viral particles, bacteriophages, baculoviruses, yeast plasmids, lipid-based mediators, polymeric microspheres, liposomes and cell-based mediators, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral mediators, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia virus, foamy virus, cytomegalovirus, Semliki Forest virus, poxvirus, pseudorabies virus, RNA viral vectors, DNA viral vectors, and vectors derived from combinations of plasmids and bacteriophage DNA, further optionally wherein the polynucleotide is operatively linked to an expression control sequence to direct peptide synthesis, and even more optionally wherein the vector contains one or more selectable marker genes to provide selectable phenotypic traits for the host cell to be transformed.

ii. Host cell

In some instances, this disclosure pertains to a host cell comprising a vector containing a nucleic acid sequence encoding a multispecific polypeptide construct of any of the foregoing examples. In some instances, the host cell comprises a cloning or expression vector as described above and/or a nucleic acid sequence as described above encoding a multispecific polypeptide construct, an antibody, or a binding fragment thereof. In some instances, the host cell of the foregoing examples comprises a cloning or expression vector configured to express a multispecific polypeptide construct as disclosed herein.

On the other hand, a nucleic acid is provided that encodes a multispecific polypeptide construct or antibody as disclosed herein.

In some instances, the host cell is any type of cell capable of being transformed or transfected by a nucleic acid or vector to produce a multispecific polypeptide construct or a binding fragment/polypeptide construct thereof encoded therefrom. In some embodiments, the host cell containing the nucleic acid or vector is used to produce a multispecific polypeptide construct or a binding fragment/polypeptide construct thereof, or a portion thereof (e.g., a heavy chain sequence or light chain sequence encoded by the nucleic acid or vector). In some instances, after the nucleic acid or vector is introduced into the cell, the cell is cultured under conditions suitable for expressing the encoding sequence. In some instances, the antibody, multispecific polypeptide construct, or fragment, or a portion of the antibody is then isolated from the cell.

In some instances, the host cell is a prokaryotic host cell (e.g., *Escherichia coli*) or a eukaryotic host cell (e.g., yeast, insect, or vertebrate cell). In some instances, when cultured under appropriate conditions, the host cell expresses an antibody or a binding fragment thereof, which is subsequently collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produced it (if it is not secreted). In some instances, the selection of an appropriate host cell depends on the desired expression level, the desired or necessary peptide modification (e.g., glycosylation or phosphorylation), the ease of folding into a biologically active molecule, or other factors conventionally considered in the art. In some instances, the selection of the host cell depends in part on whether the antibody or its binding fragment is to be post-transcriptionally modified (e.g., glycosylated and/or phosphorylated). In another embodiment, the host cell comprises bacterial cells, yeast cells, animal cells (e.g., mammalian cells), and/or plant cells.

In some instances, suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Many host cell lines are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells such as Chinese hamster ovary cells (CHO) (ATCC 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 used for antibody expression include lymphocyte lines such as NSO myeloma cells and SP2 cells, and COS cells.

iii. Cloning selection

Developing and engineering antibodies for various purposes (e.g., diagnostics or therapy) requires comprehensive characterization to determine affinity, specificity, and mechanism of action. Biolayer interferometry (BLI) is widely used to analyze interactions between two biomolecules. Therefore, it can assist antibody characterization in a relatively simple and rapid manner. In BLI, the binding between a ligand immobilized on a biosensor tip and the analyte in solution creates an increase in optical thickness at the biosensor tip, resulting in a wavelength shift proportional to the degree of binding. The sensor tip collects readings in real time as it is immersed in the analyte solution (“immersion and read”) without the need for continuous fluid flow. Therefore, this system allows for the measurement of different antibody-antigen interactions using a variety of sensors, suitable for both tagless molecules and widely used tags.

The xCELLigence platform utilizes gold microelectrodes embedded in the bottom of microtiter wells to monitor the state of adherent cells or suspended cells tethered to the bottom of a plate. The basic measurement principle is based on impedance measurement through the surface of the gold electrodes, where the attached cells act as insulators, impeding the flow of alternating microampere current between the electrodes. This impedance signal is automatically measured at a user-defined frequency (every 10 seconds, every hour, etc.) and provides highly sensitive readouts of cell number, cell size, and cell-base adhesion. In contrast to surface-attached cancer cell targets, immune effector cells are non-adherent and therefore do not directly affect the impedance signal; however, their cytotoxic activity can be detected by the reduction in the number of target cancer cells. Due to this property, the cytolytic activity of NK cells, T cells, CAR-T, oncolytic viruses, checkpoint inhibitors, bispecific antibodies, BiTE, etc., can be selectively monitored in real time.

