Antibodies and their use

Abstract

This disclosure relates to an anti-NKp46 antibody, an antigen-binding fragment, and its use. This disclosure also relates to an antibody or an antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), a second antigen-binding domain that specifically binds to an NK cell surface antigen, and an Fc region having optimized Fc effector function.

Description

[Technical Field]

[0001] Claim of priority This application claims the interests of application number PCT / CN2023 / 095161 filed on 19 May 2023, application number PCT / CN2023 / 137348 filed on 8 December 2023, application number PCT / CN2023 / 095163 filed on 19 May 2023, and application number PCT / CN2023 / 137346 filed on 8 December 2023. The entirety of the above-mentioned applications is incorporated herein by reference.

[0002] This disclosure relates to anti-NKp46 antibodies, multispecific antibodies (e.g., bispecific antibodies or their antigen-binding fragments), and antibody-drug conjugates derived therefrom. [Background technology]

[0003] Cancer is currently one of the leading causes of death in humans. According to World Health Organization statistics, in 2012, there were 14 million new cancer cases and 8.2 million cancer deaths worldwide. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million deaths. Recent clinical and commercial successes of anti-cancer antibodies have drawn considerable interest to antibody-based therapies.

[0004] Bispecific antibodies are artificial proteins capable of simultaneously binding to two different types of antigens or two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, dual-targeting different disease mediators, and delivering payloads to target sites. The approval of catumakisomab (anti-EpCAM and anti-CD3) and blinatumomab (anti-CD19 and anti-CD3) represents a major milestone in the development of bispecific antibodies.

[0005] There is a need to develop antibodies for use in various antibody-based therapies to treat cancer. Since bispecific antibodies have a wide range of applications, it is necessary to continue developing various therapeutic agents based on bispecific antibodies. [Overview of the project]

[0006] This disclosure relates to an anti-NKp46 antibody, its antigen-binding fragment, and its use.

[0007] This disclosure also relates to multispecific (e.g., bispecific) antibodies or antigen-binding fragments thereof, which specifically bind to tumor-associated antigens (TAAs) and NK cell surface antigens. In some embodiments, the antibody or antigen-binding fragment has an optimized Fc region. In some embodiments, the antibody or antigen-binding fragment has increased binding affinity to FcγRIIa receptors and / or FcγRIIIa receptors.

[0008] In one embodiment, this specification provides for an antibody or antigen-binding fragment thereof that binds to NKp46, comprising a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH CDR1, the VH CDR2 region contains an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH CDR2, and the VH CDR3 region contains an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH CDR3, and a light chain variable region (VL) including CDRs 1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL CDR1, the VL CDR2 region contains an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL CDR2, and the VL CDR3 region contains an amino acid sequence at least 80% identical to the selected VL An antibody or its antigen-binding fragment is provided, comprising a light chain variable region having an amino acid sequence at least 80% identical to that of CDR3, wherein the amino acid sequences of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 5, 7, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 6, 8, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 15, 17, and 19, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 18, and 19, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; and In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

[0009] In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 6, 8, and 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

[0010] In some embodiments, according to Kabat's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively.

[0011] In some embodiments, according to Chothia's definition, VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 16, 18, and 19, respectively, and VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 20, 21, and 22, respectively.

[0012] In some embodiments, according to the Kabat definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 25, 27, and 29, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0013] In some embodiments, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 26, 28, and 29, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0014] In some embodiments, the antibody or antigen-binding fragment specifically binds to human NKp46 or monkey NKp46.

[0015] In some embodiments, the antibody or antigen-binding fragment is a human antibody or a humanized antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0017] In one aspect, provided herein is a nucleic acid comprising a polynucleotide encoding a polypeptide comprising the following:

[0018] (1) An immunoglobulin heavy chain or a fragment thereof comprising a variable heavy chain region (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs: 5, 7, and 9, respectively, wherein the VH forms a pair with a variable light chain region (VL) comprising the amino acid sequence shown in SEQ ID NO: 4 and binds to NKp46. (2) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 3; (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 6, 8, and 9, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 4; (4) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 14; (5) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 13; (6) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 16, 18, and 19, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 14; (7) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 24; (8) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 23; (9) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 26, 28, and 29, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 24; (10) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 36; (11) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 35; (12) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 6, 8, and 9, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 36; (13) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 38; (14) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 37; (15) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 16, 18, and 19, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 38; (16) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 34; (17) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 33; (18) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 26, 28, and 29, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 34; In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH comprising CDR1, 2, and 3, which comprises the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, respectively, or SEQ ID NOs. 6, 8, and 9, respectively.

[0019] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising a VL containing an immunoglobulin light chain or fragment thereof, which includes CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

[0020] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH comprising CDR1, 2, and 3, which include the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, or SEQ ID NOs. 16, 18, and 19, respectively.

[0021] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising a VL containing an immunoglobulin light chain or fragment thereof, which includes CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively.

[0022] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH comprising CDR1, 2, and 3, which include the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, respectively, or SEQ ID NOs. 26, 28, and 29, respectively.

[0023] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, comprising a VL containing an immunoglobulin light chain or fragment thereof, which includes CDR1, 2, and 3, which contain the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0024] In some embodiments, when VH pairs with VL, it specifically binds to human NKp46 or monkey NKp46, or when VL pairs with VH, it specifically binds to human NKp46 or monkey NKp46.

[0025] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof or a humanized immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof or a humanized immunoglobulin light chain or fragment thereof.

[0026] In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

[0027] In some embodiments, the nucleic acid is cDNA.

[0028] In one embodiment, this specification provides a vector comprising one or more nucleic acids described herein.

[0029] In one embodiment, this specification provides a vector comprising two of the nucleic acids described herein, wherein the vector encodes a VH region and a VL region that bind together to NKp46.

[0030] In one embodiment, the Specified herein provides a pair of vectors, each comprising one of the nucleic acids described herein and encoding both a VH region and a VL region that bind together to NKp46.

[0031] In one embodiment, the Specified Specified provides a cell comprising any one of the vectors described herein or any pair of vectors described herein.

[0032] In some embodiments, the cells are CHO cells.

[0033] In one embodiment, this specification provides cells comprising one or more nucleic acids described herein.

[0034] In one embodiment, the Specified herein provides a cell comprising two of the nucleic acids described herein.

[0035] In some embodiments, the two nucleic acids both encode the VH region and the VL region that bind to NKp46.

[0036] In one embodiment, the present specification provides a method for producing an antibody or an antigen-binding fragment thereof, (a) Culturing any one of the cells described herein under conditions sufficient to cause the cells to produce the antibody or antigen-binding fragment, (b) Recovering the antibody or antigen-binding fragment produced by the cells, A method is provided.

[0037] In one embodiment, the Specified Reference Reference provides an antibody or antigen-binding fragment thereof that binds to NKp46, comprising a VH having an amino acid sequence identical to at least 80% a selected heavy chain variable region (VH) sequence, and a VL having an amino acid sequence identical to at least 80% a selected light chain variable region (VL) sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24; (4) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (5) The selected VH sequence is sequence number 35, and the selected VL sequence is sequence number 36; and (6) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38.

[0038] In some embodiments, VH includes the sequence of sequence number 3, and VL includes the sequence of sequence number 4.

[0039] In some embodiments, VH includes the sequence of sequence number 13, and VL includes the sequence of sequence number 14.

[0040] In some embodiments, VH includes the sequence of sequence number 23, and VL includes the sequence of sequence number 24.

[0041] In some embodiments, VH includes the sequence of sequence number 33, and VL includes the sequence of sequence number 34.

[0042] In some embodiments, VH includes the sequence of sequence number 35, and VL includes the sequence of sequence number 36.

[0043] In some embodiments, VH includes the sequence of sequence number 37, and VL includes the sequence of sequence number 38.

[0044] In some embodiments, the antibody or antigen-binding fragment specifically binds to human NKp46 or monkey NKp46.

[0045] In some embodiments, the antibody or antigen-binding fragment is a human antibody or a humanized antibody or its antigen-binding fragment.

[0046] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0047] In one embodiment, this specification provides an antibody or an antigen-binding fragment that cross-competes with any one of the antibodies or antigen-binding fragments described herein.

[0048] In one embodiment, the Specified Public Service provides an antibody or antigen-binding fragment thereof that binds to NKp46, comprising VH, which includes VH CDR1, VH CDR2, and VH CDR3 identical to the selected heavy chain variable region (VH) sequence VH CDR1, VH CDR2, and VH CDR3, and VL, which includes VL CDR1, VL CDR2, and VL CDR3 identical to the selected light chain variable region (VL) sequence VL CDR1, VL CDR2, and VL CDR3, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24; (4) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (5) The selected VH sequence is sequence number 35, and the selected VL sequence is sequence number 36; and (6) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38.

[0049] In one embodiment, the Specified provides a method for treating a subject having cancer or an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a composition comprising either an antibody or an antigen-binding fragment thereof.

[0050] In some embodiments, the antibody or its antigen-binding fragment is a bispecific antibody that targets both NK cells and tumor cells expressing tumor antigens.

[0051] In some embodiments, the method further comprises administering to a subject a therapeutically effective dose of anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

[0052] In one aspect, the Specified provides a method for reducing the rate of tumor growth, comprising contacting tumor cells with a composition comprising an effective amount of one of the antibodies or antigen-binding fragments thereof described herein.

[0053] In one embodiment, the Specified provides a method for killing tumor cells, comprising contacting tumor cells with a composition comprising an effective amount of one of the antibodies or antigen-binding fragments thereof described herein.

[0054] In one embodiment, the Specified herein provides a pharmaceutical composition comprising one of the antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier.

[0055] This specification also provides an antibody or an antigen-binding fragment thereof comprising: i) a first antigen-binding domain that specifically binds to a first antigen which is a tumor-associated antigen (TAA); ii) a second antigen-binding domain that specifically binds to a second antigen which is an NK cell surface antigen; and iii) an Fc region.

[0056] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0057] In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain containing a light chain variable domain (VL2) and a heavy chain variable domain (VH2) linked by a first linker.

[0058] In some embodiments, a second antigen-binding domain is bound to the C-terminus of the light chain of the first antigen-binding domain via a second linker.

[0059] In some embodiments, the second antigen-binding domain is ligated to the C-terminus of the Fc region.

[0060] In some embodiments, VH1 is linked to the CH1 domain, and VL1 is linked to the CL domain.

[0061] In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0062] In some embodiments, the Fc region is the Fc region of human IgG1.

[0063] In some embodiments, the antibody or its antigen-binding fragment is a bispecific antibody or its antigen-binding fragment.

[0064] In some embodiments, the Fc region includes one or more of the following (all numbering follows EU numbering): Glutamate I, ranked 332nd; Leucine (L) is ranked 330th; Alanine (A) at position 236; and / or Aspartic acid (D) ranks 293rd.

[0065] In some embodiments, the Fc region includes one of the following (all numbering follows EU numbering): i) Alanine (A) at position 236, leucine (L) at position 330, and glutamic acid I at position 332; ii) Alanine (A) at position 236, aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid I at position 332; iii) Alanine (A), ranked 236th; iv) Alanine (A) at position 236, aspartic acid (D) at position 293, and glutamic acid I at position 332; v) Aspartic acid (D) at position 293 and glutamic acid I at position 332; and vi) Aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid I at position 332.

[0066] In some embodiments, the Fc region is not fucosylated.

[0067] In some embodiments, the Fc region is the Fc region of human IgG1 and includes alanine (A) at position 236, leucine (L) at position 330, and glutamic acid I at position 332, and has an amino acid sequence that is at least 80% identical to SEQ ID NO: 43 (numbering follows EU numbering).

[0068] In some embodiments, the NK cell surface antigen is selected from NKp46, NKp30, CD16, NKG2D, and NKG2A.

[0069] In some embodiments, the NK cell surface antigen is NKp46.

[0070] In some embodiments, tumor-associated antigens (TAAs) include CD79b, EGFR, EpCAM, B-cell maturation antigen (BCMA), DLL3, PSMA, STEAP1, MUC1, c-Met, LRRC15, CCR8, OX40, perforin, granzyme B, Fas ligand (FasL), CD1d, membrane glycolipids, globotriaosylceramide (Gb3Cer / CD77), gangliosides (GD2, GD3, and GM2), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, and CLL1. , CD105, CD71, SSC, MAGE, MUC16, CD19, WT-l, B7H3, TEM8, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-fetoprotein (AFP), BAFF, B lymphoma cells, C 242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, Fibronectin Extradomain B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Scattering Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, Insulin-like Growth Factor I Receptor, Integrin a5b1, Integrin anb3, MORAb-009, MS4A1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PD-1, PD-L1, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

[0071] In some embodiments, the first antigen is CD79b and the second antigen is NKp46.

[0072] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, where the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and the first light chain variable region (VL1) includes CDRs 1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3, and the selected VH1 The amino acid sequences of CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively, and (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are the same as the amino acid sequences of the corresponding VH CDR1, 2, and 3 and VL CDR1, 2, and 3 of polatuzumab (Genentech Inc.), PRV-3279 (MacroGenics Inc.), or SHR-A1912 (Jiangsu Hengrui Pharmaceuticals Co., Ltd.).

[0073] In some embodiments, the second heavy chain variable region (VH2) includes CDR1, 2, and 3, where the VH2 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and the second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, the VL2 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3, and the selected VH2 The amino acid sequences of CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 5, 7, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 6, 8, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 15, 17, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 18, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; and (6) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0074] In some embodiments, (1) the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 5, 7, and 9, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 6, 8, and 9, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 15, 17, and 19, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 16, 18, and 19, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; or (6) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0075] In some embodiments, the first antigen is BCMA and the second antigen is NKp46.

[0076] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 88, 89, and 90, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 91, 92, and 93, respectively. (2) The amino acid sequences of the above-selected VH1 CDR1, 2, and 3 and the above-selected amino acid sequences of VL1 CDR1, 2, and 3 are used in Zevolcabtagene Autoleucel (CARsgen Therapeutics Co. Ltd.), Elranatamab (Pfizer Inc.), Teclistamag (Janssen Pharmaceutica NV), Equecabtagene Autoleucel (Nanjing IASO Biotherapeutics Co., Ltd.), Ciltacabtagene Autoleucel (Nanjing Legend Biotech Co., Ltd.), Idecabtagene Vicleucel (Bluebird bio Inc.), Verantamab (GSK Plc), Limbocertamab (Regeneron Pharmaceuticals) It is obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 sequences of alnuctamab (Celgene Corp.), TNB-383B (TeneoBio Inc.), or SEA-BCMA (Seagen Inc.).

[0077] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0078] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 88, 89, and 90, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 91, 92, and 93, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0079] In some embodiments, the first antigen is EGFR and the second antigen is NKp46.

[0080] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 94, 95, and 96, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 97, 98, and 99, respectively. (2) The amino acid sequences of the above-selected VH1 CDR1, 2, and 3 and the amino acid sequences of the above-selected VL1 CDR1, 2, and 3 are amivantamab (Janssen Biotech Inc.), cetuximab (Merck Serono SA), necitumumab (Eli Lilly & Co.), panitumumab (Amgen Inc.), nimotuzumab (Center For Molecular Immunology), BL-B01D1 (Sichuan Baili Pharmaceuticals Co.,Ltd), dempitamab (Sinocelltech Group Ltd.), depatuxizumab (Ludwig Institute for Cancer Research Ltd.), futuximab (Merck KGaA), modotuximab (Merck KGaA), JMT-101 (Shanghai JMT Biological Technology Co Ltd), MRG-003 (Shanghai Miracogen Inc.), and pimlutamab (Shanghai It is obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of Henlius Biotech Inc., QL1203 (Qilu Pharmaceutical Co., Ltd.), SI-B001 (Sichuan Baili Pharmaceuticals Co., Ltd.), or ametumumab (Academy of Military Medical Sciences).

[0081] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0082] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 94, 95, and 96, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 97, 98, and 99, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0083] In some embodiments, the first antigen is EpCAM and the second antigen is NKp46.

[0084] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 101, and 102, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 103, 104, and 105, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, 3 and the selected VL1 CDR1, 2, 3 are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of catumakisomab (Neovii Biotech GmbH), M-701 (Wuhan YZY Biopharma Co., Ltd.), adekatumumab (Amgen Inc.), edrecolomab (GSK Plc), tefivazumab (Bristol Myers Squibb Co.), VB1-008 (Sesen Bio Inc.), or VB1-050 (Sesen Bio Inc.).

[0085] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0086] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 101, and 102, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 103, 104, and 105, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0087] In some embodiments, the first antigen is DLL3 and the second antigen is NKp46.

[0088] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of lovalpituzumab tecillin (AbbVie Inc.), tarulatamab (Amgen Inc.), HPN328 (Harpoon Therapeutics Inc.), or BI 764532 (Boehringer Ingelheim GmbH).

[0089] In some embodiments, the first antigen is PSMA and the second antigen is NKp46.

[0090] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, and 3 and VL CDR1, 2, and 3 amino acid sequences of HPN-424 (Harpoon Therapeutics Inc.), LAVA-1207 (LAVA Therapeutics NV), REGN-5678 (Regeneron Pharmaceuticals Inc.), REGN4336 (Regeneron Pharmaceuticals Inc.), akapatamab (Amgen Inc.), or AMG-340 (Amgen Inc.).

[0091] In some embodiments, the first antigen is STEAP1 and the second antigen is NKp46.

[0092] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of zalritamig (Amgen Inc.), ABBV-969 (AbbVie Inc.), or vandortuzumab vedotin (Genentech Inc.).

[0093] In some embodiments, the first antigen is MUC1 and the second antigen is NKp46.

[0094] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of MAB-AR20.5 (Canariabio Inc.) or DS-3939 (Glycotope GmbH).

[0095] In some embodiments, the first antigen is c-Met and the second antigen is NKp46.

[0096] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, and VL

[0097] In some embodiments, the first antigen is LRRC15 and the second antigen is NKp46.

[0098] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of QL-315 (Qilu Pharmaceutical Co., Ltd.), RAD-502 (Radiopharm Theranostics Ltd.), or samulotamab (AbbVie Inc.).

[0099] In some embodiments, the first antigen is CCR8 and the second antigen is NKp46.

[0100] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the above-selected VH1 CDR1, 2, and 3 and the above-selected VL1 CDR1, 2, and 3 are the same as those of BMS-986340 (Bristol Myers Squibb Co.), LM-108 (LaNova Medicines Ltd.), S-531011 (Shionogi & Co., Ltd.), AMG-355 (Amgen Inc.), BAY3375968 (Bayer AG), BGB-A3055 (BeiGene Ltd.), CM369 (Keymed Biomedical Technology (Chengdu) Co., Ltd.), HBM-1022 (Harbour BioMed (Shanghai) Co., Ltd.), PSB-114 (Qilu Puget Sound Biotherapeutics Corp.), or SRF-114 (Surface Oncology Inc.) It is obtained from the amino acid sequences of CDR1, 2, 3 and VL CDR1, 2, 3.

[0101] In some embodiments, the first antigen is OX40 and the second antigen is NKp46.

[0102] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and The first light chain variable region (VL1) includes CDR1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the above-selected VH1 CDR1, 2, and 3 and the above-selected VL1 CDR1, 2, and 3 correspond to the VH CDR1, 2, 3, and VL1 CDR1, 2 It is obtained from the amino acid sequences of CDR1, 2, and 3.