In some instances, screening and/or identification of NKp80 binders involved the use of biological layer interferometry (BLI) and the XCelligence® cytotoxicity assay. In some instances, as determined by BLI, the selected binders exhibited the highest binding affinity. In some instances, as determined by xCELLigence, the selected NK binders exhibited the highest cytotoxicity profile.

iv. Humanization

Antibody and antigen-targeting domains have become effective tools for treating and diagnosing various human diseases. However, non-human antibodies and antigen-targeting domains have been shown to induce human immune responses, leading to neutralization of the administered antibody and limiting their application in treating human diseases. To overcome this problem, antibody humanization techniques have been developed. Antibody humanization is an effective method to eliminate or reduce the immunogenicity of these antibody and antigen-targeting domains. To date, researchers have innovated various methods for humanizing non-human antibodies and antigen-targeting domains, improving their affinity, specificity, and other properties. Each of these methods has its advantages and disadvantages.

A common approach to humanizing nonhuman antibodies and antigen-targeting domains is complementarity-determining region (CDR) transplantation, where a CDR of a nonhuman antibody or antigen-targeting domain is transplanted onto a human frame region. Typically, the human frame region with the highest homology to the frame region of the nonhuman antibody or antigen-targeting domain is selected as the recipient for CDR transplantation. In some cases, direct transplantation of a CDR loop from a mouse antibody or antigen-targeting domain onto a human frame does not affect the affinity of the antibody or antigen-targeting domain; however, in many other cases, it significantly reduces affinity. Certain mouse residues in the frame region (called vernier zone residues) have been shown to influence the conformation of the CDR loop and the affinity of the antibody or antigen-targeting domain. These residues are located in the β-sheet frame region, which closely forms the basis of the CDR. Therefore, after selecting the desired human frame region, these residues are retained in the humanized antibody and antigen-targeting domain.

Human germline genes can serve as alternative sources for the framework regions of humanized mouse antibodies or antigen-targeting domains. Compared to framework regions derived from IgG, germline genes exhibit fewer clonal endosomal mutations. Therefore, humanized antibodies or antigen-targeting domains with germline framework regions are expected to exhibit lower immunogenicity than those with IgG framework regions. These characteristics encourage the application of these sequences to research on the humanization of antibodies and antigen-targeting domains.

Researchers have used several methods to increase the affinity of humanized antibodies or antigen-targeting domains by altering given residues in the framework or CDR region of the engineered antibody or antigen-targeting domain. Of all CDRs, the most commonly used to increase the affinity or specificity of antibody or antigen-targeting domains is the alteration of heavy chain CDR3 (VHCDR3). This CDR is the most variable CDR, and its variations originate from somatic mutations and recombination of the coding sequences of variable (V), diverse (D), and linker (J) fragments.

Antibody surface modification is another strategy for humanizing non-human antibodies or antigen-targeting domains. This approach involves replacing potentially antigenic surface framework residues with the most common human residues at these sites. The basis of this approach is that the response of human anti-mouse antibody (HAMA) to variable regions is induced solely by surface residues. Antibodies and antigen-targeting domains humanized through this method typically exhibit little change in stability and affinity.

CDR-based humanization is based on the idea that frame regions of mouse and human antibody or antigen-targeting domains with similar CDRs can retain the CDR structures supporting each other with high affinity. In this method, human frame regions are selected regardless of frame region homology, and key mouse residues (edge region residues) are not restored in the humanized antibody or antigen-targeting domain. Using this method reduces the formation of motifs that might be recognized as foreign. Antibodies or antigen-targeting domains generated by this method have been found to maintain a good degree of affinity, relatively higher than those generated by frame homology-based humanization methods.

Fully human antibodies or antigen-targeting domains derived from transgenic animals are increasingly prevalent in novel therapeutics. Following immunization, a diverse range of high-affinity human monoclonal antibodies or antigen-targeting domains can be obtained from transgenic rodents, while larger animals (e.g., transgenic cattle) produce significant amounts of specific human immunoglobulins (Ig) in their serum. In some instances, selected multispecific polypeptide construct clones contain humanized variable regions, humanized CDRs, and/or humanized framework regions.

In some instances, the NKp80 targeting domain is humanized. In some instances, the humanized NKp80 targeting domain contains a VL domain sequence (Figure 13C):

DIQMTQSPSSVSASVGDRVTITCQASQSIGRDLAWYQQKPGKAPKLLIYGASILESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQGADRSSTPSWPFGQGTKVEIK (VL sequence of humanized anti-NKp80 domain; SEQ ID NO: 235)

In some instances, the humanized NKp80 targeting domain contains a humanized VH sequence:

QVQLVESGGGVVQPGGSLRLSCAASGFSFSGNYWICWVRQAPGKGLEWIGCIYAGSSGSTCYATWAKGRFTISKDLSKNTVYLQMNSLRAEDTAVYYCARDTGSGYWKYNIWGRGTLVTVSS (VH sequence of humanized anti-NKp80 domain; SEQ ID NO:236)

In some instances, the humanized NKp80 targeting domain contains a CL domain sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CL sequence of the anti-NKp80 domain; SEQ ID NO:237)

In some instances, the humanized NKp80 targeting domain contains the CH domain sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (CH sequence against NKp80 domain; SEQ ID NO:238)

In some instances, the humanized NKp80 targeting domain includes one, two, three, four, five, or six CDRs selected from SEQ ID NO:247-252. In some instances, the humanized NKp80 targeting domain includes one, two, three, four, five, six, seven, or eight FRs selected from SEQ ID NO:253-260.

G. Gene editing methods

In some instances, the modification of the amino acid sequence is achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

i. Site-directed mutagenesis

Mutagenesis is commonly used to understand the regulatory regions of genes and the relationship between the structure and function of polypeptide constructs. Depending on the number of sites to be mutated, site-directed mutagenesis can be categorized into two types: simple or multiplex mutagenesis. For single mutagenesis, the method is based on amplifying double-stranded DNA from a plasmid using complementary oligonucleotides carrying the mutation of interest. This is one of the most commonly used strategies for introducing mutations into DNA fragments due to its simplicity, low time consumption, and high efficiency. For multiplex mutagenesis, the method introduces the desired mutations simultaneously in the same reaction or after several rounds of mutagenesis. In some instances, site-directed mutagenesis is used to modify nucleic acid sequences encoding multispecific polypeptide constructs, or any of their domains or fragments.

ii. PCR-based mutagenesis

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering research. In this paper, we describe a rapid and efficient mutagenesis method using type II restriction enzymes. The template gene is amplified into two separate PCR fragments using two pairs of anchoring and mutagenic primers. The mutated sequence is located near the recognition site of the type II restriction enzyme. After digesting the two fragments with the type II enzyme, their complementary exposed sticky ends are ligated together to produce the mutated gene. Major strategies for PCR-based mutagenesis include base substitution, deletion, insertion, chimeric gene generation, multi-site mutagenesis, and random mutagenesis at single or multiple sites. Numerous PCR-based methods have been developed, both commercially and non-commercially. Among these methods, overlap extension, large primers, Quick Change (Stratagene, La Jolla, CA), and their modified versions are currently mainstream. In some instances, PCR-based mutagenesis is used to modify nucleic acid sequences of multispecific polypeptide constructs or any of their domains or fragments.

IV. Examples

Example 1: Screening and Identification of Novel NKp80 Bonding Agents

New Zealand white rabbits (NZW) were used for immunization via bio-ballistic DNA delivery containing the gene of interest NKp80. Titer was monitored during immunization. Peripheral whole blood was collected to isolate B cells. Briefly, B cells with positive anti-rabbit IgG staining were sorted by flow cytometry for culture. B cell culture supernatants were used for screening by ELISA followed by mRNA isolation and cDNA synthesis to identify positive NKp80 binders. Homologous VH and VL gene segments were amplified by PCR and sequenced for verification. The DNA coding sequences of VH and VL were cloned into their respective expression vectors for expression of recombinant multispecific polypeptide constructs. The multispecific polypeptide construct containing a HER2-targeting domain, a weakened ADCC-functional Fc constant region, and a specific NKp80 binder is hereinafter referred to as anti-HER2-anti-NKp80-FcX (FcX: weakened ADCC).

Multispecific peptide construct clones were recombinantly expressed in mammalian systems via transient transfection. The multispecific peptide construct candidates were purified using a peptide construct A column, and the purity was greater than 95% as assessed by SDS-PAGE under non-reducing conditions. Different formats of multispecific peptide construct candidates illustrated in this study, including a trispecific multispecific peptide 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 the same method.