[0103] In some embodiments, the Fc region exhibits increased complement-dependent cell-mediated cytotoxicity (CDC), increased antibody-dependent cell-mediated cytotoxicity (ADCC), or an extended half-life.

[0104] In one embodiment, this specification provides an antibody or an antigen-binding fragment that cross-competes with either the antibody or the antigen-binding fragment thereof.

[0105] In one embodiment, the Specified provides a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising either an antibody or an antigen-binding fragment thereof.

[0106] In some embodiments, cancer is a solid tumor or a hematological tumor.

[0107] In some embodiments, the cancers are breast cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, ovarian cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer, and liver cancer.

[0108] In some embodiments, cancers include multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prelymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, follicular lymphoma, and mantle cell lymphoma.

[0109] In some embodiments, the subject is further treated with an effective amount of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, or anti-PD-L1 antibody.

[0110] In one embodiment, this specification provides a method for reducing the rate of tumor growth, comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof as described herein.

[0111] In one embodiment, the Specified provides a method for killing tumor cells, comprising contacting tumor cells with a composition comprising an effective amount of one of the antibodies or antigen-binding fragments thereof described herein.

[0112] In one embodiment, the Specified provides a method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of a composition comprising one of the antibodies or antigen-binding fragments thereof described herein.

[0113] In one embodiment, the Specified provides a method for treating a subject having an autoimmune disease, comprising administering a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0114] In one embodiment, autoimmune diseases are selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's arteritis, cold agglutinin disease, warm autoimmune hemolytic anemia, and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (e.g., granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) or microscopic polyangiitis (MPA)).

[0115] In one embodiment, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis.

[0116] In one embodiment, the Specified herein provides a pharmaceutical composition comprising one of the antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier.

[0117] As used herein, the term “cancer” means cells capable of autonomous proliferation. Examples of such cells include cells in an abnormal state or condition characterized by rapid proliferation. The term means cancerous growth, e.g., tumors, oncogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Malignancies of various organ systems, e.g., head and neck, respiratory, cardiovascular, renal, reproductive, hematological, nervous, liver, gastrointestinal, and endocrine systems, as well as most colorectal cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestinal cancer, glioma, and small bowel cancer. “Spontaneously occurring” cancers include any cancer that is not experimentally induced by transplanting cancer cells into a subject, e.g., spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from transgenic oncogene insertion or tumor suppressor gene knockout, and cancers caused by infections, e.g., viral infections. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine glandular tissue. This term also includes carcinosarcoma, which is a malignant tumor composed of cancerous and sarcomatoid tissue. "Adenocarcinoma" refers to a carcinoma of glandular origin, or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hematopoietic hyperplasia / neoplastic cells. Hematopoietic neoplastic diseases may originate from the bone marrow, lymphoid system, or erythrocyte lineage, or their progenitor cells. Hematological cancers are cancers that begin in blood-forming tissues such as bone marrow, or in cells of the immune system. Examples of hematological cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0118] As used herein, “autoimmune disease” refers to a condition resulting from an abnormal immune response against functional body parts. Symptoms of autoimmune diseases are diverse and transient, ranging from mild to severe, but generally include a low-grade fever and fatigue. Autoimmune diseases have various causes. Some autoimmune diseases, such as lupus, can occur within families, and in certain cases, infections or other environmental factors can trigger them. Representative diseases generally considered to be autoimmune diseases include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0119] As used herein, the term “antigen-binding domain” means one or more protein domains (for example, formed from amino acids derived from a single polypeptide, or formed from amino acids derived from two or more polypeptides (e.g., identical or different polypeptides) that are specifically capable of binding to one or more different antigens (e.g., effector antigens or tumor antigens). In some examples, the antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. An example of an antigen-binding domain is an antigen-binding domain formed by VH-VL. In some embodiments, the antigen-binding domain can include an alternative scaffold. In some embodiments, the antigen-binding domain is VHH. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are well known in the art. In some examples, the antigen-binding domain can bind to a single antigen (e.g., one of effector antigens and tumor antigens).

[0120] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from an immunoglobulin light chain or any of three CDRs derived from an immunoglobulin heavy chain) and is capable of specifically binding to an antigen. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments. As used herein, the term “multispecific antibody” refers to an antibody comprising two or more different antigen-binding domains that collectively and specifically bind to two or more different epitopes. The two or more different epitopes may be epitopes on the same antigen (e.g., a single polypeptide present on the surface of a cell) or epitopes on different antigens (e.g., different proteins present on the surface of the same cell or on the surfaces of different cells). In some embodiments, a multispecific antibody binds to two different epitopes (e.g., a “bispecific antibody”). In some embodiments, a multispecific antibody binds to three different epitopes (e.g., a “triplespecific antibody”). In some embodiments, a multispecific antibody binds to four different epitopes (e.g., a “quadrispecific antibody”). In some embodiments, a multispecific antibody binds to five different epitopes (e.g., a “quintuplespecific antibody”). Each binding specificity can exist at any appropriate valency. Non-limiting examples of multispecific antibodies are described herein.

[0121] As used herein, the term “bispecific antibody” means an antibody that binds to two different epitopes. Epitopes may be present on the same antigen or on different antigens.

[0122] As used herein, the term “chimeric antibody” means an antibody containing sequences present in at least two different species (e.g., an antibody derived from two different mammalian species, such as a human antibody and a mouse antibody, or a human antibody and a rabbit antibody). Non-limiting examples of chimeric antibodies include antibodies containing a variable domain sequence (e.g., all or part of the light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse or rabbit) antibody, as well as a constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0123] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse or rabbit) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse or rabbit) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains substantially all, at least one, and typically two, variable domains, where all or substantially all of the hypervariable loop (CDR) corresponds to the hypervariable loop of a non-human (e.g., mouse or rabbit) immunoglobulin, and all or substantially all of the framework region is a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0124] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0125] Where used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to an animal, human, or non-human being to be treated according to the methods of the present invention. Veterinary and non-veterinary uses are conceived in this disclosure. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0126] As used herein, when referring to an antibody, the terms "specifically binding" and "specifically binds" mean that the antibody interacts more with its target molecule (e.g., NKp46) than with other molecules, because the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to a molecule containing a specific structure, rather than to all general molecules. An antibody that specifically binds to a target molecule may also be called a target-specific antibody. For example, an antibody that specifically binds to the NKp46 molecule may be called an NKp46-specific antibody or an anti-NKp46 antibody.

[0127] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and mean polymers of at least two amino acids of any length.

[0128] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and mean, but are not limited to, polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0129] As used herein, the term “NK cell surface antigen” means a molecule on an NK cell, such as a surface receptor, that is involved in the regulation and / or induction of NK cell function. In some embodiments, the NK cell surface antigen is an NK cell activation receptor.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0131] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0132] [Figure 1] This shows the affinity of an anti-NKp46 antibody for human NKp46. [Figure 2A] This shows the ELISA binding of chimeric and humanized anti-NKp46 antibodies. [Figure 2B] This shows the ELISA binding of chimeric and humanized anti-NKp46 antibodies. [Figure 2C] This shows the ELISA binding of chimeric and humanized anti-NKp46 antibodies. [Figure 3A]This shows BLI binding of chimeric and humanized anti-NKp46 antibodies. [Figure 3B] This shows BLI binding of chimeric and humanized anti-NKp46 antibodies. [Figure 3C] This shows BLI binding of chimeric and humanized anti-NKp46 antibodies. [Figure 4A] This is a schematic diagram showing an example of a bispecific antibody described herein. [Figure 4B] This is a schematic diagram showing an example of a bispecific antibody described herein. [Figure 5] This demonstrates the activation of NK cells by a CD79b / NKp46 bispecific antibody. [Figure 6] This demonstrates the binding ability of a CD79b / NKp46 bispecific antibody with silent Fc to effector NK cells. [Figure 7] This study demonstrates the binding ability of a CD79b / NKp46 bispecific antibody with silent Fc to target Ramos cells. [Figure 8] This study demonstrates that a CD79b / NKp46 bispecific antibody with silencing Fc cells mediates the killing of B cells in target cancers by NK cells. [Figure 9] This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of Ramos cells by NK cells. [Figure 10] This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of Daudi cells by NK cells. [Figure 11A] This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of endogenous B cells by PBMCs. [Figure 11B] This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of endogenous B cells by PBMCs. [Figure 11C] This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of endogenous B cells by PBMCs. [Figure 11D]This study demonstrates that a CD79b / NKp46 bispecific antibody possessing wild-type Fc mediates the killing of endogenous B cells by PBMCs. [Figure 12A] This demonstrates the activation of NK cells by a CD79b / NKp46 bispecific antibody with optimized Fc. [Figure 12B] This demonstrates the activation of NK cells by a CD79b / NKp46 bispecific antibody with optimized Fc. [Figure 13] This demonstrates the activation of NK cells by a CD79b / NKp46 bispecific antibody with optimized Fc and a CD79b antibody with optimized Fc. [Figure 14A] This study demonstrates that a CD79b / NKp46 bispecific antibody with optimized Fc mediates the killing of Ramos cells by NK cells. [Figure 14B] This study demonstrates that a CD79b / NKp46 bispecific antibody with optimized Fc mediates the killing of Ramos cells by NK cells. [Figure 15] This study demonstrates that a CD79b / NKp46 bispecific antibody with optimized Fc mediates the killing of Ramos cells by PBMCs. [Figure 16] This study demonstrates that a CD79b / NKp46 bispecific antibody with optimized Fc mediates the killing of Nalm-6 cells by PBMCs. [Figure 17A] We demonstrate that NK cell engagers with optimized Fc levels mediate the killing of Ramos cells by PBMCs. [Figure 17B] We demonstrate that NK cell engagers with optimized Fc levels mediate the killing of Ramos cells by PBMCs. [Figure 18A] This demonstrates the cytokine release activity of NK cell engagers with optimized Fc. [Figure 18B] This demonstrates the cytokine release activity of NK cell engagers with optimized Fc. [Figure 18C] This demonstrates the cytokine release activity of NK cell engagers with optimized Fc. [Figure 19] We demonstrate that NK cell engagers with optimized Fc levels mediate the killing of Nalm-6 cells by PBMCs. [Figure 20] This shows the number of NK cells during target cell killing with a CD79b / NKp46 bispecific antibody having an optimized Fc. [Figure 21] This shows the number of monocytes during target cell killing with a CD79b / NKp46 bispecific antibody having an optimized Fc. [Figure 22A] We demonstrated that NK cell engagers with optimized Fc levels mediate endogenous B cell killing by PBMCs (Study 1). [Figure 22B] We demonstrated that NK cell engagers with optimized Fc levels mediate endogenous B cell killing by PBMCs (Study 1). [Figure 23] We demonstrated that NK cell engagers with optimized Fc levels mediate endogenous B cell killing by PBMCs (Study 2). [Figure 24A] This demonstrates the cytokine release activity of NK cell engagers with optimized Fc. [Figure 24B] This demonstrates the cytokine release activity of NK cell engagers with optimized Fc. [Figure 25A] This study demonstrates that NK cell engagers kill B cells in target cancers via monocytes and NK cells. [Figure 25B] This study demonstrates that NK cell engagers kill B cells in target cancers via monocytes and NK cells. [Figure 25C] This study demonstrates that NK cell engagers kill B cells in target cancers via monocytes and NK cells. [Figure 25D] This study demonstrates that NK cell engagers kill B cells in target cancers via monocytes and NK cells. [Figure 26] This demonstrates the binding ability of humanized NK cell engagers to NKp46-expressing cells. [Figure 27] This demonstrates the binding ability of humanized NK cell engagers to CD79b-expressing cells. [Figure 28A] Humanized NK cell engagers mediate the killing of B cells in target cancers by PBMCs (Study 1). [Figure 28B] Humanized NK cell engagers mediate the killing of B cells in target cancers by PBMCs (Study 1). [Figure 29A] Humanized NK cell engagers demonstrate that PBMCs mediate the killing of B cells in target cancers (Study 2). [Figure 29B] Humanized NK cell engagers demonstrate that PBMCs mediate the killing of B cells in target cancers (Study 2). [Figure 30] This study demonstrates that NK cell engagers added to plasma mediate the killing of target cancer B cells by PBMCs. [Figure 31] This shows the quantification of BCMA antigen density on the surface of multiple myeloma tumor cells and normal B cells. [Figure 32] This study demonstrates that a BCMA / NKp46 bispecific antibody mediates the killing of targeted multiple myeloma cells by PBMCs. [Figure 33A] The following are some of the amino acid sequences described in this disclosure. [Figure 33B] The following are some of the amino acid sequences described in this disclosure. [Figure 33C] The following are some of the amino acid sequences described in this disclosure. [Figure 33D] The following are some of the amino acid sequences described in this disclosure. [Figure 33E] The following are some of the amino acid sequences described in this disclosure. [Figure 33F] The following are some of the amino acid sequences described in this disclosure. [Figure 33G] The following are some of the amino acid sequences described in this disclosure. [Figure 33H] The following are some of the amino acid sequences described in this disclosure. [Figure 33I] The following are some of the amino acid sequences described in this disclosure. [Figure 33J] The following are some of the amino acid sequences described in this disclosure. [Figure 33K] The following are some of the amino acid sequences described in this disclosure. [Figure 33L] The following are some of the amino acid sequences described in this disclosure. [Figure 33M] The following are some of the amino acid sequences described in this disclosure. [Figure 33N] The following are some of the amino acid sequences described in this disclosure. [Figure 33O] The following are some of the amino acid sequences described in this disclosure. [Figure 33P] The following are some of the amino acid sequences described in this disclosure. [Figure 33Q] The following are some of the amino acid sequences described in this disclosure. [Figure 33R] The following are some of the amino acid sequences described in this disclosure. [Figure 33S] The following are some of the amino acid sequences described in this disclosure. [Figure 33T] The following are some of the amino acid sequences described in this disclosure. [Figure 33U] The following are some of the amino acid sequences described in this disclosure. [Figure 33V] The following are some of the amino acid sequences described in this disclosure. [Figure 33W] The following are some of the amino acid sequences described in this disclosure. [Figure 33X] The following are some of the amino acid sequences described in this disclosure. [Figure 33Y] The following are some of the amino acid sequences described in this disclosure. [Figure 33Z] The following are some of the amino acid sequences described in this disclosure. [Modes for carrying out the invention]

[0133] Natural killer (NK) cells play a crucial role in the first line of defense against tumors and virus-infected cells. NK cell activity is tightly regulated by a repertoire of inhibitory and activating receptors expressed on the cell surface. NKp46 is a major NK cell activating receptor involved in the elimination of target cells. NK cells form various types of synapses that result in different functional outcomes: cytotoxic, inhibitory, and regulatory. Recent research has revealed that the complex integration of NK receptor signaling controls cytoskeletal reorganization and other immunosynaptic events. Therefore, anti-NKp46 antibodies may have potential applications in treating cancer and other diseases such as autoimmune disorders.

[0134] This disclosure provides examples of antibodies that bind to NKp46 and their antigen-binding fragments.

[0135] A multispecific antibody or its antigen-binding fragment is an artificial protein capable of simultaneously binding to two or more different epitopes (e.g., on two different antigens). In some embodiments, the multispecific antibody is a bispecific antibody. The bispecific antibody or its antigen-binding fragment may have two arms. Each arm has one heavy chain variable region and one light chain variable region, and can form an antigen-binding domain (or antigen-binding region). The two arms can bind to two different antigens. In some embodiments, an additional antigen-binding domain can be added to a monoclonal antibody (e.g., to the C-terminus of the light or heavy chain).

[0136] This disclosure relates to a multispecific (e.g., bispecific) antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to tumor-associated antigens (TAAs) and a second antigen-binding domain that specifically binds to NK cell surface antigens.

[0137] NKp46 Natural killer (NK) cells are granular lymphocytes that were initially recognized for their ability to efficiently eliminate tumor cells without prior sensitization. Viral infections can also induce NK cell toxicity. In fact, NK cells are a crucial component of the innate immune response to viruses, as evidenced by the high incidence of infections in patients with primary NK cell deficiency and in mice lacking NK cells. The regulation of NK cell function is primarily controlled by the recognition of autologous MHC class I molecules via a set of inhibitory receptors (human killer cell immunoglobulin-like receptors (KIRs) and the mouse Ly49 receptor family). NK cells do not express variable antigen-specific receptors. Instead, their activation is controlled by a repertoire of germline-encoded activating receptors that recognize various ligands present on the surface of sensitive target cells. Key NK activating receptors include NKG2D and three members of the innate cytotoxic receptor group (NCRs): NKp46, NKp44, and NKp30.

[0138] Among NCRs, NKp46 (NCR1) stands out for its evolutionary conservation between mice and humans, and its activity has been studied in multiple mouse models. NKp46 is uniquely expressed in all NK cell subsets and has been proposed as a candidate target for NK cell elimination and as a general-purpose NK cell marker. NKp46 is a 46kDa type I transmembrane glycoprotein characterized by a 30-amino acid intracellular tail, a 20-amino acid transmembrane domain, and two Ig-like extracellular domains linked by short peptides of 25 amino acids. The transmembrane domain contains Arg residues that can bind to the signaling adapter proteins FcεRI and CD3ζ. Two NKp46 extracellular (D1-D2) crystal structures have been elucidated and shown to be similar to the extracellular domains of various leukocyte immunoglobulin-like receptors (LIRs) and KIR receptors. The proximal membrane domain (D2) was identified as the ligand-binding domain of the NKp46 receptor.

[0139] To date, few pathogen-associated ligands and cell co-ligands of NKp46 have been documented. Viral hemagglutinin (HA) from influenza virus and Sendai virus, as well as HA-neuraminidase from Newcastle disease, bind to NKp46 and induce NK activation. Vimentin, an intermediate filament protein expressed on monocytes infected with Mycobacterium tuberculosis, is involved in binding to the NKp46 receptor. Heparan sulfate (HS), expressed on target cells, binds to NKp46 and other NK receptors and functions as an auxiliary molecule for NKp46 recognition of cell ligands. On the other hand, HS proteoglycans expressed on the NK cell membrane may regulate the function and fate of NKp46 and other HS-binding NK receptors.

[0140] In addition to its role in cancer cell recognition, several studies have suggested that NKp46 is also involved in other pathological conditions. NKp46 has been shown to be important in the development of type 1 diabetes by recognizing an unknown ligand on pancreatic β-cells. Furthermore, NKp46 has been demonstrated to interact with infected alveolar macrophages to contribute to the clearance of Streptococcus pneumoniae. NKp46 also mediates astrocytosis in reduced hepatic fibrosis and hepatocyte toxicity in HCV-infected cells. In a random mutant mouse model (Ncr1No▲e▼ / No▲e▼), the absence of NKp46 cell surface expression resulted in an overreaction of NK cells after MCMV infection. NKp46 also regulates graft-versus-host disease and allergic reactions.