The DNA coding sequences of the NKp80 binding agents were then isolated and cloned into a rabbit IgG format for validation using ELISA screening. A total of 108 NKp80 binding agent DNA coding sequences were inserted into a mammalian expression vector for expression in a trispecific format (anti-HER2-anti-NKp80-FcX) in a mammalian system (Figure 1, point 1). The Fc constant region of the bispecific polypeptide construct possesses attenuated ADCC function (denoted here as FcX). Including the Fc domain with attenuated function during validation is important: it demonstrates that any detected cytotoxicity is due to NKp80 binding.

Example 2: NKp80 binder cloning: Binding and cytotoxicity screening assays

Potential NKp80 binding clones were identified using the ForteBio Octet Biolayer Interference System (BLI). Streptomycin avidin biosensors were loaded with different biotinylated antigens, including human and cynomolgus monkey NKp80, to determine the binding kinetics of these candidates.

Of the 108 clones with reduced ADCC function (trispecific, anti-HER2-anti-NKp80-FcX), 78 clones and 67 clones were validated for binding to human and cynomolgus monkey NKp80 using biolayer interference (BLI) (Fig. 1, point a).

The ability of NKp80 binders to redirect NK cell cytotoxicity against the HER2-positive target cell line N87 was determined using the Xcelligence cytotoxicity assay, which tracks cell death in real time. Single-dose concentrations of clones of interest were incubated with primary PBMCs (from healthy donors) at the described effector cell to target cell ratio. Effector cells were primary PBMCs isolated from healthy donors.

Cytotoxicity assays were performed on a total of 92 clones in this manner (70 NKp80 binders, 22 non-binding agents). The non-binding agents were included to determine baseline activity in conjunction with the anti-HER2 antibody (anti-HER2-FcX).

Activating binders were defined as clones that exhibited consistent killing activity above the median in two independent assays using two different PBMC donors. A total of 20 NKp80 clones (out of 70 binders) were identified as activating binders (Figure 2 shows the killing activity of all 20 activating binders). Importantly, the 22 non-binding binders did not induce significantly higher cytotoxicity than baseline (data not shown). In summary, these data reveal a unique subset of NKp80 binders that conjugate and enhance NK cell cytotoxicity.

Example 3: Enhanced cytotoxicity mediated by co-conjugation of NKp80 and CD16

Xcelligence real-time assays were performed to evaluate the cytotoxic potential of various multispecific peptide construct candidates. Briefly, on day 0, target cells were seeded onto Xcelligence microtiter plates. On day 1, PBMCs from healthy donors, along with the NKp80-binding candidate or conjugate to be studied, were added at a predetermined effector cell:target cell (ET) ratio. Cell lysis of target cells over time was tracked based on electrical impedance detected by the Xcelligence instrument. The output, cell index, is a measure of the detected electrical impedance and is proportional to the number of adherent target cells.

For hit screening, potential multispecific NKp80-binding peptide construct candidates in a single concentration trispecific format (anti-HER2-anti-NKp80-FcX) were used to identify NKp80 activating binders in the presence of N87 cancer cells.

In some instances, the innate immune cell cytotoxicity of multispecific peptide constructs is examined using cytotoxicity assays such as, but not limited to, the Xcelligence® cytotoxicity assay.

Not wanting to be bound by theory, the real-time Xcelligence® cytotoxicity assay examines the ability of treatments to redirect the cytotoxicity of innate immune cells against target antigen-positive cells. For example, it can assess the ability of NK cell cytotoxicity redirected by NK cell conjugates containing an anti-HER2 arm against HER2-positive target cells (such as N87).

As the experimental data of this disclosure show, NKp80 activating binders exhibited greater than median cytotoxicity in cytotoxicity assays compared to the remainder of the population. Of the 108 clones containing an Fc domain with attenuated ADCC function (trispecific, anti-HER2-anti-NKp80-FcX), 78 clones and 67 clones, respectively, were validated for binding to NKp80 in humans and cynomolgus monkeys using biolayer interference (BLI) (Fig. 1, point 2a). A total of 92 trispecific clones (70 NKp80 binders and 22 non-binding clones) were subjected to cytotoxicity assays, and a total of 20 NKp80 activating binder clones were identified.

Of the 20 activating binders, 13 were humanized and constructed as trispecific binders with a fully functional Fc domain (trispecific anti-HER2-anti-NKp80-Fc). All clones were again validated as human NKp80 binders in single-point measurements using BLI, showing dissociation constants (KD) ranging from low nM to sub-pM.