[0141] Early studies on NK cells clearly showed that their cytolytic activity is contact-dependent, but it wasn't until 25 years later that the intercellular complex between NK cells and target cells was thoroughly elucidated and the term "NK cell immunosynapse" was coined. The formation and function of NK cell immunosynapses consist of several strictly controlled stages. We studied the role of human NKp46 in the formation and function of NK immunosynapses. It has been reported that NKp46 accumulates on the cell membrane, particularly at immunosynaptic sites, when NK cell-target cell interaction begins. At immunosynapses, NKp46 mediates cytoskeletal reorganization and cell polarization.

[0142] Further information regarding NKp46 can be found, for example, in Hadad et al., Front Immunol. 2015; 6:495; Sivori et al., J Exp Med (1997) 186:1129; Pessino et al., J Exp Med (1998) 188:953; and Westgaard et al., J Leukoc Biol (2004) 76:1200-6, the entirety of each reference is incorporated herein by reference.

[0143] Anti-NKp46 antibody and antigen-binding fragment This disclosure provides antibodies and antigen-binding fragments that specifically bind to NKp46 (e.g., human NKp46). The antibodies and antigen-binding fragments described herein are capable of binding to NKp46. In some embodiments, these antibodies activate NK cells and enhance NK cell-mediated killing of target cells (e.g., tumor cells). In some embodiments, these antibodies can initiate complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, these antibodies bind to cells expressing NKp46 (e.g., NK cells).

[0144] This disclosure provides modified antibodies, such as anti-NKp46 antibodies ch3-7 (also known as "3-7"), ch5-13 (also known as "5-13"), and ch6-1 (also known as "6-1"), including chimeric antibodies, humanized antibodies, and human antibodies.

[0145] This disclosure also provides humanized anti-NKp46 antibodies h3-7 (humanized antibody of ch3-7), h5-13 (humanized antibody of ch5-13), and h6-1 (humanized antibody of ch6-1).

[0146] CDR sequences for ch3-7 and antibodies derived from ch3-7 (e.g., humanized antibodies such as h3-7) include the heavy chain variable domain CDRs (sequences 5, 7, and 9) and the light chain variable domain CDRs (sequences 10, 11, and 12), as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the heavy chain variable domain CDR sequences are shown in sequence numbers 6, 8, and 9, and the light chain variable domain CDR sequences are shown in sequence numbers 10, 11, and 12.

[0147] CDR sequences for ch5-13 and antibodies derived from ch5-13 (e.g., humanized antibodies such as h5-13) include the heavy chain variable domain CDRs (sequences 15, 17, and 19) and the light chain variable domain CDRs (sequences 20, 21, and 22), as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in sequence numbers 16, 18, and 19, and the light chain variable domain CDRs are shown in sequence numbers 20, 21, and 22.

[0148] CDR sequences for ch6-1 and antibodies derived from ch6-1 (e.g., humanized antibodies such as h6-1) include the heavy chain variable domain CDRs (sequences 25, 27, and 29) and the light chain variable domain CDRs (sequences 30, 31, and 32), as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in sequence numbers 26, 28, and 29, and the light chain variable domain CDRs are shown in sequence numbers 30, 31, and 32.

[0149] The amino acid sequence for the heavy chain variable region of the CH3-7 antibody is shown in SEQ ID NO: 3. The amino acid sequence for the light chain variable region of the CH3-7 antibody is shown in SEQ ID NO: 4.

[0150] The amino acid sequence for the heavy chain variable region of the CH5-13 antibody is shown in SEQ ID NO: 13. The amino acid sequence for the light chain variable region of the CH5-13 antibody is shown in SEQ ID NO: 14.

[0151] The amino acid sequence for the heavy chain variable region of the CH6-1 antibody is shown in SEQ ID NO: 23. The amino acid sequence for the light chain variable region of the CH6-1 antibody is shown in SEQ ID NO: 24.

[0152] The amino acid sequence for the heavy chain variable region of the H3-7 antibody is shown in SEQ ID NO: 35. The amino acid sequence for the light chain variable region of the H3-7 antibody is shown in SEQ ID NO: 36.

[0153] The amino acid sequence for the heavy chain variable region of the h5-13 antibody is shown in SEQ ID NO: 37. The amino acid sequence for the light chain variable region of the h5-13 antibody is shown in SEQ ID NO: 38.

[0154] The amino acid sequence for the heavy chain variable region of the h6-1 antibody is shown in SEQ ID NO: 33. The amino acid sequence for the light chain variable region of the h6-1 antibody is shown in SEQ ID NO: 34.

[0155] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3, 13, 23, 33, 35, or 37. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 4, 14, 24, 34, 36, or 38. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, which together bind to NKp46.

[0156] In some embodiments, the variable region is entirely human, derived from, for example, human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and IGKJ genes).

[0157] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs. 5, 7, and 9, SEQ ID NOs. 6, 8, and 9, SEQ ID NOs. 15, 17, and 19, SEQ ID NOs. 16, 18, and 19, SEQ ID NOs. 25, 27, and 29, and SEQ ID NOs. 26, 28, and 29, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs. 10, 11, and 12, SEQ ID NOs. 20, 21, and 22, and SEQ ID NOs. 30, 31, and 32.

[0158] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR1; the CDR2 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR2; and the CDR3 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of a selected VH CDR3. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH and VH CDR1, 2, and 3, as well as the amino acid sequences of the selected VL and VL CDR1, 2, and 3, are shown in Figure 33.

[0159] In some embodiments, the antibody or antigen-binding fragment described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

[0160] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9

[0161] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 9, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 9

[0162] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19

[0163] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19

[0164] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 29, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0165] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 29, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0166] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0167] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 22 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0168] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 30 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 31 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, and SEQ ID NO: 32 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0169] Insertions, deletions, and substitutions can be located within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on a Kabat-defined scheme. In some embodiments, the CDR is determined based on a Chothia-defined scheme. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia-defined schemes. In some embodiments, the CDR is determined based on the IMGT definition. In some embodiments, the CDR is determined based on the contact definition.

[0170] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to NKp46. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising, or consisting thereof, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising, or consisting thereof, an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 3, and the selected VL sequence is SEQ ID NO: 4. In some embodiments, the selected VH sequence is SEQ ID NO: 13, and the selected VL sequence is SEQ ID NO: 14. In some embodiments, the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 24. In some embodiments, the selected VH sequence is SEQ ID NO: 33, and the selected VL sequence is SEQ ID NO: 34. In some embodiments, the selected VH sequence is SEQ ID NO: 35, and the selected VL sequence is SEQ ID NO: 36. In some embodiments, the selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38.

[0171] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to optimally align them for comparison, and non-homologous sequences may be ignored). The length of the reference sequence aligned for comparison is at least 80% of the reference sequence length, and in some embodiments, at least 90%, 95%, or 100%. Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of identity percentage between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0172] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing immunoglobulin heavy chains or immunoglobulin light chains. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to NKp46.

[0173] Anti-NKp46 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multimer, multispecific (e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric or human-rabbit chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0174] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to NKp46 retains its ability to bind to NKp46. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site. A single-stranded Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0175] This disclosure also provides an antibody or its antigen-binding fragment that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of Virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one embodiment, this disclosure also provides an antibody or its antigen-binding fragment that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0176] Method for producing anti-NKp46 antibodies Isolated fragments of human NKp46 (e.g., extracellular regions) can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of the antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected with at least one adjuvant. In some embodiments, the antigen peptide or protein can be conjugated with a drug that is immunogenic in the immunized species. Animals may be injected with the antigen peptide or protein two or more times (e.g., two, three, or four times).

[0177] Full-length polypeptides or proteins can be used, or their antigen peptide fragments can be used as immunogens. The protein antigen peptide contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the NKp46 amino acid sequence and includes an epitope of the protein such that the antibody produced against the peptide forms a specific immune complex with the protein. As previously mentioned, the full-length sequence of human NKp46 is known in the art (the sequence of amino acids 22 to 254 of human NKp46 is shown in SEQ ID NO: 1). The full-length sequence of monkey NKp46 is known in the art (the sequence of amino acids 22 to 257 of monkey NKp46 is shown in SEQ ID NO: 2). In some embodiments, His-tagged human NKp46 protein is used as an immunogen.

[0178] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., a fragment of human NKp46). The preparation may further contain an adjuvant, such as a Freund complete or incomplete adjuvant, or a similar immunostimulant.

[0179] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with the NKp46 polypeptide or its antigenic peptide (e.g., a part of NKp46, such as the extracellular region) as an immunogen. The antibody titer in the immunized target can be monitored over time using standard techniques such as enzyme-linked immunosorbent assay (ELISA) with the immobilized NKp46 polypeptide or peptide. If desired, the antibody molecule can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein G or protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells can be obtained from the target population and used to prepare monoclonal antibodies using standard techniques such as the hybridoma technique first described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, hybridoma cells that produce monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to a target polypeptide or epitope using a standard ELISA assay.

[0180] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human antibody, a humanized antibody, or a chimeric antibody, or an antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that produces the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments exhibit increased affinity for a target protein, such as NKp46. Any combination of deletions, insertions, and / or combinations can be realized in an antibody or antigen-binding fragment with increased binding affinity to the target. Antibodies or antigen-binding fragments can be modified, or novel post-translational modifications can be introduced, by changing the amino acids introduced into the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing novel glycosylation sites.

[0181] The antibodies disclosed herein may originate from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically modified to produce human antibodies.

[0182] Examples of human antibodies and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as) human germline immunoglobulin sequences. Examples of human antibodies include amino acid residues within the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0183] Humanized antibodies typically have a human framework (FR) into which a non-human CDR has been transplanted. Therefore, a humanized antibody has one or more amino acid sequences that have been introduced into humans from a non-human source. These non-human amino acid residues are often called “import” residues, and typically, these are derived from “import” variable domains. Humanization can essentially be carried out by substituting, for example, a rodent CDR or CDR sequence with the corresponding sequence in a human antibody. These methods are described, for example, in Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069(1986):522, Riechmann et al. "Reshaping human antibodies for therapy." Nature 332.6162(1988):323, and Dall'Acqua et al. "Antibody humanization by framework shuffling." Methods 36.1(2005):43-60, each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which a portion considerably smaller than the intact human V domain is substituted with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically non-human (e.g., mouse or rabbit) antibodies in which several CDR residues and several FR residues are substituted with residues derived from similar sites within the human antibody.

[0184] Furthermore, it is important to humanize antibodies while maintaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanization products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By observing these displays, it is possible to analyze the roles that residues can play in the functionalization of candidate immunoglobulin sequences, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0185] Typically, amino acid sequence variants of human antibodies, humanized antibodies, or chimeric anti-NKp46 antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0186] Identity or homology to the original sequence is typically the percentage of amino acid residues present in a candidate sequence that is identical to a sequence present in a human antibody or fragment, humanized antibody or fragment, or chimeric anti-NKp46 antibody or fragment, after aligning the sequences, introducing gaps where necessary, and achieving the maximum percentage of sequence identity, without considering conservative substitutions as part of the sequence identity.

[0187] Further modifications can be made to the anti-NKp46 antibody or antigen-binding fragment. For example, a cysteine ​​residue can be introduced into the Fc region to enable the formation of interchain disulfide bonds within this region. The homodimer antibody thus produced may have an extended half-life in vitro and / or in vivo. For example, homodimer antibodies with extended half-lives in vitro and / or in vivo can also be prepared using heterobifunctional crosslinking agents, as described in Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer Research 53.11(1993):2560-2565). Alternatively, antibodies with a double Fc region can be recombinant.

[0188] In some embodiments, covalent modifications can be added to anti-NKp46 antibodies or their antigen-binding fragments. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N or C-terminal residues.

[0189] In some embodiments, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans located at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (position 314 in the Eu numbering or Kabat numbering of the Fc region residues). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further recombined, and asparagine at position 297 can be replaced with alanine (N297A).

[0190] In some embodiments, to enhance production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombined, replacing serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is, for example, found in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0191] Tumor-associated antigens Tumor-associated antigens (TAAs) are antigenic substances produced by tumor cells. They can trigger an immune response in the host. Tumor antigens are useful tumor markers for identifying tumor cells in diagnostic tests and may be candidates for use in cancer treatment. Numerous tumor-associated antigens are known in the art (see, for example, Yu et al., Cancers (Basel). 2023 Apr;15(8):2323; and Tong et al., Mol Cancer, 2022 Nov 1;21(1):206; each reference is incorporated herein in its entirety by reference).

[0192] In some embodiments, TAA is CD79b, EpCAM, B cell maturation antigen (BCMA), c-Met, LRRC15, MUC1, OX40, STEAP1, PSMA, DLL3, EGFR, perforin, granzyme B, Fas ligand (FasL), CD1d, membrane glycolipid, globotriaosylceramide (Gb3Cer / CD77), gangliosides (GD2, GD3, and GM2), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD123, CD38, CLL1, CD105 , CD71, SSC, MAGE, MUC16, CD19, WT-l, B7H3, TEM8, CD22, LI-CAM, ROR-l, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-fetoprotein (AFP), BAFF, B lymphoma cells, C242 Antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, Fibronectin Extradomain-B, Folate Receptor 1, GD2, GD3 Ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Scattering Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, Insulin-like Growth Factor I Receptor, Integrin a5b1, Integrin anb3, MORAb-009, MS4A1, Mucin CanAg, N-Glycolylneuraminic Acid, NPC-1C, PD-1, PD-L1, PDGF-R a, PDL192, Phosphatidylserine, Prostate Cancer Cells, RANKL, RON, SCH The group is selected from 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0193] In some embodiments, a multispecific (e.g., bispecific) antibody or its antigen-binding fragment includes a first antigen-binding domain that specifically binds to a tumor-associated antigen (TAA) and a second antigen-binding domain that specifically binds to an NK cell surface antigen (e.g., NKp46). In some embodiments, the first antigen-binding domain specifically binds to CD79b. In some embodiments, the first antigen-binding domain specifically binds to BCMA. In some embodiments, the first antigen-binding domain specifically binds to EGFR. In some embodiments, the first antigen-binding domain specifically binds to EpCAM. In some embodiments, the first antigen-binding domain specifically binds to DLL3. In some embodiments, the first antigen-binding domain specifically binds to PSMA. In some embodiments, the first antigen-binding domain specifically binds to STEAP1. In some embodiments, the first antigen-binding domain specifically binds to MUC1. In some embodiments, the first antigen-binding domain specifically binds to c-Met. In some embodiments, the first antigen-binding domain specifically binds to LRRC15. In some embodiments, the first antigen-binding domain specifically binds to OX40.

[0194] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH1) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is approximately or at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of a selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR2, the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH1 CDR3, and The first antigen-binding domain comprises a variable light chain region (VL1) containing CDR1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence that is about or at least 80%, 85%, 90%, 95%, or 100% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence.

[0195] In some embodiments, the first antigen-binding domain comprises VH1 comprising an amino acid sequence that is about or at least 80%, 85%, 90%, 95% or 100% identical to the selected heavy chain variable region (VH1) amino acid sequence, and the first antigen-binding domain comprises VL1 comprising an amino acid sequence that is about or at least 80%, 85%, 90%, 95% or 100% identical to the selected light chain variable region (VL1) amino acid sequence.

[0196] In some embodiments, the first antigen-binding domain specifically binds to DLL3 and the second antigen-binding domain specifically binds to NKp46.

[0197] In some embodiments, the selected CDRs of the DLL3 antigen-binding fragment are obtained from the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 amino acid sequences of lobapizumab tesirine (AbbVie Inc.), talquetamab (Amgen Inc.), HPN328 (Harpoon Therapeutics Inc.) or BI 764532 (Boehringer Ingelheim GmbH). In some embodiments, the selected VH1 or VL1 sequence of the DLL3 antigen-binding fragment is obtained from the corresponding VH or VL amino acid sequence of lobapizumab tesirine (AbbVie Inc.), talquetamab (Amgen Inc.), HPN328 (Harpoon Therapeutics Inc.), or BI764532 (Boehringer Ingelheim GmbH).

[0198] In some embodiments, the first antigen-binding domain specifically binds to PSMA and the second antigen-binding domain specifically binds to NKp46.

[0199] In some embodiments, the selected CDRs of the PSMA antigen-binding fragment are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of HPN-424 (Harpoon Therapeutics Inc.), LAVA-1207 (LAVA Therapeutics NV), REGN-5678 (Regeneron Pharmaceuticals Inc.), REGN4336 (Regeneron Pharmaceuticals Inc.), acapatamab (Amgen Inc.), or AMG-340 (Amgen Inc.). In some embodiments, the selected VH1 or VL1 sequences of the PSMA antigen-binding fragment are obtained from the corresponding VH or VL amino acid sequences of HPN-424 (Harpoon Therapeutics Inc.), LAVA-1207 (LAVA Therapeutics NV), REGN-5678 (Regeneron Pharmaceuticals Inc.), REGN4336 (Regeneron Pharmaceuticals Inc.), acapatamab (Amgen Inc.), or AMG-340 (Amgen Inc.).

[0200] In some embodiments, the first antigen-binding domain specifically binds to STEAP1 and the second antigen-binding domain specifically binds to NKp46.

[0201] In some embodiments, selected CDRs for the STEAP1 antigen-binding fragment are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of zarritamig (Amgen Inc.), ABBV-969 (AbbVie Inc.), or bundletuzumab vedotin (Genentech Inc.). In some embodiments, selected VH1 or VL1 sequences for the STEAP1 antigen-binding fragment are obtained from the corresponding VH or VL amino acid sequences of zarritamig (Amgen Inc.), ABBV-969 (AbbVie Inc.), or bundletuzumab vedotin (Genentech Inc.).

[0202] In some embodiments, the first antigen-binding domain specifically binds to MUC1, and the second antigen-binding domain specifically binds to NKp46.

[0203] In some embodiments, selected CDRs of the MUC1 antigen-binding fragment are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 from MAB-AR20.5 (Canariabio Inc.) or DS-3939 (Glycotope GmbH). In some embodiments, selected VH1 or VL1 sequences of the MUC1 antigen-binding fragment are obtained from the corresponding VH or VL amino acid sequences from MAB-AR20.5 (Canariabio Inc.) or DS-3939 (Glycotope GmbH).

[0204] In some embodiments, the first antigen-binding domain specifically binds to c-Met, and the second antigen-binding domain specifically binds to NKp46.

[0205] In some embodiments, selected CDRs of c-Met antigen-binding fragments are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amivantamab-VMJM (Janssen Biotech Inc.), terisotuzumab vedotin (AbbVie Inc.), ABBV-400 (AbbVie Inc.), bafisontamab (EpimAb Biotherapeutics Inc.), CKD-702 (Chong Kun Dang Pharmaceutical Corp.), emibetuzumab (Eli Lilly & Co.), RC-108 (RemeGen Co., Ltd.), MCLA-129 (Merus NV), REGN-5093 (Regeneron Pharmaceuticals Inc.), or REGN5093-M114 (Regeneron Pharmaceuticals Inc.). In some embodiments, selected VH1 or VL1 sequences of c-Met antigen-binding fragments are obtained from the corresponding VH and VL amino acid sequences of amivantamab-VMJM (Janssen Biotech Inc.), terisotuzumab vedotin (AbbVie Inc.), ABBV-400 (AbbVie Inc.), bafisontamab (EpimAb Biotherapeutics Inc.), CKD-702 (Chong Kun Dang Pharmaceutical Corp.), emibetuzumab (Eli Lilly & Co.), RC-108 (RemeGen Co., Ltd.), MCLA-129 (Merus NV), REGN-5093 (Regeneron Pharmaceuticals Inc.), or REGN5093-M114 (Regeneron Pharmaceuticals Inc.).