To validate the synergistic effect of NKp80 and CD16 co-conjugation, anti-NKp80 clones identified as activating binders were humanized and formatted as trispecific binders (anti-HER2-anti-NKp80-Fc) containing a fully functional ADCC Fc region. Binding to these clones was tested again, and four clones were selected for further investigation. Cytotoxicity assays were performed on these four clones using OVCAR3 cells as target cells in a dose-dependent manner. All clones showed improved cytotoxicity relative to the trastuzumab control (Figure 3A). In the absence of Fc function, these four clones were able to induce cytotoxicity, but at a lower level than trastuzumab-induced (Figure 3B), indicating that co-conjugation of NKp80 and CD16 is necessary to enhance the synergistic cytotoxicity of NK cells. Dose-response curves and EC50 values were plotted using normalized cell indices (to PBMCs and points where binders were added) in Prism. From these four clones, one was selected for further evaluation in four cell lines representing different cancer origins, using 3-4 independent PBMC donors for each cell line. Dose-response curves and EC50 values were calculated similarly to those described above.

Example 4: Efficacy of anti-NKp80 clone 87-2 in multiple target cell lines and PBMC donors

Of the four conjugates containing anti-NKp80 clones that showed improved efficacy in combined targeting of NKp80 and CD16, the conjugate containing clone 87-2 (trispecific anti-HER2-anti-NKp80(87-2)-Fc) was selected for further illustration. The conjugate was cytotoxically tested using the Xcelligence assay in four different cell lines from different cancer origins using 3–4 independent PBMC batches from healthy donors (Table 1, column 3). Trastuzumab was used as a baseline in these experiments. The selected cell lines and their tumor antigen copy numbers/cells are shown. The selected cell lines and their target copy numbers/cells are shown (Table 1, columns 1 and 2).

EC50 values were used as a measure of potency for these experiments. The fold change in EC50 values (compared to trastuzumab) was then calculated for each experiment and averaged across independent experiments to obtain the overall fold change in EC50 value. The conjugate containing anti-NKp80 clone 87-2 consistently showed improved potency (mean EC50) across different cell lines compared to trastuzumab (Table 1, column 4; Figure 3). Interestingly, it also showed an increasing fold change in potency with decreasing target antigen copy number expression (Table 1, column 4). This is likely due to the diminishing potency of trastuzumab 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).

The increased cytotoxic potency may be supported by the increased NK cell activation following co-conjugation of NKp80 and CD16 (Figure 5). As determined by flow cytometry, the conjugate containing anti-NKp80 clone 87-2 increased NK activation marker expression and cytokine secretion (compared to trastuzumab). Importantly, the conjugate did not induce T cell activation.

Next, the anti-NKp80 clone 87-2 was cloned into a trispecific format, where anti-HER2 was replaced by anti-EGFR. As seen in all previous experiments, this version of the trispecific conjugate targets EGFR instead of HER2. Xcelligence cytotoxicity assays confirmed that this conjugate was able to induce cytotoxicity in EGFR+ cell lines, exhibiting greater potency compared to cetuximab (anti-EGFR-Fc) (Figure 6). As the experimental data show, anti-EGFR-anti-NKp80(87-2)-Fc was more effective than cetuximab (anti-EGFR-Fc) in the cytotoxicity killing assay. The control in the cytotoxicity killing assay (isotype-anti-NKp80(87-2)-Fc) did not show NK cell cytotoxicity killing in HER2-positive cell lines, indicating that cytotoxicity is antigen-dependent.

Table 1: Mean EC50 fold change of anti-HER2-anti-NKp80(87-2)-Fc relative to trastuzumab.

Example 5: Off-target activation of NK cells in antigen-target-negative cell lines

To assess the potential for on-target-off-tumor effects, a trispecific conjugate containing clone 87-2 (anti-HER2-anti-NKp80-Fc) was tested against normal, healthy fibroblast cell line MRC-5. No cell killing was observed in these cells, indicating no on-target-off-tumor effect (Figure 7). Furthermore, no cell killing was observed in the absence of the HER2-targeting antibody arm (isotype-anti-NKp80-Fc, where an isotype control was used instead of HER2) compared to the positive control trastuzumab, demonstrating that NK cell cytotoxicity is target antigen-dependent (Figure 7).