[0206] In some embodiments, the first antigen-binding domain specifically binds to LRRC15, and the second antigen-binding domain specifically binds to NKp46.

[0207] In some embodiments, selected CDRs for the LRRC15 antigen-binding fragment are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of QL-315 (Qilu Pharmaceutical Co., Ltd.), RAD-502 (Radiopharm Theranostics Ltd.), or samrotamab (AbbVie Inc.). In some embodiments, selected VH1 or VL1 sequences for the LRRC5 antigen-binding fragment are obtained from the corresponding VH or VL amino acid sequences of QL-315 (Qilu Pharmaceutical Co., Ltd.), RAD-502 (Radiopharm Theranostics Ltd.), or samrotamab (AbbVie Inc.).

[0208] In some embodiments, the first antigen-binding domain specifically binds to CCR8, and the second antigen-binding domain specifically binds to NKp46.

[0209] In some embodiments, selected CDRs of the CCR8 antigen-binding fragment are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of BMS-986340 (Bristol Myers Squibb Co.), LM-108 (LaNova Medicines Ltd.), S-531011 (Shionogi & Co., Ltd.), AMG-355 (Amgen Inc.), BAY3375968 (Bayer AG), BGB-A3055 (BeiGene Ltd.), CM369 (Keymed Biomedical Technology (Chengdu) Co., Ltd.), HBM-1022 (Harbour BioMed (Shanghai) Co., Ltd.), PSB-114 (Qilu Puget Sound Biotherapeutics Corp.), or SRF-114 (Surface Oncology Inc.). In some embodiments, selected VH1 or VL1 sequences of CCR8 antigen-binding fragments are obtained from the corresponding VH or VL amino acid sequences of BMS-986340 (Bristol Myers Squibb Co.), LM-108 (LaNova Medicines Ltd.), S-531011 (Shionogi & Co., Ltd.), AMG-355 (Amgen Inc.), BAY3375968 (Bayer AG), BGB-A3055 (BeiGene Ltd.), CM369 (Keymed Biomedical Technology (Chengdu) Co., Ltd.), HBM-1022 (Harbour BioMed (Shanghai) Co., Ltd.), PSB-114 (Qilu Puget Sound Biotherapeutics Corp.), or SRF-114 (Surface Oncology Inc.).

[0210] In some embodiments, the first antigen-binding domain specifically binds to OX40, and the second antigen-binding domain specifically binds to NKp46.

[0211] In some embodiments, selected CDRs of OX40 antigen-binding fragments are obtained from the amino acid sequences of the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amritelimab (Kymab Ltd.), locatinlimab (Kyowa Kirin Co., Ltd.), INBRX-106 (Inhibrx Inc.), BGB-A445 (BeiGene Ltd.), INCAGN-1949, ibuxolimab (Pfizer Inc.), MEDI-6469 (Providence Cancer Center), BAT-6026 (Bio-Thera Solutions, Ltd.), IMG-007 (HUTCHMED (China) Ltd.), ABBV-368 (AbbVie Inc.), or BMS-986178 (Bristol Myers Squibb Co.). In some embodiments, selected VH1 or VL1 sequences of OX40 antigen-binding fragments are obtained from the corresponding VH or VL amino acid sequences of amritelimab (Kymab Ltd.), locatinlimab (Kyowa Kirin Co., Ltd.), INBRX-106 (Inhibrx Inc.), BGB-A445 (BeiGene Ltd.), INCAGN-1949, ibuxolimab (Pfizer Inc.), MEDI-6469 (Providence Cancer Center), BAT-6026 (Bio-Thera Solutions, Ltd.), IMG-007 (HUTCHMED (China) Ltd.), ABBV-368 (AbbVie Inc.), or BMS-986178 (Bristol Myers Squibb Co.).

[0212] The sequences of commercially available antibodies are known in the relevant field.

[0213] NK cell surface antigen "NK cell surface antigen" refers to molecules on NK cells (e.g., surface receptors) that are involved in regulating and / or inducing NK cell function.

[0214] Natural killer (NK) cells play a crucial role in innate immune defense against viruses, as well as in regulating tumor growth and metastasis. The regulation / induction of NK cell function is mediated by a range of activating or inhibitory surface receptors. In humans, the major activating receptors involved in target cell killing are innate cytotoxic receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or denominated during cellular stress, viral infection, or tumor transformation. HLA class I-specific inhibitory receptors, namely KIRs that recognize HLA class I allotypic determinants and CD94 / NKG2A that recognize class Ib HLA-E, constitute a fail-safe mechanism to avoid undesirable NK cell-mediated killing of healthy cells. Other receptors, such as PD-1, are primarily expressed by activated T lymphocytes and are important inhibitory checkpoints in the immune response that ensure T cell tolerance. PD-1 can also be expressed in NK cells of cancer patients. Because PD-1 ligand (PD-L1) can be expressed by various tumors, the PD-1 / PD-L1 interaction inactivates both T cells and NK cells. Therefore, reliable assessment of PD-L1 expression in tumors is a crucial challenge in selecting patients who may benefit from mAb therapy that inhibits the PD-1 / PD-L1 interaction. Recently, NKG2A has been identified as a key checkpoint regulating the activation of both NK cells and T cells.

[0215] Human NK cells express multiple receptors that induce their function through interaction with specific ligands present on the surface of transformed cells, virus-infected cells, or stressed cells. The NCR is one of the major activated NK receptors and consists of three components: NKp46 (also known as NCR1 / CD335), NKp44 (also known as NCR2 / CD33623), and NKp30 (also known as NCR3 / CD337). These molecules have traditionally been described as germ cell-encoded receptors and are important for inducing the cytotoxic function of NK cells against tumor and infected cells. NKp46 and NKp30 are expressed in almost all resting human NK cells, are upregulated in activated NK cells, and downregulated in "adaptive" NK cells found in CMV+ individuals. Unlike NKp46 and NKp30, NKp44 is constitutively expressed only in CD56-bright NK cells, but becomes acquired in virtually all NK cells after cytokine-induced activation. NCRs are type I transmembrane molecules belonging to the immunoglobulin-like family. These receptors were named according to their molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (NKp46, NKp30, and NKp44). Their transmembrane domains contain positively charged amino acids, enabling binding to the transmembrane regions of adapter proteins such as TCR-ζ and / or FcεRI-γ (for NKp30 and NKp46) or KARAP / DAP1266 (for NKp44). Physical binding of NKp44 to KARAP / DAP12 is essential for NKp44 surface expression, and reduced surface density of NKp30 and NKp46 on "adaptive" NK cells is associated with the lack of FcεRI-γ expression in these cells. Another important activated NK receptor is NKG2D, a type II transmembrane and type C lectin-like receptor, which can be expressed on cytotoxic T cells. NKG2D ligands are represented by ULBP and MICA / B,83, which are HLA-cl I structural homologs upregulated in infected cells, stress cells, and tumor cells. In particular, the release of NKG2D ligands by tumor cells may represent a mechanism of immune evasion in tumors.

[0216] Human NK cells express two types of inhibitory receptors that specifically recognize HLA class I molecules of the KIR / CD158 family members and the CD94 / NKG2A (CD94 / CD159a) heterodimer. KIR is a type I transmembrane receptor specific for polymorphic HLA-A, B, C molecules, while NKG2A is a type II transmembrane receptor that recognizes HLA-E, a non-classical HLA molecule characterized by limited polymorphism. To transmit inhibitory signals, both types of these inhibitory receptors contain an ITIM motif in the cytoplasmic tail. Furthermore, the activating form of KIR has also been identified. Unlike inhibitory KIR, activating KIR does not have an ITIM motif in the cytoplasmic tail, but instead has a transmembrane domain containing charged amino acid residues, which mediates the binding to KARAP / DAP12, a molecule having an ITAM. The role of activating KIR in the immune response has not yet been fully elucidated. Only a very small part of those that have demonstrated specificity for HLA-cl I molecules are limited.

[0217] For a detailed overview of NK cell surface antigens, see, for example, Sivori et al., Cellular & Molecular Immunology, volume 16, pages 430-441 (2019), the entire content of which is incorporated herein by reference.

[0218] In some embodiments, the NK cell surface antigen is an NK cell activating receptor.

[0219] In some embodiments, the NK cell surface antigen is selected from NKp46, NKp30, CD16, NKG2D and NKG2A.

[0220] Multispecific antibody or antigen-binding fragment thereof In one embodiment, the Specified Reference Indicators provide an antibody or an antigen-binding fragment thereof comprising: i) a first antigen-binding domain that specifically binds to a first antigen which is a tumor-associated antigen (TAA); and ii) a second antigen-binding domain that specifically binds to a second antigen which is an NK cell surface antigen. In some embodiments, the antibody comprises a fragment crystallizable region (Fc region). In some embodiments, the antibody is a bispecific antibody.

[0221] In some embodiments, a bispecific antibody or its antigen-binding fragment (e.g., anti-CD79b / NKp46 antibody) specifically binds to a tumor-associated antigen (e.g., CD79b) and an NK cell surface antigen (e.g., NKp46), and such a bispecific antibody has an optimized Fc region.

[0222] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0223] In some embodiments, the first antigen-binding domain is a human or humanized antigen-binding domain, and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0224] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0225] In some embodiments, the multispecific antibodies described herein are designed to include one of the following (all numbering follows EU numbering): i) Alanine (A) at position 236, leucine (L) at position 330, and glutamic acid I at position 332; ii) Alanine (A) at position 236, aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332; iii) Alanine (A), ranked 236th; iv) Alanine at position 236 (A), aspartic acid at position 293 (D), and glutamic acid at position 332 (E); v) Aspartic acid (D) at position 293 and glutamic acid (E) at position 332; and vi) Aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332.

[0226] In some embodiments, the antibody or its antigen-binding fragment comprises a fragment crystallizable region (Fc region). In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0227] In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure containing the GASDALIE mutation (EU numbering G236A, S293D, A330L, and I332E mutations). In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 236, aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332, according to EU numbering.

[0228] In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure with the G236A mutation in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region with alanine (A) at position 236 in EU numbering.

[0229] In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure having the G236A, S293D, and I332E mutations in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having alanine (A) at position 236, aspartic acid (D) at position 293, and glutamic acid (E) at position 332 in EU numbering.

[0230] In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure with S293D and I332E mutations in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region with aspartic acid (D) at position 293 and glutamic acid (E) at position 332 in EU numbering.

[0231] In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 subtype structure having the S293D, A330L, and I332E mutations in EU numbering. In some embodiments, the multispecific antibodies described herein are designed to have an IgG1 Fc region having aspartic acid (A) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering.

[0232] In some embodiments, the multispecific antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the multispecific antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or a control multispecific antibody. In some embodiments, the antibody or its antigen-binding fragment has increased binding affinity to the FcγRIIa receptor and / or FcγRIIIa receptor.

[0233] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0234] In some embodiments, a second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. In some embodiments, VH1 is linked to the CH1 domain and VL1 is linked to the CL domain. A schematic diagram of this configuration is shown in Figures 4A and 4B.

[0235] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the Fc region. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. A schematic diagram of this configuration is shown in Figure 4B. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region via a third linker.

[0236] In some embodiments, the multispecific antibody has a heavy chain sequence containing the wild-type IgG1 Fc region (SEQ ID NO: 41). In some embodiments, the multispecific antibody has a heavy chain sequence containing an Fc region that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41.

[0237] In some embodiments, the multispecific antibody has a heavy chain sequence containing an optimized mutation in the IgG1 Fc region (SEQ ID NO: 43). In some embodiments, the multispecific antibody has a heavy chain sequence containing an Fc region that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43.

[0238] In some embodiments, the Fc region of the multispecific antibody is not fucosylated.

[0239] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 87.

[0240] Anti-CD79b / NKp46 antibody and its antigen-binding fragment CD79b is a cell surface antigen expressed on mature B cells but absent on plasma cells. Immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form a signaling component of the B cell antigen receptor complex (BCR). Given the crucial role of Ig beta in BCR signaling, its variable expression in CLL suggests it may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong surface Ig expression, advanced clinical stage, and shortened overall survival in B-CLL. Furthermore, high CD38 expression correlates with high CD40, CD69, and CD79b expression, which is consistent with the activated phenotype. Additionally, the ZAP-70+ CLL subgroup has been shown to exhibit rapid disease progression and low overall survival. Since BCR involvement has been reported to significantly influence B-CLL cell survival, activation, and cell cycle progression, CD79b is an important prognostic parameter and can be used to identify and stratify patients with progressive B-CLL at an early stage.

[0241] Most mature B-cell-derived malignancies, including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, express CD79b and exhibit rapid internalization of anti-CD79b antibodies. Studies on polatuzumab vedotin, an antibody-drug conjugate (ADC) that targets CD79b to deliver the microtubule inhibitor monomethyl auristatin E (MMAE), have clinically validated CD79b as a useful target for delivering cytotoxic agents to malignant cells.

[0242] For a detailed overview of CD79b and its function, see Del Poeta et al., Blood (2008) 112(11):1054; Herrera et al., Clin Cancer Res. 2022 Apr1;28(7):1294-1301; and Huang et al., Pathol Int 2011;61:122-9, the entire contents of which are incorporated herein by reference.

[0243] Natural killer (NK) cells are granular lymphocytes that were initially recognized for their ability to efficiently eliminate tumor cells without prior sensitization. Viral infections can also induce NK cell cytotoxicity. In fact, NK cells are a crucial component of the innate immune response to viruses, as evidenced by the high incidence of infections in patients with primary NK cell deficiency and in mice lacking NK cells. In healthy adults, NK cells account for 5–15% of peripheral blood lymphocytes. The majority of NK cells (about 90%) are CD56dim CD16bright and can induce potent cytolytic reactions, while 10% are CD56bright-CD16null / dim and possess rapid cytokine secretion capabilities. The regulation of NK cell function is primarily regulated by the recognition of auto-MHC class I molecules via a series of inhibitory receptors (human killer cell immunoglobulin-like receptors (KIRs) and the mouse Ly49 receptor family). NK cells do not express variable antigen-specific receptors. Instead, their activation is controlled by a repertoire of germline-encoded activating receptors that recognize various ligands present on the surface of sensitive target cells. Key NK activating receptors include NKG2D and three members of the innate cytotoxic receptor group (NCR): NKp46, NKp44, and NKp30.

[0244] Among NCRs, NKp46 (NCR1) stands out for its evolutionary conservation between mice and humans, and its activity has been studied in multiple mouse models. NKp46 is uniquely expressed in all NK cell subsets and has been proposed as a candidate target for NK cell elimination and as a general-purpose NK cell marker. NKp46 is a 46kDa type I transmembrane glycoprotein characterized by a 30-amino acid intracellular tail, a 20-amino acid transmembrane domain, and two Ig-like extracellular domains linked by short peptides of 25 amino acids. The transmembrane domain contains Arg residues that can bind to the signaling adapter proteins FcεRI and CD3ζ. Two NKp46 extracellular (D1-D2) crystal structures have been elucidated and shown to be similar to the extracellular domains of various leukocyte immunoglobulin-like receptors (LIRs) and KIR receptors. The proximal membrane domain (D2) was identified as the ligand-binding domain of the NKp46 receptor.

[0245] In addition to its role in cancer cell recognition, several studies have suggested that NKp46 is also involved in other pathological conditions. NKp46 has been shown to be important in the development of type 1 diabetes by recognizing an unknown ligand on pancreatic β-cells. Furthermore, NKp46 has been demonstrated to interact with infected alveolar macrophages to contribute to the clearance of Streptococcus pneumoniae. NKp46 also mediates astrocytosis in reduced hepatic fibrosis and hepatocyte toxicity in HCV-infected cells. In a random mutant mouse model (Ncr1No▲e▼ / No▲e▼), the absence of NKp46 cell surface expression resulted in an overreaction of NK cells after MCMV infection. NKp46 also regulates graft-versus-host disease and allergic reactions.

[0246] Further information regarding NKp46 can be found, for example, in Hadad et al., Front Immunol. 2015; 6:495; Sivori et al., J Exp Med (1997) 186:1129; Pessino et al., J Exp Med (1998) 188:953; and Westgaard et al., J Leukoc Biol (2004) 76:1200-6, the entirety of each reference is incorporated herein by reference.

[0247] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibodies described herein have agonist activity for NK cell activation. In some embodiments, NK cell activation upon contact with the anti-CD79b / NKp46 antibodies described herein is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to NK cell activation without contact with such antibodies. In some embodiments, NK cell activation is measured by the expression of an activation marker such as CD56. In some embodiments, the anti-CD79b / NKp46 antibodies described herein bind to NK cells having mutations optimized for the Fc region (SEQ ID NO: 43).

[0248] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody described herein binds to NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-CD79b / NKp46 antibody described herein binds to target cells (e.g., Ramos cells) having a silent mutation (e.g., LALA-PG mutation) in the Fc region.

[0249] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody described herein activates NK cells (e.g., through the involvement of NKp46) and mediates the killing of target cells (e.g., B cells or B cell lines such as Ramos cells or Daudi cells). In some embodiments, the anti-CD79b / NKp46 antibody described herein activates NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-CD79b / NKp46 antibody described herein contains the wild-type human IgG1 Fc region. In some embodiments, the anti-CD79b / NKp46 antibody described herein binds to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43).

[0250] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibodies described herein activate NK cells in a population of peripheral blood mononuclear cells (PBMCs) (e.g., through the involvement of NKp46). In some embodiments, PBMCs mediate the killing of target cells (e.g., B cells). In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the PBMCs are NK cells.

[0251] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody described herein promotes monocyte-mediated killing of target cells (e.g., B cells or B cell lines such as Ramos cells). In some embodiments, the anti-CD79b / NKp46 antibody described herein contains the wild-type human IgG1 Fc region. In some embodiments, the anti-CD79b / NKp46 antibody described herein binds to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43). In some embodiments, the target cells are CD79+ cancer cells. In some embodiments, the target cells are Ramos cells. In some embodiments, the killing of target cells is carried out by either phagocytic and / or trogocytotic cancer cell cytotoxicity mechanisms. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the target cells are killed.

[0252] In some embodiments, there is no significant change in cell count (e.g., NK cell count or monocyte cell count) after killing the target cells.

[0253] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to CD79b / NKp46 (e.g., human CD79b / NKp46). In one embodiment, this disclosure provides an anti-CD79b / NKp46 multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to CD79b and a second antigen-binding domain that specifically binds to NKp46.

[0254] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0255] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 above and the selected VL1 CDR1, 2, and 3 above are the corresponding VH CDR1, 2, and 3 and VL CDR1, 2, and 3 of polatuzumab, PRV-3279, and SHR-A1912.

[0256] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 5, 7, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 6, 8, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 15, 17, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 18, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; and (6) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0257] In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 5, 7, and 9, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 6, 8, and 9, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 15, 17, and 19, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 16, 18, and 19, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; In some embodiments, the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0258] In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 45, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 46, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 3, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 4.

[0259] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 45, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 46, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 13, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 14.

[0260] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 45, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 46, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 23, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 24.

[0261] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 47, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 48, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 33, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 34.

[0262] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 47, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 48, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 35, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 36.

[0263] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 47, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 48, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 37, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 38.

[0264] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, the selected VH sequence being shown in SEQ ID NO: 45, and the selected VL sequence being shown in SEQ ID NO: 46. In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, the selected VH sequence and the selected VL sequence being one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; and (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24.

[0265] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34.

[0266] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; and (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24.

[0267] In some embodiments, VH1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 48.

[0268] In some embodiments, VH2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VH sequence, and VL2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (2) The selected VH sequence is sequence number 35, and the selected VL sequence is sequence number 36; and (3) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38.

[0269] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 47, and the selected VL sequence is sequence number 48.

[0270] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, and the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (2) The selected VH sequence is sequence number 35, and the selected VL sequence is sequence number 36; and (3) The selected VH sequence is sequence number 37, and the selected VL sequence is sequence number 38.

[0271] In some embodiments, the first antigen-binding domain specifically binds to human, mouse, monkey, or dog CD79b, and / or the second antigen-binding domain specifically binds to human, mouse, monkey, or dog NKp46.

[0272] In some embodiments, the first antigen-binding domain is a human or humanized antigen-binding domain, and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0273] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0274] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region). In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody described herein can be designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-CD79b / NKp46 antibody described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0275] In some embodiments, the multispecific (bispecific) anti-CD79b / NKp46 antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-CD79b / NKp46 antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0276] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0277] In some embodiments, a second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. Schematic diagrams of this configuration are shown in Figures 4A and 4B.

[0278] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains the ch44G2 heavy chain sequence. In some embodiments, the ch44G2 heavy chain contains an IgG1 Fc region (SEQ ID NO: 42) containing the LALA-PG mutation. In some embodiments, the ch44G2 heavy chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 57, 59, 61, or 63.

[0279] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch3-7 Fc silencing light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42.

[0280] In some embodiments, the heavy chain (e.g., ch44G2 / ch3-7 Fc silencing heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 59, and the light chain (e.g., ch44G2 / ch3-7 The Fc silencing light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60.

[0281] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch5-13 Fc silencing light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62.

[0282] In some embodiments, the heavy chain (e.g., ch44G2 / ch5-13 Fc silencing heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 61, and the light chain (e.g., ch44G2 / ch5-13 The Fc silent light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62.

[0283] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch6-1 Fc silencing light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64.

[0284] In some embodiments, the heavy chain (e.g., ch44G2 / ch6-1 Fc silencing heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 63, and the light chain (e.g., ch44G2 / ch6-1 The Fc silent light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64.

[0285] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains the ch44G2 heavy chain sequence. In some embodiments, the ch44G2 heavy chain contains the wild-type IgG1 Fc region (SEQ ID NO: 41). In some embodiments, the ch44G2 heavy chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 65, 67, and 69.

[0286] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch3-7 Fc-WT light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66.

[0287] In some embodiments, the heavy chain (e.g., ch44G2 / ch3-7 Fc-WT heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 65, and the light chain (e.g., ch44G2 / ch3-7) of the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately. The Fc-WT light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66.

[0288] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch5-13 Fc-WT light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68.

[0289] In some embodiments, the heavy chain (e.g., ch44G2 / ch5-13 Fc-WT heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 67, and the light chain (e.g., ch44G2 / ch5-13 The Fc-WT light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 68.

[0290] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch6-1 Fc-WT light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70.

[0291] In some embodiments, the heavy chain (e.g., ch44G2 / ch6-1 Fc-WT heavy chain) of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 69, and the light chain (e.g., ch44G2 / ch6-1) of the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains approximately. The Fc-WT light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70.

[0292] In some embodiments, the multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody contains the ch44G2 heavy chain sequence. In some embodiments, the ch44G2 heavy chain contains an IgG1 Fc region (SEQ ID NO: 43) with optimized mutations. In some embodiments, the ch44G2 heavy chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 75, 77, 79, 81, 83, or 85.

[0293] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., a ch44G2 / ch6-1(LC)Fc optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74.

[0294] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch6-1(LC)Fc optimized heavy chain) is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids to SEQ ID NO: 73. The light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody containing an acid sequence (e.g., a ch44G2 / ch6-1(LC)Fc optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 74.

[0295] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., a ch44G2 / ch6-1(HC)Fc optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76.

[0296] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., ch44G2 / ch6-1(HC)Fc optimized heavy chain) is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids to SEQ ID NO: 75. The light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody containing an acid sequence (e.g., a ch44G2 / ch6-1(HC)Fc optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 76.

[0297] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., an h44G2 / h6-1(LC)Fc optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78.

[0298] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., h44G2 / h6-1(LC)Fc optimized heavy chain) is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids to SEQ ID NO: 77. The light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody containing an acid sequence (e.g., an h44G2 / h6-1(LC)Fc optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 78.

[0299] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., an h44G2 / h6-1(HC)Fc optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80.

[0300] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody (e.g., h44G2 / h6-1(HC)Fc optimized heavy chain) is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids to SEQ ID NO: 79. The light chain of a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody containing an acid sequence (e.g., an h44G2 / h6-1(HC)Fc optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 80.

[0301] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the Fc region. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. A schematic diagram of this configuration is shown in Figure 4B. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region via a third linker.

[0302] In some embodiments, the anti-CD79b / NKp46 antibody contains the ch44G2 light chain sequence. In some embodiments, the ch44G2 light chain contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.

[0303] The linkers described herein may be any suitable linkers known in the art. In some embodiments, the linker contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 87.

[0304] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0305] This disclosure provides, for example, anti-CD79b / NKp46 antibodies ch44G2 / ch3-7 (also referred to as "44G2 / 3-7" or "44G2 / ch3-7"), ch44G2 / ch5-13 (also referred to as "44G2 / 5-13" or "44G2 / ch5-13"), and ch44G2 / ch6-1 (also referred to as "44G2 / 6-1" or "44G2 / ch6-1"), as well as modified antibodies thereof, including, for example, chimeric antibodies, humanized antibodies, and human antibodies.

[0306] In some embodiments, a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody, or an antibody fragment thereof, includes a combination of an anti-CD79b antigen-binding domain and an anti-NKp46 antigen-binding domain as shown in Figure 33.

[0307] In some embodiments, ch44G2 / ch3-7 refers to a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody comprising a first anti-CD79b antigen-binding domain derived from ch44G2 and a second anti-NKp46 antigen-binding domain derived from ch3-7. In some embodiments, the first anti-CD79b antigen-binding domain comprises the CDR of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of ch3-7. In some embodiments, the first anti-CD79b antigen-binding domain comprises the VH and VL of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of ch3-7.

[0308] In some embodiments, ch44G2 / ch5-13 refers to a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody comprising a first anti-CD79b antigen-binding domain derived from ch44G2 and a second anti-NKp46 antigen-binding domain derived from ch5-13. In some embodiments, the first anti-CD79b antigen-binding domain comprises the CDR of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of ch5-13. In some embodiments, the first anti-CD79b antigen-binding domain comprises the VH and VL of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of ch5-13.

[0309] In some embodiments, ch44G2 / ch6-1 refers to a multispecific (e.g., bispecific) anti-CD79b / NKp46 antibody comprising a first anti-CD79b antigen-binding domain derived from ch44G2 and a second anti-NKp46 antigen-binding domain derived from ch6-1. In some embodiments, the first anti-CD79b antigen-binding domain comprises the CDR of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of ch6-1. In some embodiments, the first anti-CD79b antigen-binding domain comprises the VH and VL of ch44G2. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of ch6-1.

[0310] In some embodiments, anti-CD79b / NKp46 antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0311] Either the anti-CD79b / NKp46 antibody or its antigen-binding fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-CD79b / NKp46 antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0312] This disclosure also provides nucleic acids comprising polynucleotides encoding an anti-CD79b / NKp46 antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-CD79b / NKp46 antibody comprises a CDR as shown in Figure 33. When a polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to NKp46 and / or CD79b.

[0313] Anti-CD79b / NKp46 antibodies may be anti-CD79b / NKp46 antibody variants (including derivatives and conjugates) of anti-CD79b / NKp46 antibodies or antibody fragments. Additional anti-CD79b / NKp46 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Anti-CD79b / NKp46 antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-CD79b / NKp46 antibody or antigen-binding fragment is an IgG antibody (e.g., the IgG1 Fc region is shown in SEQ ID NO: 41) or its antigen-binding fragment.

[0314] The fragments of the anti-CD79b / NKp46 antibody are suitable for use in the provided method, provided that they retain the desired affinity and specificity for both NKp46 and CD79b. Thus, the fragments of the anti-CD79b / NKp46 antibody retain the ability to bind to both NKp46 and CD79b.

[0315] Anti-BCMA / NKp46 antibody and its antigen-binding fragment B-cell maturation antigen (BCMA) is encoded by the 2.92 kb TNFRSF17 gene located on the short arm of chromosome 16 (16p13.13), and consists of three exons separated by two introns. BCMA is a 184-amino acid, 20.2 kDa type III transmembrane glycoprotein with a conserved motif of six cysteine ​​units at its extracellular N-terminus. BCMA has been identified as a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily. Human BCMA has four native splice variants exhibiting different receptor binding affinities, membrane fixation capabilities, and intracellular domain signaling. Because BCMA is highly selectively expressed in malignant plasma cells (PCs), it has become a novel therapeutic target for multiple myeloma (MM).

[0316] A detailed overview of BCMA and its function is provided in Yu et al., J Hematol Oncol. 2020;13:125, the entire content of which is incorporated herein by reference.

[0317] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibodies described herein have agonist activity for NK cell activation. In some embodiments, NK cell activation upon contact with the anti-BCMA / NKp46 antibodies described herein is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to NK cell activation without contact with such antibodies. In some embodiments, NK cell activation is measured by the expression of an activation marker such as CD56. In some embodiments, the anti-BCMA / NKp46 antibodies described herein bind to NK cells having mutations optimized for the Fc region (SEQ ID NO: 43).

[0318] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody described herein binds to NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-BCMA / NKp46 antibody described herein binds to target cells (e.g., Ramos cells) having a silent mutation (e.g., LALA-PG mutation) in the Fc region.

[0319] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibodies described herein activate NK cells (e.g., through the involvement of NKp46) and mediate the killing of target cells (e.g., B cells or B cell lines such as Ramos cells or Daudi cells). In some embodiments, the anti-BCMA / NKp46 antibodies described herein activate NK cells having a silent mutation (e.g., an LALA-PG mutation) in the Fc region. In some embodiments, the anti-BCMA / NKp46 antibodies described herein contain the wild-type human IgG1 Fc region. In some embodiments, the anti-BCMA / NKp46 antibodies described herein bind to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43).

[0320] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibodies described herein activate NK cells in a population of peripheral blood mononuclear cells (PBMCs) (e.g., through the involvement of NKp46). In some embodiments, PBMCs mediate the killing of target cells (e.g., B cells). In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the PBMCs are NK cells.

[0321] In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody described herein promotes monocyte-mediated killing of target cells (e.g., B cells or B cell lines such as Ramos cells). In some embodiments, the anti-BCMA / NKp46 antibody described herein contains the wild-type human IgG1 Fc region. In some embodiments, the anti-BCMA / NKp46 antibody described herein binds to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43). In some embodiments, the target cells are Ramos cells. In some embodiments, target cell killing is carried out by either phagocytic and / or trogocytosis-type cancer cell killing mechanisms. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the target cells are killed.

[0322] In some embodiments, there is no significant change in cell count (e.g., NK cell count or monocyte cell count) after killing the target cells.

[0323] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to BCMA / NKp46 (e.g., human BCMA / NKp46). In one embodiment, this disclosure provides an anti-BCMA / NKp46 multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to BCMA and a second antigen-binding domain that specifically binds to NKp46.

[0324] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0325] In some embodiments, the first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and the first light chain variable region (VL1) comprises CDRs 1, 2, and 3, wherein the VL1 CDR1 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR2, and the VL1 CDR3 region comprises an amino acid sequence at least 80% identical to the amino acid sequence of the selected VL1 CDR3. The amino acid sequences of the selected VH1 CDR1, 2, and 3 and the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the above-selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 88, 89, and 90, respectively, and the amino acid sequences of the above-selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 91, 92, and 93, respectively; or (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 above and the selected VL1 CDR1, 2, and 3 above are the corresponding VH CDR1, 2, and 3 and VL CDR1, 2, and 3 of zevolcabutagen autolucel, erranatamab, tecristamab, echecabutagen autolucel, siltakabutagen autolucel, idekabutagen vicleucel, verantamab, limbocertamab, alnuctamab, TNB-383B, and SEA-BCMA.

[0326] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0327] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 88, 89, and 90, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 91, 92, and 93, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 26, 28, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively.

[0328] In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 106, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 107, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 33, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 34.

[0329] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 106, and the selected VL sequence is sequence number 107.

[0330] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34.

[0331] In some embodiments, the first antigen-binding domain specifically binds to human, mouse, monkey, or canine BCMA, and / or the second antigen-binding domain specifically binds to human, mouse, monkey, or canine NKp46.

[0332] In some embodiments, the first antigen-binding domain is a human or humanized antigen-binding domain, and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0333] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0334] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region). In some embodiments, the multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody described herein can be designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-BCMA / NKp46 antibody described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0335] In some embodiments, the multispecific (bispecific) anti-BCMA / NKp46 antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-BCMA / NKp46 antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0336] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0337] In some embodiments, a second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. Schematic diagrams of this configuration are shown in Figures 4A and 4B.

[0338] In some embodiments, a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody comprises a heavy chain sequence (SEQ ID NO: 43) containing an optimized mutation in the IgG1 Fc region.

[0339] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody (e.g., BCMA / NKp46 Fc optimized heavy chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81.

[0340] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody (e.g., BCMA / NKp46 Fc-optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82.

[0341] In some embodiments, the heavy chain (e.g., BCMA / NKp46 Fc-optimized heavy chain) of a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 81, and the light chain (e.g., BCMA / NKp46 Fc-optimized light chain) of a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 82.

[0342] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the Fc region. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. A schematic diagram of this configuration is shown in Figure 4B. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region via a third linker.

[0343] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0344] This disclosure provides modified antibodies, such as anti-BCMA / NKp46 antibodies, BCMA / NKp46 Fc-optimized antibodies, and human antibodies, including, for example, chimeric antibodies, humanized antibodies, and human antibodies.

[0345] In some embodiments, a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody, or an antibody fragment thereof, includes a combination of an anti-BCMA antigen-binding domain and an anti-NKp46 antigen-binding domain as shown in Figure 33.

[0346] In some embodiments, BCMA / NKp46 refers to a multispecific (e.g., bispecific) anti-BCMA / NKp46 antibody comprising a first anti-BCMA antigen-binding domain derived from a BCMA antibody and a second anti-NKp46 antigen-binding domain derived from an NKp46 antibody. In some embodiments, the first anti-BCMA antigen-binding domain comprises the CDR of the BCMA antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of the NKp46 antibody. In some embodiments, the first anti-BCMA antigen-binding domain comprises the VH and VL of the BCMA antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of the NKp46 antibody.

[0347] In some embodiments, anti-BCMA / NKp46 antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0348] Either the anti-BCMA / NKp46 antibody or its antigen-binding fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-BCMA / NKp46 antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0349] The present invention also provides nucleic acids comprising polynucleotides encoding an anti-BCMA / NKp46 antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-BCMA / NKp46 antibody comprises a CDR as shown in Figure 33. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to NKp46 and / or BCMA.

[0350] Anti-BCMA / NKp46 antibodies may be anti-BCMA / NKp46 antibody variants (including derivatives and conjugates) of anti-BCMA / NKp46 antibodies or antibody fragments. Additional anti-BCMA / NKp46 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Anti-BCMA / NKp46 antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-BCMA / NKp46 antibody or antigen-binding fragment is an IgG (e.g., IgG1 Fc region shown in SEQ ID NO: 41) antibody or its antigen-binding fragment.

[0351] The fragments of the anti-BCMA / NKp46 antibody are suitable for use in the provided method, provided that they retain the desired affinity and specificity for both NKp46 and BCMA. Therefore, the fragments of the anti-BCMA / NKp46 antibody retain the ability to bind to NKp46 and BCMA.

[0352] Anti-EGFR / NKp46 antibody and its antigen-binding fragment The epidermal growth factor receptor (EGFR) is positioned as a fundamental member of the large growth factor receptor family with endogenous tyrosine kinase function. Large amounts of EGFR and large internal deletions are frequently observed in brain tumors, while point mutations and small insertion mutations within the kinase domain are common in lung cancer. For these reasons, EGFR and its preferred heterodimer partner, HER2 / ERBB2, have become widely used as targets for anti-cancer therapy. Nevertheless, research on EGFR has revealed a series of unexpected observations, which are discussed here. When activated by its ligand, EGFR controls genes that collectively determine phenotype by initiating a chain of time-dependent molecular switches, including the downregulation of large microRNA cohorts, the upregulation of newly synthesized mRNA, and covalent modification of proteins. In addition to microRNAs, long non-coding RNAs and circular RNAs also play important roles in EGFR signaling. EGFR, along with driver mutations, promotes metastasis in various ways. The paracrine loop, which includes tumor cells and stromal cells, enables EGFR to invade tissue barriers, survive circulating tumor cell masses, and even colonize distant organs.

[0353] Epidermal growth factor receptor (EGFR) is highly expressed in various solid tumors and is a rational target in cancer therapy because it is involved in regulating cell survival, proliferation, metastasis, and angiogenesis. However, although there is evidence that EGFR expression is associated with poor prognosis in some tumors (such as breast cancer and head and neck cancer), the association is not always clear.

[0354] A detailed overview of EGFR and its function is provided in Uribe et al, Cancers (Basel), 2021 Jun 1;13(11):2748, and Ciardiello et al., Eur J Cancer, 2003 Jul;39(10):1348-54, the entire contents of which are incorporated herein by reference.

[0355] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibodies described herein have agonist activity for NK cell activation. In some embodiments, NK cell activation upon contact with the anti-EGFR / NKp46 antibodies described herein is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to NK cell activation without contact with such antibodies. In some embodiments, NK cell activation is measured by the expression of an activation marker such as CD56. In some embodiments, the anti-EGFR / NKp46 antibodies described herein bind to NK cells having mutations optimized for the Fc region (SEQ ID NO: 43).

[0356] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody described herein binds to NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-EGFR / NKp46 antibody described herein binds to target cells (e.g., Ramos cells) having a silent mutation (e.g., LALA-PG mutation) in the Fc region.

[0357] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibodies described herein activate NK cells (e.g., through the involvement of NKp46) and mediate the killing of target cells (e.g., B cells or B cell lines such as Ramos cells or Daudi cells). In some embodiments, the anti-EGFR / NKp46 antibodies described herein activate NK cells having a silent mutation (e.g., an LALA-PG mutation) in the Fc region. In some embodiments, the anti-EGFR / NKp46 antibodies described herein contain the wild-type human IgG1 Fc region. In some embodiments, the anti-EGFR / NKp46 antibodies described herein bind to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43).