Example 6: Binding sites and sequence similarity of identified NKp80 binding clones

To analyze the epitope binding patterns of NKp80, BLI epitope binning experiments were performed using four anti-NKp80 clones selected for cytotoxicity studies from Figure 3A: humanized clones 45-2, 87-2, 94-1, and 101-1 (Table 2). In these experiments, the target antigen was first immobilized onto the biosensor, and (potentially) competing antibodies were added in successive steps. If the second antibody still generated a signal after the addition of the first antibody, it meant that the second antibody bound to an epitope different from that of the first antibody. The binding index of the epitope binning assays is visualized in Figure 8: clones 45-2 and 87-2 bound to similar epitopes, while clones 94-1 and 101-1 bound to different epitopes.

Table 2: Standardized binding index generated for each clone through tandem binning experiments.

To assess the overall diversity of the 20 activating binders, including the four clones analyzed in Figure 8 (Figure 1, point 2b), a sequence identity matrix was generated using the heavy chain CDR3 region sequences of the 20 NKp80 binders (Figure 14). Subsequent output clustering analysis revealed five distinct clusters (Figure 9), confirming the diversity of the 20 multispecific peptide constructs. Consistent with the epitope binning data in Figure 8, clones 45 and 87 fell into the same cluster (C2), while clone 94 fell into cluster 3 (C3) and clone 101 fell into cluster 5 (C5), indicating that the similar base sequences of clones 45 and 87 led to their binding to similar epitopes of NKp80.

Example 7: Domain arrangement in vivo of multispecific polypeptide constructs

Figure 13D shows different possible arrangements of the domains in a multispecific peptide construct. Selected exemplary constructs are shown below:

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

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

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

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

As shown in Figure 13E, various arrangements of the trispecific binder containing anti-EGFR, wild-type Fc, and anti-NKp80 (87-2) consistently exhibited superior cytotoxicity against breast cancer MDA-MB-231 cells compared to cetuximab, indicating that the superior activity conferred by the additional anti-NKp80 targeting domain is not limited to a specific format. Normalized cell indices (to PBMCs and binder addition points) were plotted using Prism to obtain dose-response curves and EC50 values.

Example 8: Using CD20 to further illustrate the flexibility of antigen-targeting domains

To further illustrate the flexibility of NKp80 targeting, CD20 was used as another target antigen. Anti-CD20 (the Fab portion of rituximab) was cloned into a trispecific format by directly replacing the trastuzumab Fab in the corresponding pcDNA-based VL and VH anti-HER2-anti-NKp80-Fc expression plasmids while keeping everything else identical.

These plasmids were recombinantly expressed in the EXPI-CHO cell mammalian system. Expression vectors containing different fragments of each multispecific polypeptide construct (one containing VH and the other VL) were co-transfected into EXPI-CHO cells according to the manufacturer's instructions and purified using Protein A column chromatography, eluted with 0.2 M Tris-glycine at pH 2.7 and neutralized with 1 M Tris at pH 8.0. Protein purity was assessed by SDS-PAGE under non-reducing conditions (ideally, greater than 95%). The construct buffers were exchanged in ultracentrifuge tubes with 1x PBS, and their concentrations were measured using Nanodrop.

To assess cytotoxic potential, the trispecific conjugate anti-CD20-anti-NKp80-Fc was tested in cytotoxicity assays (e.g., the Xcelligence® assay or a CalceinAM-based staining assay), with rituximab as a control. Treatment was the same as that performed with anti-HER2-anti-NKp80-Fc.

Claims (27)

1. A multispecific polypeptide construct comprising:

(a) One or more antigen targeting domains that bind one or more cancer-associated antigens, and

(B) One or more NK cell targeting domains, wherein binding to NK cells can stimulate and/or inhibit innate immune cell function.

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

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

(1) A heavy chain variable domain (VH) comprising 1,2 or 3 Complementarity Determining Regions (CDRs) selected from the group consisting of 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 about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(2) A light chain variable domain (VL) comprising 1,2 or 3 CDRs selected from the group consisting of 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 to the amino acid sequence thereof, or 2 or 3 amino acid substitutions thereof.