[0358] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibodies described herein activate NK cells in a population of peripheral blood mononuclear cells (PBMCs) (e.g., through the involvement of NKp46). In some embodiments, PBMCs mediate the killing of target cells (e.g., B cells). In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the PBMCs are NK cells.

[0359] In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody described herein promotes monocyte-mediated killing of target cells (e.g., B cells or B cell lines such as Ramos cells). In some embodiments, the anti-EGFR / NKp46 antibody described herein contains the wild-type human IgG1 Fc region. In some embodiments, the anti-EGFR / NKp46 antibody described herein binds to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43). In some embodiments, the target cells are Ramos cells. In some embodiments, the killing of target cells is carried out by either phagocytic and / or trogocytotic cancer cell cytotoxicity mechanisms. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the target cells are killed.

[0360] In some embodiments, there is no significant change in cell count (e.g., NK cell count or monocyte cell count) after killing the target cells.

[0361] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to EGFR / NKp46 (e.g., human EGFR / NKp46). In one embodiment, this disclosure provides an anti-EGFR / NKp46 multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to NKp46.

[0362] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0363] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, where the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and the first light chain variable region (VL1) includes CDRs 1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3, and the selected VH1 The amino acid sequences of CDR1, 2, and 3, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 94, 95, and 96, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 97, 98, and 99, respectively; or (2) The amino acid sequences of the selected VH1 CDR1, 2, 3 and the selected VL1 CDR1, 2, 3 are the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amibantamab, cetuximab, necitumumab, panitumumab, nimotuzumab, BL-B01D1, dempitamab, depatuxizumab, futuximab, modotuximab, JMT-101, MRG-003, pimrutamab, QL1203, SI-B001, or ametumumab.

[0364] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0365] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 94, 95, and 96, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 97, 98, and 99, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; In some embodiments, the first heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 108, the first light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 109, the second heavy chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 33, and the second light chain variable region contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 34.

[0366] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 108, and the selected VL sequence is sequence number 109.

[0367] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34.

[0368] In some embodiments, the first antigen-binding domain specifically binds to human, mouse, monkey, or dog EGFR, and / or the second antigen-binding domain specifically binds to human, mouse, monkey, or dog NKp46.

[0369] In some embodiments, the first antigen-binding domain is a human or humanized antigen-binding domain, and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0370] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0371] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region). In some embodiments, the multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody described herein can be designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-EGFR / NKp46 antibody described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0372] In some embodiments, the multispecific (bispecific) anti-EGFR / NKp46 antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-EGFR / NKp46 antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0373] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0374] In some embodiments, a second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. Schematic diagrams of this configuration are shown in Figures 4A and 4B.

[0375] In some embodiments, a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody comprises a heavy chain sequence (SEQ ID NO: 43) containing an optimized mutation in the IgG1 Fc region.

[0376] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody (e.g., an EGFR / NKp46 Fc optimized heavy chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 83.

[0377] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody (e.g., an EGFR / NKp46 Fc optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 84.

[0378] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody (e.g., EGFR / NKp46 Fc-optimized heavy chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 83, and the light chain of a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody (e.g., EGFR / NKp46 Fc-optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 84.

[0379] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the Fc region. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. A schematic diagram of this configuration is shown in Figure 4B. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region via a third linker.

[0380] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0381] This disclosure provides modified antibodies, such as anti-EGFR / NKp46 antibodies, EGFR / NKp46 Fc-optimized antibodies, and human antibodies, including, for example, chimeric antibodies, humanized antibodies, and human antibodies.

[0382] In some embodiments, a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody, or an antibody fragment thereof, includes a combination of an anti-EGFR antigen-binding domain and an anti-NKp46 antigen-binding domain as shown in Figure 33.

[0383] In some embodiments, EGFR / NKp46 refers to a multispecific (e.g., bispecific) anti-EGFR / NKp46 antibody comprising a first anti-EGFR antigen-binding domain derived from an EGFR antibody and a second anti-NKp46 antigen-binding domain derived from an NKp46 antibody. In some embodiments, the first anti-EGFR antigen-binding domain comprises the CDR of the EGFR antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of the NKp46 antibody. In some embodiments, the first anti-EGFR antigen-binding domain comprises the VH and VL of the EGFR antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of the NKp46 antibody.

[0384] In some embodiments, anti-EGFR / NKp46 antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0385] Either the anti-EGFR / NKp46 antibody or its antigen-binding fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-EGFR / NKp46 antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0386] This disclosure also provides nucleic acids comprising polynucleotides encoding an anti-EGFR / NKp46 antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-EGFR / NKp46 antibody comprises a CDR as shown in Figure 33. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to NKp46 and / or EGFR.

[0387] Anti-EGFR / NKp46 antibodies may be anti-EGFR / NKp46 antibody variants (including derivatives and conjugates) of anti-EGFR / NKp46 antibodies or antibody fragments. Additional anti-EGFR / NKp46 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Anti-EGFR / NKp46 antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-EGFR / NKp46 antibody or antigen-binding fragment is an IgG (e.g., IgG1 Fc region shown in SEQ ID NO: 41) antibody or its antigen-binding fragment.

[0388] The fragments of the anti-EGFR / NKp46 antibody are suitable for use in the provided method, provided that they retain the desired affinity and specificity for both NKp46 and EGFR. Therefore, the fragments of the anti-EGFR / NKp46 antibody retain the ability to bind to NKp46 and EGFR.

[0389] Anti-EPCAM / NKp46 antibody and its antigen-binding fragment EpCAM is a homophilic type I transmembrane glycoprotein belonging to the small GA733 protein family. Previous studies have shown that EpCAM is involved in cell adhesion. However, other reports have revealed that EpCAM functions as a negative regulator of classical cadherin-mediated adhesion. These conflicting results suggest a complex function of EpCAM in cancer. Apart from its function in mediating cell adhesion, the extracellular domain (EpEX) and intracellular domain (EpICD) of EpCAM are released when membrane-bound EpCAM is cleaved via TNF-α-converting enzyme (TACE) and presenilin 2 (PS-2), and function as ligands or transcriptional co-factors for signaling receptors. These molecular mechanisms further restrict EpCAM's function and complicate its role in cancer progression.

[0390] A detailed overview of EPCAM and its functions is provided in Liu et al., Exp Hematol Oncol. 2022;11:97, the entire content of which is incorporated herein by reference.

[0391] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibodies described herein have agonist activity for NK cell activation. In some embodiments, NK cell activation upon contact with the anti-EpCAM / NKp46 antibodies described herein is increased by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to NK cell activation without contact with such antibodies. In some embodiments, NK cell activation is measured by the expression of an activation marker such as CD56. In some embodiments, the anti-EpCAM / NKp46 antibodies described herein bind to NK cells having mutations optimized for the Fc region (SEQ ID NO: 43).

[0392] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody described herein binds to NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-EpCAM / NKp46 antibody described herein binds to target cells (e.g., Ramos cells) having a silent mutation (e.g., LALA-PG mutation) in the Fc region.

[0393] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibodies described herein activate NK cells (e.g., through the involvement of NKp46) and mediate the killing of target cells (e.g., B cells or B cell lines such as Ramos cells or Daudi cells). In some embodiments, the anti-EpCAM / NKp46 antibodies described herein activate NK cells having a silent mutation (e.g., LALA-PG mutation) in the Fc region. In some embodiments, the anti-EpCAM / NKp46 antibodies described herein contain the wild-type human IgG1 Fc region. In some embodiments, the anti-EpCAM / NKp46 antibodies described herein bind to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43).

[0394] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibodies described herein activate NK cells in a population of peripheral blood mononuclear cells (PBMCs) (e.g., through the involvement of NKp46). In some embodiments, PBMCs mediate the killing of target cells (e.g., B cells). In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the PBMCs are NK cells.

[0395] In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody described herein promotes monocyte-mediated killing of target cells (e.g., B cells or B cell lines such as Ramos cells). In some embodiments, the anti-EpCAM / NKp46 antibody described herein contains the wild-type human IgG1 Fc region. In some embodiments, the anti-EpCAM / NKp46 antibody described herein binds to NK cells having a mutation optimized for the Fc region (SEQ ID NO: 43). In some embodiments, the target cells are Ramos cells. In some embodiments, target cell killing is carried out by either phagocytic and / or tropocytotic cancer cell killing mechanisms. In some embodiments, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more of the target cells are killed.

[0396] In some embodiments, there is no significant change in cell count (e.g., NK cell count or monocyte cell count) after killing the target cells.

[0397] This disclosure provides a multispecific (e.g., bispecific) antibody and its antigen-binding fragment that specifically bind to EpCAM / NKp46 (e.g., human EpCAM / NKp46). In one embodiment, this disclosure provides an anti-EpCAM / NKp46 multispecific (e.g., bispecific) antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to EpCAM and a second antigen-binding domain that specifically binds to NKp46.

[0398] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0399] In some embodiments, the first heavy chain variable region (VH1) includes complementarity-determining regions (CDRs) 1, 2, and 3, where the VH1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR1, the VH1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR2, the VH1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH1 CDR3, and the first light chain variable region (VL1) includes CDRs 1, 2, and 3, where the VL1 CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR1, the VL1 CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR2, the VL1 CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL1 CDR3, and the selected VH1 The amino acid sequences of CDR1, 2, and 3 and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 100, 101, and 102, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 103, 104, and 105, respectively; or The amino acid sequences of the selected VH1 CDR1, 2, and 3 above, and the selected VL1 CDR1, 2, and 3 above, are the corresponding VH CDR1, 2, and 3 and VL CDR1, 2, and 4 for catumakisomab, M-701, adekatumumab, edrecolomab, tefibazumab, VB1-008, and VB1-050.

[0400] In some embodiments, the second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR1, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR2, the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH2 CDR3, and The second light chain variable region (VL2) includes CDR1, 2, and 3, where the VL2 CDR1 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR1, the VL2 CDR2 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR2, and the VL2 CDR3 region contains an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL2 CDR3. The amino acid sequences of the selected VH2 CDR1, 2, and 3 and the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0401] In some embodiments, the amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 101, and 102, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 103, 104, and 105, respectively; the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; In some embodiments, the first heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 114, the first light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 111, the second heavy chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 33, and the second light chain variable region includes a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 34.

[0402] In some embodiments, VH1 includes VH1 CDR1, VH1 CDR2, and VH1 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL1 includes VL1 CDR1, VL1 CDR2, and VL1 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 110, and the selected VL sequence is sequence number 111.

[0403] In some embodiments, VH2 includes VH2 CDR1, VH2 CDR2, and VH2 CDR3 which are the same as VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, and VL2 includes VL2 CDR1, VL2 CDR2, and VL2 CDR3 which are the same as VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, the selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34.

[0404] In some embodiments, the first antigen-binding domain specifically binds to human, mouse, monkey, or canine EpCAM, and / or the second antigen-binding domain specifically binds to human, mouse, monkey, or canine NKp46.

[0405] In some embodiments, the first antigen-binding domain is a human or humanized antigen-binding domain, and / or the second antigen-binding domain is a human or humanized antigen-binding domain.

[0406] In some embodiments, the first antigen-binding domain is a single-stranded variable fragment (scFv), and / or the second antigen-binding domain is an scFv.

[0407] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region). In some embodiments, the multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody described herein can be designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the anti-EpCAM / NKp46 antibody described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0408] In some embodiments, the multispecific (bispecific) anti-EpCAM / NKp46 antibodies described herein can be designed to have an IgG1 subtype structure with a knob-into-hole (KIH) mutation, thereby promoting heterodimerization and avoiding mispairing between the two heavy chains. In some embodiments, the anti-EpCAM / NKp46 antibody has a higher endocytosis ratio than the corresponding monoclonal antibody or control bispecific antibody.

[0409] The first and second antigen-binding fragments of the bispecific antibody or antigen-binding fragment described herein can be any preferred configuration. In some embodiments, the second antigen-binding domain is a single-stranded variable fragment (scFv) domain comprising a light-chain variable domain (VL) and a heavy-chain variable domain (VH) linked by a first linker.

[0410] In some embodiments, a second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. Schematic diagrams of this configuration are shown in Figures 4A and 4B.

[0411] In some embodiments, the multispecific (bispecific) anti-EpCAM / NKp46 antibody comprises a heavy chain sequence containing an optimized mutation in the IgG1 Fc region (SEQ ID NO: 43).

[0412] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody (e.g., EpCAM / NKp46 Fc-optimized heavy chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 85.

[0413] In some embodiments, the light chain of a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody (e.g., EpCAM / NKp46 Fc-optimized light chain) contains an amino acid sequence that is approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 86.

[0414] In some embodiments, the heavy chain of a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody (e.g., EpCAM / NKp46 Fc-optimized heavy chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 85, and the light chain of a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody (e.g., EpCAM / NKp46 Fc-optimized light chain) contains approximately or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 86.

[0415] In some embodiments, the second antigen-binding domain is linked to the C-terminus of the Fc region. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region. A schematic diagram of this configuration is shown in Figure 4B. In some embodiments, the heavy chain variable domain of the first antigen-binding domain is linked to the Fc region via a third linker.

[0416] In some embodiments, a knob-into-hole mutation was introduced into the Fc region of a multispecific (e.g., bispecific) antibody to reduce the possibility of mispairing between two heavy chains.

[0417] This disclosure provides modified antibodies, such as anti-EpCAM / NKp46 antibodies, EpCAM / NKp46 Fc-optimized antibodies, and human antibodies, including, for example, chimeric antibodies, humanized antibodies, and human antibodies.

[0418] In some embodiments, a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody, or an antibody fragment thereof, includes a combination of an anti-EpCAM antigen-binding domain and an anti-NKp46 antigen-binding domain as shown in Figure 33.

[0419] In some embodiments, EpCAM / NKp46 refers to a multispecific (e.g., bispecific) anti-EpCAM / NKp46 antibody comprising a first anti-EpCAM antigen-binding domain derived from an EpCAM antibody and a second anti-NKp46 antigen-binding domain derived from an NKp46 antibody. In some embodiments, the first anti-EpCAM antigen-binding domain comprises the CDR of the EpCAM antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the CDR of the NKp46 antibody. In some embodiments, the first anti-EpCAM antigen-binding domain comprises the VH and VL of the EpCAM antibody. In some embodiments, the second anti-NKp46 antigen-binding domain comprises the VH and VL of the NKp46 antibody.

[0420] In some embodiments, anti-EpCAM / NKp46 antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0421] Either the anti-EpCAM / NKp46 antibody or its antigen-binding fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-EpCAM / NKp46 antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0422] This disclosure also provides nucleic acids comprising polynucleotides encoding an anti-EpCAM / NKp46 antibody. The immunoglobulin heavy chain or immunoglobulin light chain of the anti-EpCAM / NKp46 antibody comprises a CDR as shown in Figure 33. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to NKp46 and / or EpCAM.

[0423] Anti-EpCAM / NKp46 antibodies may be anti-EpCAM / NKp46 antibody variants (including derivatives and conjugates) of anti-EpCAM / NKp46 antibodies or antibody fragments. Additional anti-EpCAM / NKp46 antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Anti-EpCAM / NKp46 antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the anti-EpCAM / NKp46 antibody or antigen-binding fragment is an IgG (e.g., IgG1 Fc region shown in SEQ ID NO: 41) antibody or its antigen-binding fragment.

[0424] The fragments of the anti-EpCAM / NKp46 antibody are suitable for use in the provided method, provided that they retain the desired affinity and specificity for both NKp46 and EpCAM. Thus, the fragments of the anti-EpCAM / NKp46 antibody retain the ability to bind to both NKp46 and EpCAM.

[0425] Antibodies and their antigen-binding fragments In some embodiments, the antibodies described herein (monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies)) and their antigen-binding fragments can have various forms.

[0426] Generally, antibodies (also called immunoglobulins) can consist of two classes of polypeptide chains: a light chain and a heavy chain. Non-limiting antibodies of this disclosure (anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, or anti-EpCAM / NKp46 antibodies) may be intact four-unit immunoglobulin chain antibodies containing two heavy chains and two light chains. The heavy chain of the antibody may be any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a kappa light chain (the amino acid sequence of the human kappa light chain is shown in SEQ ID NO: 44) or a lambda light chain.

[0427] The hypervariable region, known as the complementarity-determining region (CDR), forms a loop containing the antibody's principle antigen-binding surface. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form linked loops, sometimes even forming part of the β-sheet structure. The CDR of each chain is held in close proximity to the framework region and, together with the CDRs of other chains, contributes to the formation of the antigen-binding domain.

[0428] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422~“39;Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et This is described in al., Nature 342(6252):877-83(Dec.1989); and Ponomarenko and Bourne, BMC Structural Biology 7:64(2007), and the entirety of each of these is incorporated herein by reference.

[0429] CDRs are important for recognizing the epitopes of antigens. As used herein, “epitope” refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0430] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology “5(2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015):171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, and the entirety of each of these is incorporated herein by reference.

[0431] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, or camelids). The antigen-binding domain or antigen-binding fragment is any portion of the antibody that retains the specific binding activity of the intact antibody, i.e., any portion of the antibody that is specifically capable of binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may include, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide comprising a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0432] In some embodiments, the scFv of a multispecific (e.g., bispecific) antibody has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFv has two antigen-binding regions (antigen-binding regions: A and B), the two antigen-binding regions capable of binding to their respective target antigens having different affinities.

[0433] In some embodiments, a multispecific (e.g., bispecific) antibody or its antigen-binding fragment may include one, two, or three heavy chain variable regions (CDRs) selected from Figure 33.

[0434] In some embodiments, the antibodies described herein may be conjugated with a therapeutic agent. The antibody-drug conjugate, comprising the antibody or its antigen-binding fragment, may be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mytoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as analogues). In some embodiments, the therapeutic agent is MMAE or MMAF. In some embodiments, the therapeutic agent is conjugated via a linker, such as a VC linker. Details of the linkers used in ADCs are described, for example, in Su, Z. et al. “Antibody-drug conjugates: Recent advances in linker chemistry.” Acta Pharmaceutica Sinica B (2021), which is incorporated in its entirety by reference.

[0435] Multiple specific (or bispecific) antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture media. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated in its entirety by reference.

[0436] Either the antibody or its antigen-binding fragment described herein can be conjugated with a stabilizing molecule (e.g., a molecule that extends the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can extend the half-life of the anti-CD79b / NKp46 antibody or antigen-binding fragment or sustain its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0437] Antibodies or their antigen-binding fragments can also exist in various forms. Many different formats of antibodies or their antigen-binding fragments are known in the art, for example, as described in Suurs, et al., “A review of bispecific antibodies and antibody constructs in oncology and clinical challenges,” Pharmacology & Therapeutics (2019), which is incorporated herein by reference in its entirety.

[0438] In some embodiments, the antibody is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the anti-CD79b / NKp46 antibody is VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, Diabody, crossMab, DAF (2in1), DAF(4in1), DutaMab, DT-IgG, Knob-in-hole common light chain, Knob-in-hole assembly, Charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH These include IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Mini-antibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0439] In some embodiments, the antibody may be a TrioMab. In a TrioMab, the two heavy chains originate from different species, and their different sequences limit the formation of heavy-light chain pairs.

[0440] In some embodiments, the antibody has two distinct heavy chains and one common light chain. Heterodimerization of the heavy chains can be based on knob-into-hole or some other heavy chain pairing technique.

[0441] In several embodiments, antibodies can be produced using CrossMAb technology. CrossMAb technology can enhance correct light chain association in bispecific heterodimeric IgG antibodies, enabling the production of various bispecific antibody formats, including bivalent (1+1), trivalent (2+1), and tetravalent (2+2) bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) based antibodies. These formats can be derived from any existing antibody pair using domain crossover without requiring recognition of a common light chain, post-translational processing / extracorporeal chemical assembly, or the introduction of a series of mutations to enhance correct light chain association. This method is described in Klein et al., “The use of CrossMAb technology for the generation of bi- and multispecific antibodies.” MAbs. Vol.8. No.6. Taylor & Francis, 2016, and is incorporated in its entirety by reference. In some embodiments, the CH1 in the heavy chain is exchanged with the CL domain in the light chain.

[0442] Bispecific antibodies may also be duobodies. The Fab exchange mechanism naturally present in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between heavy and light chains.

[0443] In the bivariable domain antibody (DVD-Ig), additional VH and variable light chain (VL) domains are added to each N-terminus for bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind individually to the corresponding N-terminus of each heavy chain, instead of the scFv.

[0444] In scFv-IgG, two scFv molecules are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two distinct divalent binding sites and is therefore also called tetravalent. There is no problem with heavy-light chain pairing in scFv-IgG.

[0445] In some embodiments, bispecific antibodies may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically bonding the C-terminuses of their heavy chains.

[0446] Bispecific antibodies may also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, a light chain, a heavy chain, and a third chain containing the Fc region and scFv are assembled. This ensures efficient preparation and purification.

[0447] In some embodiments, the bispecific antibody may be a TF. Three Fab fragments are linked by disulfide crosslinks. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format does not have an Fc region.

[0448] ADAPTIR has two scFvs bound to both sides of a certain Fc region. It discards the intact IgG that serves as the basis for its construct, but preserves the Fc region, extending its half-life and facilitating purification.

[0449] Biaffinity retargeting (DART) involves two peptide chains linking opposite fragments (i.e., VLA to VHB and VLB to VHA), and a sulfur bond fusing them together at the C-terminus. In DART, the sulfur bond can improve stability compared to BiTE.

[0450] In DART-Fc, the Fc region is bound to DART. This can be generated by assembling three chains (two via disulfide bonds, similar to DART). One chain contains half of the Fc region, which dimerizes with the third chain, expressing only the Fc region. The addition of the Fc region extends the half-life, resulting in a longer effective concentration and avoiding consecutive IV injections.

[0451] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are created by half of the Fc region and then dimerized. This format has divalent bonds to both targets, and therefore, it is a tetravalent molecule.

[0452] A tandem diabody (TandAb) contains two diabodies. Each diabody consists of a VHA and a VLB fragment, and another VHA and a VLB fragment, associated by covalent bonds. The two diabodies are linked by a peptide chain. This can improve stability compared to a diabody composed of two scFvs. It has two divalent bonding sites.

[0453] The scFv-scFv-toxin comprises a toxin and two scFvs containing a stabilizing linker. This can be used for the specific delivery of the payload.

[0454] In some embodiments, the bispecific antibodies in this disclosure are designed to be 1+1 (monovalent for each target) and have an IgG1 subtype structure. This allows for reduced avidity to cells with low expression levels of the first and second epitopes and increased avidity to cells co-expressing the first and second epitopes, thereby achieving improved targeting function.

[0455] In some embodiments, the bispecific antibody contains a KIH mutation. In some embodiments, the bispecific antibody contains a first antigen-binding domain that specifically binds to a first epitope and a second antigen-binding domain that specifically binds to a second epitope. In some embodiments, the first antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations, and the second antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations. In some embodiments, the first antigen-binding domain contains a heavy chain (hole heavy chain) containing one or more hole mutations, and the second antigen-binding domain contains a heavy chain (knob heavy chain) containing one or more knob mutations.

[0456] Characteristics of antibodies In some embodiments, an antibody or its antigen-binding fragment can initiate NK cell-mediated target cell (tumor cell) killing, CDC, or ADCC.

[0457] This disclosure provides antibodies that specifically bind to NKp46 and antigen-binding fragments thereof. The antibodies or antigen-binding fragments described herein can block the binding between NKp46 and its ligand.

[0458] This disclosure provides antibodies and antigen-binding fragments that can specifically bind to TAAs and NK cell engagers. The antibodies or antigen-binding fragments described herein can block the binding between TAAs and their ligands.

[0459] Common techniques available for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). Affinity can be estimated from the quotient of the velocity constant (KD = koff / kon). In some embodiments, the antibody or its antigen-binding fragment can bind to the TAA with a dissociation rate (koff) of less than 0.1s⁻¹, less than 0.01s⁻¹, less than 0.001s⁻¹, less than 0.0001s⁻¹, or less than 0.00001s⁻¹. In some embodiments, the dissociation rate (koff) is greater than 0.01s⁻¹, greater than 0.001s⁻¹, greater than 0.0001s⁻¹, greater than 0.00001s⁻¹, or greater than 0.000001s⁻¹.

[0460] In some embodiments, the kinetic coupling velocity (kon) is greater than 1 × 10² / Ms, greater than 1 × 10³ / Ms, greater than 1 × 10⁴ / Ms, greater than 1 × 10⁵ / Ms, or greater than 1 × 10⁶ / Ms. In some embodiments, the kinetic coupling velocity (kon) is less than 1 × 10⁵ / Ms, less than 1 × 10⁶ / Ms, or less than 1 × 10⁷ / Ms.

[0461] In some embodiments, the antibody or its antigen-binding fragment may bind to the TAA with a KD of less than 1 × 10⁻⁶ M, less than 1 × 10⁻⁷ M, less than 1 × 10⁻⁸ M, less than 1 × 10⁻⁹ M, or less than 1 × 10⁻¹⁰ M. In some embodiments, the KD is 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or less than 1 nM. In some embodiments, the KD is greater than 1 × 10⁻⁷ M, greater than 1 × 10⁻⁸ M, greater than 1 × 10⁻⁹ M, or greater than 1 × 10⁻¹⁰.

[0462] The antibody or its antigen-binding fragment may also include an antigen-binding domain that can specifically bind to an NK cell surface antigen. In some embodiments, the antibody or its antigen-binding fragment described herein activates NK cells and enhances cell killing mediated by NK cells (e.g., killing of tumor cells). In some embodiments, the antibody can reduce tumor volume in an animal. In some embodiments, the antibody can reduce or delay the progression of an autoimmune disease.

[0463] In some embodiments, the antibody or its antigen-binding fragment described herein has agonist activity with respect to the activation of NK cells. In some embodiments, the antibody or its antigen-binding fragment described herein increases the expression of activation markers (e.g., CD69) on NK cells. In some embodiments, the expression of the activation marker (e.g., CD69) increases by about or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0464] Thus, in some embodiments, the antibody described herein is an NK cell surface antigen agonist. In some embodiments, the antibody described herein is an NK cell surface antigen antagonist.

[0465] In some embodiments, the antibody or its antigen-binding fragment has a dissociation rate (koff) of less than 0.1 s -1 less than 0.01 s -1 less than 0.001 s -1 less than 0.0001 s -1 less than, or 0.00001 s -1 less than and specifically binds to the NK cell surface antigen. In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 greater than 0.001 s -1 greater than 0.0001 s -1 greater than 0.00001 s -1 greater than, or 0.000001 s -1 greater than.

[0466] In some embodiments, the kinetic coupling velocity (kon) is 1 × 10⁻⁶ 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms greater than, or 1 × 10⁻⁶ 6 It is greater than / Ms. In some embodiments, the kinetic coupling velocity (kon) is 1 × 10⁻⁶. 5 / Ms less than 1 × 10 6 Less than / Ms, or 1 × 10 7 It is less than / Ms.

[0467] The affinity can be estimated from the quotient of the velocity constant (KD = koff / kon). In some embodiments, KD is 1 × 10⁻⁶ -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, KD is 50nM, 40nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, KD is 1 × 10⁻¹⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 M or 1 × 10 -10 It is greater than M.

[0468] Multispecific antibodies (e.g., bispecific antibodies) bind to both TAA and NK cell surface antigens, and therefore, for cells expressing both of these molecules, the antibody has a higher binding affinity to these cells. Binding activity can be used to measure the binding affinity of an antibody to these cells. Binding activity is the cumulative strength of multiple affinities of individual non-covalent interactions.

[0469] In some embodiments, the antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate or percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the anti-CD79b / NKp46 antibody, antigen-binding fragment, or ADC has a tumor growth inhibition rate less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI (%) can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 days after the start of treatment. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula.

[0470] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100% Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0471] In some embodiments, the antibody, antigen-binding fragment, or ADC has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0472] In some embodiments, the antibody, antigen-binding fragment, or ADC does not have a functional Fc region. For example, the antibody or its antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment. In some embodiments, the anti-CD79b / NKp46 antibody or its antigen-binding fragment described herein has an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbering) or an LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering). The amino acid sequence of the human IgG1 LALA-PG variant is shown in SEQ ID NO: 42.

[0473] In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region). In some embodiments, the antibodies described herein can be designed to have an IgG1 subtype structure with optimized mutations (G236A, A330L, and I332E mutations in EU numbering). In some embodiments, the antibodies described herein can be designed to have an IgG1 Fc region having alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332 in EU numbering. In some embodiments, the Fc region contains an amino acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43.

[0474] Several other modifications can be made to the Fc region. For example, a cysteine ​​residue can be introduced into the Fc region to enable interchain disulfide bond formation within this region. The homodimeric fusion protein thus produced may have any, increased in vitro and / or in vivo half-lives.

[0475] In some embodiments, IgG4 has the S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm replacement both in vivo and in vitro.

[0476] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans located at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (position 314 in the Eu numbering or Kabat numbering of Fc region residues). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further recombined, and asparagine at position 297 can be replaced with alanine (N297A).

[0477] Recombinant vectors This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such host cells contain polynucleotides and / or the polynucleotide-containing vectors), and the production of antibody polypeptides or fragments thereof by recombinant technology.

[0478] In some embodiments, the antibody is an anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, or anti-EpCAM / NKp46 antibody. The full-length sequence of human NKp46 is known in the art (the sequence of amino acids 22 to 254 of human NKp46 is shown in SEQ ID NO: 1). The full-length sequence of monkey NKp46 is known in the art (amino acids 22 to 257 of monkey NKp46 are shown in SEQ ID NO: 2). In some embodiments, His-tagged human NKp46 protein is used as an immunogen.

[0479] As used herein, “vector” is any construct that, when introduced into a host cell, can deliver one or more polynucleotides of interest to the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably bound to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the host cell genome, at, near, or adjacent to the integration site of the polynucleotides of interest, so that the polynucleotides of interest are translated within the host cell into which the expression vector has been introduced.

[0480] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0481] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., a smallpox or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (deficient) replicable virus or a non-replicable virus. In the latter case, viral replication generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321;Flexner et al.,1989,Ann.NYAcad Sci.569:86-103;Flexner et al. al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; 91 / 02805;Berkner-Biotechniques,6:616-627,1988;Rosenfeld et al. Preferred systems are disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA with biodegradable beads that are efficiently transported into cells.

[0482] For expression, a DNA insert containing a polynucleotide encoding the polypeptide disclosed herein can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as, to name a few, the phage λPL promoter, the E. coli lac, trp and tac promoters, the SV40 early promoter and the SV40 late promoter, and the promoter of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may further contain sites for transcription start and end, and within the transcription region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start at the beginning and a stop codon (UAA, UGA, or UAG) located approximately at the end of the polypeptide being translated.

[0483] As shown, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for Escherichia coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella tihumurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0484] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0485] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV pre-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0486] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used. For an overview, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997).

[0487] The construct can be introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference.

[0488] Transcription of the antibody-encoding DNA in more eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, approximately 10–300 bp in length, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer, located behind the origin of replication at base pairs 100–270, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0489] Appropriate secretory signals can be incorporated into expressed polypeptides to induce the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals can be endogenous to the polypeptide, or they can be heterologous signals.

[0490] Polypeptides can be expressed in modified forms, such as fusion proteins (e.g., GST fusions), or by histidine tagging, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a polypeptide to improve stability and endurance in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to induce secretion or excretion, improve stability, and facilitate purification is a well-known and common technique, particularly in the art.

[0491] This disclosure includes nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence described herein, and We also provide amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to any amino acid sequence described herein.

[0492] This disclosure includes nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology to any nucleotide sequence described herein, and We also provide amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology to any amino acid sequence described herein.

[0493] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0494] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence, or (ii) consists of an amino acid sequence, the amino acid sequence being one of the sequences described herein.

[0495] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence, or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is one of the sequences described herein.

[0496] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to ensure optimal alignment for comparison, and non-homologous sequences may be ignored). Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are considered identical at that position (as used herein, "identity" of amino acids or nucleic acids corresponds to "homology" of amino acids or nucleic acids). The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of the percentage of identity between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty 12, a gap extension penalty 4, and a frameshift gap penalty 5.

[0497] This disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy-chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a light-chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding an scFv polypeptide described herein.

[0498] In some embodiments, the vector may have two of the nucleic acids described herein, and the vectors may both encode a VL region and a VH region that bind to a TAA (e.g., CD79b). In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, and the pair of vectors may both encode a VL region and a VH region that bind to a TAA (e.g., CD79b, BCMA, EGFR, or EpCAM).

[0499] In some embodiments, the vector may have two of the nucleic acids described herein, and the vector encodes a VL region and a VH region that co-bind with an NK cell surface antigen (e.g., NKp46). In some embodiments, a pair of vectors is provided, each vector comprising one of the nucleic acids described herein, and the pair of vectors together encodes a VL region and a VH region that co-bind with an NK cell surface antigen (e.g., NKp46).

[0500] Treatment method The methods described herein include methods for treating cancer-related disorders. Generally, these methods involve administering a therapeutically effective dose of one of the antibodies described herein (e.g., anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, or anti-EpCAM / NKp46 antibody) to a subject who needs or is determined to need such treatment.

[0501] As used in this context, “treatment” means alleviating at least one symptom of a cancer-associated disorder. Often, cancer is fatal. Therefore, treatment can result in an increase in life expectancy (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). For the treatment of a cancer-associated condition, administering a therapeutically effective amount of the agents described herein results in a reduction in the number of cancer cells and / or alleviation of symptoms.

[0502] As used herein, the term “cancer” means an abnormal situation or condition characterized by autonomously proliferating cells, i.e., rapidly growing cell proliferation. This term includes all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. As used herein, the term “tumor” means cancerous cells, e.g., a mass of cancerous cells. Cancers treatable or diagnoseable using the methods described herein include malignant tumors of various organ systems, such as those affecting the lungs, chest, thyroid, lymphatic system, gastrointestinal tract, and genitourinary tract, as well as adenocarcinomas, including most colorectal cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestinal cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose carcinomas in subjects. The term "carcinoma" is recognized in the art and means a malignant tumor of epithelial or endocrine glandular tissue, including respiratory cancers, digestive cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, the cancer is renal cancer or melanoma. Exemplary carcinomas include carcinomas formed from the tissues of the cervix, lungs, prostate, chest, head and neck, colon, and ovaries. The term also includes carcinosarcomas, which include, for example, malignant tumors composed of cancerous and sarcomatous tissues. "Adenocarcinoma" means a carcinoma of glandular tissue originating from glandular tissue, or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and means a malignant tumor of mesenchymal origin.

[0503] In some embodiments, the cancer is chemotherapy-resistant.

[0504] In one embodiment, the disclosure also provides a method for treating cancer in a subject, a method for slowing the rate of increase of tumor volume in a subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can interrupt, slow, stop, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0505] In one embodiment, the Disclosure features a method comprising administering a therapeutically effective dose of one of the antibodies described herein (e.g., anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, anti-EpCAM / NKp46 antibody, or antibody-drug conjugate) to a subject in need thereof, for example, a subject having, or identified or diagnosed with, cancer, such as B-cell lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer (NSCLC), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, oral cancer, or kidney cancer. In some embodiments, the cancer is a solid tumor, lung cancer, head and neck cancer, thyroid cancer, central nervous system (CNS) cancer, liver cancer, or brain cancer.

[0506] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to animals, humans, or non-humans to which treatment according to the methods of the present invention is provided. Veterinary and non-veterinary uses are conceived by the present invention. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0507] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients with cancer can be identified by various methods well known in the art.

[0508] As used herein, “effective dose” means an amount or dose sufficient to produce a beneficial or desired outcome, including interrupting, slowing, blocking, or inhibiting the progression of a disease, such as cancer. The effective dose varies depending on the age and weight of the person to whom the antibody, antigen-binding fragment, antibody-drug conjugate, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the dose may be determined on an individual basis.

[0509] An effective dose can be administered in one or more doses. For example, an effective dose of antibody or antigen-binding fragment is an amount sufficient to alleviate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of autoimmune disease or cancer in a patient, or an amount sufficient to alleviate, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro.

[0510] The effective doses and schedules for administering the antibodies, antigen-binding fragments, antibody-drug conjugates, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary, for example, depending on the mammal to which the antibodies, antigen-binding fragments, antibody-drug conjugates, and / or compositions disclosed herein are administered, the route of administration, the specific type of drug or composition disclosed herein, and other drugs administered to the mammal.

[0511] The typical daily effective dose of an antibody, antigen-binding fragment, or antibody-drug conjugate (e.g., anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, anti-EpCAM / NKp46 antibody, or antibody-drug conjugate) is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose may be 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dose may be about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0512] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of B-Raf inhibitors, CD79b inhibitors, BCMA inhibitors, EGFR inhibitors, EpCAM inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, NKp46 inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0513] In some embodiments, additional therapeutic agents may include one or more inhibitors selected from the group consisting of CD79b inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0514] In some embodiments, additional therapeutic agents include trabectedin, nab-paclitaxel, trevananib, pazopanib, sediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolisin, alimta, jikaida, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, and AGS-00. 3. The treatment may include one or more therapeutic agents selected from the group consisting of cabozantinib, vinflunin, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, salomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0515] In some embodiments, additional therapeutic agents may include one or more agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0516] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0517] In some embodiments, additional therapeutic agents include anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-4-1BB antibody, anti-TIM3 antibody, or anti-GITR antibody.

[0518] Pharmaceutical composition and route of administration This specification also provides pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein, or antibody-drug conjugates (e.g., anti-NKp46, anti-CD79b / NKp46, anti-BCMA / NKp46, anti-EGFR / NKp46, anti-EpCAM / NKp46 antibodies, or antibody-drug conjugates). The pharmaceutical compositions can be formulated in any form well known in the art.

[0519] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antimicrobial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g.,...