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

(1) 1, 2, 3 or 4 VH Framework Regions (FRs) of VHFR1 selected from the group consisting 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 about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(2) 1, 2, 3 Or 4 VL FRs selected from the group consisting of 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 to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

(1) VH comprising 1,2 or 3 CDRs selected from VH CDR1 of SEQ ID NO 51-67, 250, VH CDR2 of SEQ ID NO 68-85, 251 and/or VH CDR3 of SEQ ID NO 86-104, 252, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VL comprising 1, 2 or 3 CDRs selected from the group consisting of VLCDR1 of SEQ ID NOS.1-16, 247, VLCDR2 of SEQ ID NOS.17-31, 248 and VLCDR3 of SEQ ID NOS.32-50, 249, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(3) 1, 2, 3 or 4 VH Framework Regions (FRs) of VHFR1 selected from the group consisting 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 about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(4) 1, 2, 3 Or 4 VL FRs selected from the group consisting of 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 to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

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

(I) The native/wild-type Fc domain (FcWT) or the attenuated Fc (FcX) domain of SEQ ID NO 224, or having at least about 80% sequence identity to its amino acid sequence, or a2 or 3 amino acid substitution thereof;

(ii) An enhanced Fc domain (FcE) of SEQ ID NO 226, or having at least about 80% sequence identity to its amino acid sequence, or a2 or 3 amino acid substitution thereof;

(iii) The silent Fc domain/inactive mutant Fc domain (FcLALA) of SEQ ID NO 225, or has at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

(a) A first domain targeting NKp 80;

(b) A second domain that targets CD 16;

(c) One or more antigen targeting domains that bind to one or more tumor-associated antigens.

9. The multispecific polypeptide construct of claims 1-8, wherein one or more antigen-targeting domains bind to a member selected from HER-2, EGFR, and CD 20.

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

(1) VH of amino acid sequence SEQ ID No. 231 (VH cetuximab), VL of amino acid sequence SEQ ID No. 232 (VL cetuximab), CH of amino acid sequence SEQ ID No. 233 and/or CL of amino acid sequence SEQ ID No. 234, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VH of amino acid sequence SEQ ID No. 227 (VH trastuzumab), VL of amino acid sequence SEQ ID No. 228 (VL trastuzumab), CH of amino acid sequence SEQ ID No. 229 and/or CL of amino acid sequence SEQ ID No. 230, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(3) VH of amino acid sequence SEQ ID No. 244 (VH rituximab), VL of amino acid sequence SEQ ID No. 243 (VL rituximab), CH of amino acid sequence SEQ ID No. 246 (CH rituximab) and/or CL of amino acid sequence SEQ ID No. 245 (CL rituximab), or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

(A) A NKp80 targeting domain comprising:

(1) VH comprising 1,2 or 3 CDRs selected from VH CDR1 of SEQ ID NO 51-67, 250, VH CDR2 of SEQ ID NO 68-85, 251 and/or VH CDR3 of SEQ ID NO 86-104, 252, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VL comprising 1, 2 or 3 CDRs selected from the group consisting of VLCDR1 of SEQ ID NOS.1-16, 247, VLCDR2 of SEQ ID NOS.17-31, 248 and VLCDR3 of SEQ ID NOS.32-50, 249, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(3) 1, 2, 3 or 4 VH Framework Regions (FRs) of VHFR1 selected from the group consisting 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 about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

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

(B) One or more antigen targeting domains comprising:

(1) VH of amino acid sequence SEQ ID No. 231 (VH cetuximab), VL of amino acid sequence SEQ ID No. 232 (VL cetuximab), CH of amino acid sequence SEQ ID No. 233 and/or CL of amino acid sequence SEQ ID No. 234, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VH of amino acid sequence SEQ ID No. 227 (VH trastuzumab), VL of amino acid sequence SEQ ID No. 228 (VL trastuzumab), CH of amino acid sequence SEQ ID No. 229 and/or CL of amino acid sequence SEQ ID No. 230, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(3) VH of amino acid sequence SEQ ID No. 244 (VH rituximab), VL of amino acid sequence SEQ ID No. 243 (VL rituximab), CH of amino acid sequence SEQ ID No. 246 (CH rituximab) and/or CL of amino acid sequence SEQ ID No. 245 (CL rituximab), or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

(A) A NKp80 targeting domain comprising:

(1) VH comprising 1,2 or 3 CDRs selected from VH CDR1 of SEQ ID NO 51-67, 250, VH CDR2 of SEQ ID NO 68-85, 251 and/or VH CDR3 of SEQ ID NO 86-104, 252, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VL comprising 1, 2 or 3 CDRs selected from the group consisting of VLCDR1 of SEQ ID NOS.1-16, 247, VLCDR2 of SEQ ID NOS.17-31, 248 and VLCDR3 of SEQ ID NOS.32-50, 249, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(3) 1, 2, 3 or 4 VH Framework Regions (FRs) of VHFR1 selected from the group consisting 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 about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

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

(B) One or more antigen targeting domains comprising:

(1) VH of amino acid sequence SEQ ID No. 231 (VH cetuximab), VL of amino acid sequence SEQ ID No. 232 (VL cetuximab), CH of amino acid sequence SEQ ID No. 233 and/or CL of amino acid sequence SEQ ID No. 234, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VH of amino acid sequence SEQ ID No. 227 (VH trastuzumab), VL of amino acid sequence SEQ ID No. 228 (VL trastuzumab), CH of amino acid sequence SEQ ID No. 229 and/or CL of amino acid sequence SEQ ID No. 230, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof, and/or

(3) VH of amino acid sequence SEQ ID NO. 244 (VH rituximab), VL of amino acid sequence SEQ ID NO. 243 (VL rituximab), CH of amino acid sequence SEQ ID NO. 246 (CH rituximab) and/or CL of amino acid sequence SEQ ID NO. 245 (CL rituximab), or having at least about 80% sequence identity to its amino acid sequence, or2 or3 amino acid substitutions thereof, and

(C) An Fc domain having an amino acid sequence selected from the group consisting of SEQ ID 224-226, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

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

(a) A first targeting domain that binds NKp 80;

(b) A second targeting domain that binds CD16, and

(C) A third targeting domain that binds to a target antigen,

Wherein the targeting domain is selected from the group consisting of 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), secreted T cell bispecific Ab (STAb), single domain Ab (sdAb), single domain CH antibodies, single domain CL antibodies, VHH, variable domains of neoantigen receptors (VNAR), sdabs of shark-based VNAR structures, and surrogate scaffold-based binding domains, including but not limited to ankyrin-based domains, fynomer, avimer, anticalin, fibronectin, and binding sites are constructed into the constant region of the antibody.

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

(a) A first targeting domain that binds NKp80, wherein the targeting domain is selected from the group consisting of a 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 of a shark-based VNAR structure;

(b) A first targeting domain that binds 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/inactivating mutant Fc domain (FcLALA) (SEQ ID NO: 225) or FcE (SEQ ID NO: 226), and

(C) A third targeting domain that binds a tumor-associated antigen, optionally HER2, EGFR or CD20, wherein the targeting domain is selected from the group consisting of 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), secreted T cell bispecific Ab (STAb), single domain Ab (sdAb), single domain CH antibodies, single domain CL antibodies, VHH, variable domains of neoantigen receptors (VNAR), sdAb of shark-based VNAR structures, and surrogate scaffold-based binding domains, including but not limited to an ankyrin-based domain, fynomer, avimer, anticalin, fibronectin, and binding sites are constructed into the constant region of the antibody.

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

(1) VH comprising an amino acid sequence selected from SEQ ID NOs 203-222, 236, or having at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof;

(2) VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 183-202, 235;

Wherein the VH and VL pair 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 have at least about 80% sequence identity to its amino acid sequence, or 2 or 3 amino acid substitutions thereof.

16. The multi-specific polypeptide construct of claims 7-15, comprising:

(i) An antigen targeting domain consisting of 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 an scFv comprising a VH, a second [ (G4S) n ] linker, and a VL;

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

(Iv) An antigen targeting domain consisting of an Fd fragment (comprising VH and CH 1) or a Fab fragment, a first [ (G4S) n ] linker, a first NK cell targeting domain consisting of an scFv comprising VH, a second [ (G4S) n ] linker and VL, and a second NK cell targeting domain consisting of an Fc domain comprising CH2 and CH3.

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

(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 an antigen binding fragment thereof, comprising a CDR sequence selected from the group consisting of:

(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),

Wherein the CDR sequence has at least about 90% homology with an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-104, 247-252, and/or

Wherein the CDR sequence selected from SEQ ID NO 1-104, 247-252 comprises 2 or 3 amino acid substitutions.

19. An antigen binding protein, or antigen binding fragment thereof, comprising CDR and FR sequences selected from the group consisting of:

(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),

Wherein the FR and CDR sequences have at least about 90% homology with amino acid sequences selected from the group consisting of SEQ ID NOS: 1-104, 247-260, and/or

Wherein the FR and CDR sequences selected from SEQ ID NO 1-104, 247-260 comprise 2 or 3 amino acid substitutions.

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

21. A vector comprising a sequence for the multispecific polypeptide construct or antibody of claims 1-19.

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

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

24. A method of screening and/or identifying the multi-specific polypeptide construct or antibody of claims 1-19, wherein the NK cell targeting domain is anti-NKp 80.

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

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

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