Claims

1. An antibody or antigen-binding fragment that binds to NKp46, It comprises a heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence; and It comprises light chain variable regions (VLs) including CDR1, 2, and 3, wherein the VL CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence. Here, the amino acid sequences of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 5, 7, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 6, 8, and 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 15, 17, and 19, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 18, and 19, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; (6) An antibody or antigen-binding fragment wherein the amino acid sequences of selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively, and the amino acid sequences of selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively.

2. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Kabat definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

3. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Chothia definition, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 6, 8, and 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively.

4. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Kabat definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively.

5. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Chothia definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 16, 18, and 19, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, respectively.

6. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Kabat definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively.

7. An antibody or antigen-binding fragment according to claim 1, wherein, according to the Chothia definition, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 26, 28, and 29, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively.

8. An antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment specifically binds to human NKp46 or monkey NKp46.

9. An antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a human antibody or a humanized antibody, or an antigen-binding fragment thereof.

10. An antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment is a single-chain mutant fragment (scFv) or a polyspecific antibody (e.g., a bispecific antibody).

11. A nucleic acid comprising a polynucleotide encoding a polypeptide, wherein the polypeptide is: (1) comprising an immunoglobulin heavy chain or fragment thereof, comprising heavy chain variable regions (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO. 4; (2) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, wherein the VL binds to NKp46 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 3; (3) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs: 6, 8, and 9, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO: 4; (4) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, respectively, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) which includes the amino acid sequence shown in SEQ ID NO. 14; (5) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, wherein the VL binds to NKp46 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 13; (6) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 16, 18, and 19, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 14; (7) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 24; (8) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, wherein the VL binds to NKp46 when paired with a VH containing the amino acid sequence shown in SEQ ID NO. 23; (9) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 26, 28, and 29, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 24; (10) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 36; (11) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 35; (12) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs: 6, 8, and 9, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 36; (13) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 38; (14) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each having the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22, wherein the VL binds to NKp46 when paired with a VH having the amino acid sequence shown in SEQ ID NO. 37; (15) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 16, 18, and 19, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 38; (16) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 34; (17) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively, wherein the VL binds to NKp46 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO. 33; (18) A nucleic acid comprising an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NOs. 26, 28, and 29, wherein the VH binds to NKp46 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO.

34.

12. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each having the amino acid sequences shown in SEQ ID NOs. 5, 7, and 9.

13. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12.

14. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 19.

15. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 20, 21, and 22.

16. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof, comprising a VH containing CDR1, 2, and 3, each having the amino acid sequences shown in SEQ ID NOs. 25, 27, and 29.

17. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof, comprising a VL containing CDR1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32.

18. A nucleic acid according to any one of claims 11 to 17, wherein VH specifically binds to human NKp46 or monkey NKp46 when paired with VL, or VL specifically binds to human NKp46 or monkey NKp46 when paired with VH.

19. A nucleic acid according to any one of claims 11 to 18, wherein the immunoglobulin heavy chain or fragment thereof is a human or humanized immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human or humanized immunoglobulin light chain or fragment thereof.

20. A nucleic acid according to any one of claims 11 to 19, wherein the nucleic acid encodes a single-chain mutant fragment (scFv), a polyspecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

21. A nucleic acid according to any one of claims 11 to 20, wherein the nucleic acid is cDNA.

22. A vector comprising one or more nucleic acids as described in any one of claims 11 to 21.

23. A vector comprising two nucleic acids according to any one of claims 11 to 21, wherein the vector encodes a VH region and a VL region that bind together to NKp46.

24. A vector pair wherein each vector comprises one nucleic acid as described in any one of claims 11 to 21, wherein the vector pair together encodes a VH region and a VL region that bind together to NKp46.

25. A cell comprising the vector according to claim 22 or 23, or the vector pair according to claim 24.

26. A cell according to claim 25, wherein the cell is a CHO cell.

27. A cell comprising one or more nucleic acids according to any one of claims 11 to 21.

28. A cell comprising two nucleic acids according to any one of claims 11 to 21.

29. A cell according to claim 28, wherein the two nucleic acids together encode a VH region and a VL region that bind together to NKp46.

30. A method for producing an antibody or its antigen-binding fragment, (a) A step of culturing the cells according to any one of claims 25 to 29 under conditions sufficient to cause the cells to produce antibodies or antigen-binding fragments; and (b) A method comprising the step of recovering an antibody or antigen-binding fragment produced by the cell.

31. An antibody or antigen-binding fragment that binds to NKp46, It comprises a heavy chain variable region (VH) containing an amino acid sequence that is at least 80% identical to the selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 80% identical to the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24; (4) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (5) The selected VH sequence is sequence number 35 and the selected VL sequence is sequence number 36; and (6) The selected VH sequence is sequence number 37 and the selected VL sequence is sequence number 38; an antibody or its antigen-binding fragment.

32. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 3 and VL comprises the sequence of SEQ ID NO:

4.

33. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 13 and VL comprises the sequence of SEQ ID NO:

14.

34. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 23 and VL comprises the sequence of SEQ ID NO:

24.

35. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 33 and VL comprises the sequence of SEQ ID NO:

34.

36. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 35 and VL comprises the sequence of SEQ ID NO:

36.

37. An antibody or antigen-binding fragment according to claim 31, wherein VH comprises the sequence of SEQ ID NO: 37 and VL comprises the sequence of SEQ ID NO:

38.

38. An antibody or antigen-binding fragment according to any one of claims 31 to 37, wherein the antibody or antigen-binding fragment specifically binds to human NKp46 or monkey NKp46.

39. An antibody or antigen-binding fragment according to any one of claims 31 to 38, wherein the antibody or antigen-binding fragment is a human antibody or a humanized antibody, or an antigen-binding fragment thereof.

40. An antibody or antigen-binding fragment according to any one of claims 31 to 39, wherein the antibody or antigen-binding fragment is a single-chain mutant fragment (scFv) or a polyspecific antibody (e.g., a bispecific antibody).

41. An antibody or antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment according to any one of claims 1 to 10 and 31 to 40.

42. An antibody or antigen-binding fragment that binds to NKp46, A heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3, which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; The selected VH sequence and the selected VL sequence include a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3, which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is sequence number 3, and the selected VL sequence is sequence number 4; (2) The selected VH sequence is sequence number 13, and the selected VL sequence is sequence number 14; (3) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 24; (4) The selected VH sequence is sequence number 33, and the selected VL sequence is sequence number 34; (5) The selected VH sequence is sequence number 35 and the selected VL sequence is sequence number 36; and (6) The selected VH sequence is sequence number 37 and the selected VL sequence is sequence number 38; an antibody or its antigen-binding fragment.

43. A method for treating a subject having cancer or an autoimmune disease, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10 and 31 to 42.

44. A method according to claim 43, wherein the antibody or its antigen-binding fragment is a bispecific antibody that targets both NK cells and tumor cells expressing a tumor antigen.

45. A method according to claim 43 or 44, further comprising the step of administering to a subject a therapeutically effective amount of anti-OX40 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

46. A method for reducing the rate of tumor growth, A method comprising the step of contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10 and 31 to 42.

47. A method to kill tumor cells, A method comprising the step of contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10 and 31 to 42.

48. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10 and 31 to 42, and a pharmaceutically acceptable carrier.

49. An antibody or its antigen-binding fragment, i) comprising a first antigen-binding domain that specifically binds to a first antigen, wherein the first antigen is a tumor-associated antigen (TAA); ii) comprising a second antigen-binding domain that specifically binds to a second antigen, wherein the second antigen is an NK cell surface antigen; iii) Containing an Fc region; an antibody or its antigen-binding fragment.

50. An antibody or antigen-binding fragment according to claim 49, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1); and the second antigen-binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

51. An antibody or antigen-binding fragment according to claim 49, wherein the second antigen-binding domain is a single-chain fragment mutation (scFv) domain in which a light chain variable domain (VL2) and a heavy chain variable domain (VH2) are linked by a first linker.

52. An antibody or antigen-binding fragment according to claim 51, wherein the second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain via a second linker.

53. An antibody or antigen-binding fragment according to claim 51, wherein the second antigen-binding domain is linked to the C-terminus of the Fc region.

54. An antibody or antigen-binding fragment according to any one of claims 49 to 53, wherein VH1 is linked to the CH1 domain and VL1 is linked to the CL domain.

55. An antibody or antigen-binding fragment according to any one of claims 49 to 54, wherein the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

56. An antibody or antigen-binding fragment according to claim 55, wherein the Fc region is the Fc region of human IgG1.

57. An antibody or antigen-binding fragment according to claim 55 or 56, wherein the antibody or antigen-binding fragment is a bispecific antibody or its antigen-binding fragment.

58. An antibody or antigen-binding fragment according to any one of claims 53 to 57, wherein the Fc region comprises one or more of the following (all numbered according to EU numbering): Glutamic acid (E) at position 332; Leucine (L) at position 330; Alanine (A) at position 236; and / or Aspartic acid (D) at position 293, an antibody, or its antigen-binding fragment.

59. An antibody or antigen-binding fragment according to any one of claims 53 to 57, wherein the Fc region comprises any of the following (all numbered according to EU numbering): i) Alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332; ii) Alanine (A) at position 236, aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332; iii) Alanine (A) at position 236; iv) Alanine at position 236 (A), aspartic acid at position 293 (D), and glutamic acid at position 332 (E); v) Aspartic acid (D) at position 293 and glutamic acid (E) at position 332; and vi) Aspartic acid (D) at position 293, leucine (L) at position 330, and glutamic acid (E) at position 332, an antibody or its antigen-binding fragment.

60. An antibody or antigen-binding fragment according to any one of claims 53 to 57, wherein the Fc region is not fucosylated.

61. An antibody or antigen-binding fragment according to claim 59, wherein the Fc region is the Fc region of human IgG1, and the Fc region comprises alanine (A) at position 236, leucine (L) at position 330, and glutamic acid (E) at position 332, wherein the Fc region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 43 (numbering according to EU numbering).

62. An antibody or antigen-binding fragment according to any one of claims 49 to 61, wherein the NK cell surface antigen is selected from NKp46, NKp30, CD16, NKG2D, and NKG2A.

63. An antibody or antigen-binding fragment according to claim 62, wherein the NK cell surface antigen is NKp46.

64. An antibody or antigen-binding fragment according to any one of claims 49 to 63, wherein the tumor-associated antigen (TAA) is CD79b, EGFR, EpCAM, B-cell maturation antigen (BCMA), DLL3, PSMA, STEAP1, MUC1, c-Met, LRRC15, CCR8, OX40, perforin, granzyme B, Fas ligand (FasL), CD1d, membrane glycolipid, globotriaosylceramide (Gb3Cer / CD77), ganglioside (GD2, GD3, GM2), CD34, CD45, human leukocyte antigen-DR (HLA-DR), CD 123, CD38, CLL1, CD105, CD71, SSC, MAGE, MUC16, CD19, WT-1, B7H3, TEM8, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, α-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), CCR4, CD133, CD152, CD20, CD125, CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, CD3, FAP, Fibronectin excess domain B, Folate receptor 1, GD2, GD3 ganglioside, Glycoprotein 75, GPNMB, HER2 / neu, HGF, Human scattering factor receptor kinase, IGF-I receptor, IGF-I, IgG1, IL-5, IL-13, IL-6, IL-15, Insulin-like growth factor I receptor, Integrin a5b1, Integrin avb3, MORAb-009, MS4A1, Mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PD-1, PD-L1, PDGF-R a. An antibody or its antigen-binding fragment, which is PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, Tenacin C, TGF beta 2, TGF-b, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGF-1, VEGF-2, or vimentin.

65. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is CD79b; and the second antigen is NKp46.

66. The antibody or antigen-binding fragment according to claim 65, The first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) comprises CDR1, 2, and 3, where the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence. Here, the amino acid sequences of the selected VH1 CDR1, 2, and 3, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; and (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and (3) The amino acid sequences of selected VH1 CDR1, 2, 3 and selected VL1 CDR1, 2, 3 are selected from polatuzumab, PRV-3279, and SHR-A1912; antibodies or their antigen-binding fragments.

67. An antibody or antigen-binding fragment according to claim 65 or 66, The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) comprises CDR1, 2, and 3, where the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. Here, the amino acid sequences of the selected VH2 CDR1, 2, and 3, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are one of the following: (1) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 5, 7, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 6, 8, and 9, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 15, 17, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 16, 18, and 19, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; (6) The amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively, and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; an antibody or its antigen-binding fragment.

68. An antibody or antigen-binding fragment according to any one of claims 65 to 67, (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 5, 7, and 9, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 10, 11, and 12, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 6, 8, and 9, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 10, 11, and 12, respectively; (3) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 49, 51, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 15, 17, and 19, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 20, 21, and 22, respectively; (4) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 16, 18, and 19, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 20, 21, and 22, respectively; (5) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 49, 51, and 53, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; or (6) The amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs. 50, 52, and 53, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs. 54, 55, and 56, respectively; and the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 26, 28, and 29, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; an antibody or its antigen-binding fragment.

69. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is BCMA; and the second antigen is NKp46.

70. The antibody or antigen-binding fragment according to claim 69, The first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) comprises CDR1, 2, and 3, where the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence. The amino acid sequences of the selected VH1 CDR1, 2, and 3, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 88, 89, and 90, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 91, 92, and 93, respectively. (2) The amino acid sequences of the selected VH1 CDR1, 2, 3 and the selected VL1 CDR1, 2, 3 are the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of zevolcabutagen autoleucil, erranatamab, tecristamab, equecabutagen autoleus, siltakabutagen autoleucel, idekabutagen vicreucel, verantamab, limbocerutamab, alnuctamab, TNB-383B, or SEA-BCMA; an antibody or its antigen-binding fragment.

71. An antibody or antigen-binding fragment according to claim 69 or 70, The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) comprises CDR1, 2, and 3, where the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. The amino acid sequences of the selected VH2 CDR1, 2, and 3, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; antibodies or their antigen-binding fragments.

72. An antibody or antigen-binding fragment according to any one of claims 69 to 71, wherein the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 88, 89, and 90, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 91, 92, and 93, respectively; and the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; an antibody or antigen-binding fragment.

73. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is EGFR; and the second antigen is NKp46.

74. The antibody or antigen-binding fragment according to claim 73, The first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) comprises CDR1, 2, and 3, where the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence. The amino acid sequences of the selected VH1 CDR1, 2, and 3, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 94, 95, and 96, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 97, 98, and 99, respectively. (2) The amino acid sequences of the selected VH1 CDR1, 2, 3 and the selected VL1 CDR1, 2, 3 are the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amibantamab, cetuximab, nesitumumab, panitumumab, nimotuzumab, BL-B01D1, dempitamab, depatuxizumab, futuximab, modetuximab, JMT-101, MRG-003, pimrutamab, QL1203, SI-B001, and ametumumab; antibodies or their antigen-binding fragments.

75. An antibody or antigen-binding fragment according to claim 73 or 74, The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) comprises CDR1, 2, and 3, where the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. The amino acid sequences of the selected VH2 CDR1, 2, and 3, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; An antibody or its antigen-binding fragment.

76. An antibody or antigen-binding fragment according to any one of claims 73 to 75, The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 94, 95, and 96, respectively; the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 97, 98, and 99, respectively; and the amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; An antibody or its antigen-binding fragment.

77. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is EpCAM; and the second antigen is NKp46.

78. The antibody or antigen-binding fragment according to claim 77, The first heavy chain variable region (VH1) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR1 amino acid sequence, the VH1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR2 amino acid sequence, and the VH1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH1 CDR3 amino acid sequence; and The first light chain variable region (VL1) comprises CDR1, 2, and 3, where the VL1 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR1 amino acid sequence, the VL1 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR2 amino acid sequence, and the VL1 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL1 CDR3 amino acid sequence. The amino acid sequences of the selected VH1 CDR1, 2, and 3, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 101, and 102, respectively, and the amino acid sequences of the selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 103, 104, and 105, respectively; (2) The amino acid sequences of the selected VH1 CDR1, 2, 3 and the selected VL1 CDR1, 2, 3 are the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of catumakisomab, M-701, adecatumumab, edrecolomab, tefivazumab, VB1-008, or VB1-050; An antibody or its antigen-binding fragment.

79. An antibody or antigen-binding fragment according to claim 77 or 78, The second heavy chain variable region (VH2) comprises CDR1, 2, and 3, wherein the VH2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR1 amino acid sequence, the VH2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR2 amino acid sequence, and the VH2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VH2 CDR3 amino acid sequence; and The second light chain variable region (VL2) comprises CDR1, 2, and 3, where the VL2 CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR1 amino acid sequence, the VL2 CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR2 amino acid sequence, and the VL2 CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL2 CDR3 amino acid sequence. The amino acid sequences of the selected VH2 CDR1, 2, and 3, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are as follows: The amino acid sequences of the selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs. 30, 31, and 32, respectively; An antibody or its antigen-binding fragment.

80. An antibody or antigen-binding fragment according to any one of claims 77 to 79, wherein the amino acid sequences of selected VH1 CDR1, 2, and 3 are shown in SEQ ID NOs: 100, 101, and 102, respectively; the amino acid sequences of selected VL1 CDR1, 2, and 3 are shown in SEQ ID NOs: 103, 104, and 105, respectively; and the amino acid sequences of selected VH2 CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 27, and 29, respectively; and the amino acid sequences of selected VL2 CDR1, 2, and 3 are shown in SEQ ID NOs: 30, 31, and 32, respectively; An antibody or its antigen-binding fragment.

81. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is DLL3; and the second antigen is NKp46.

82. An antibody or antigen-binding fragment according to claim 81, wherein the CDR of the DLL3 antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of lovalpituzumab tecilin, tarlatamab, HPN328, or BI 764532.

83. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is PSMA; and the second antigen is NKp46.

84. An antibody or antigen-binding fragment according to claim 83, wherein the CDR of the PSMA antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of HPN-424, LAVA-1207, REGN-5678, REGN-4336, akapatamab, or AMG-340.

85. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is STEAP1; and the second antigen is NKp46.

86. An antibody or antigen-binding fragment according to claim 85, wherein the CDR of the STEAP1 antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of zalritamig, ABBV-969, or bundletozumab vedotin.

87. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is MUC1; and the second antigen is NKp46.

88. An antibody or antigen-binding fragment according to claim 87, wherein the CDR of the MUC1 antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of MAB-AR20.5 or DS-3939.

89. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is c-MET; and the second antigen is NKp46.

90. An antibody or antigen-binding fragment according to claim 89, wherein the CDR of the c-Met antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amivantamab-VMJM, terisotuzumab vedotin, ABBV-400, bafisontamab, CKD-702, emibetuzumab, RC-108, MCLA-129, REGN-5093, or REGN5093-M114.

91. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is LRRC15; and the second antigen is NKp46.

92. An antibody or antigen-binding fragment according to claim 91, wherein the CDR of the LRRC15 antigen-binding fragment is QL-315, RAD-502, or the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of samulotamab.

93. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is CCR8; and the second antigen is NKp46.

94. An antibody or antigen-binding fragment according to claim 93, wherein the CDR of the CCR8 antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of BMS-986340, LM-108, S-531011, AMG-355, BAY3375968, BGB-A3055, CM369, HBM-1022, PSB-114, or SRF-114.

95. An antibody or antigen-binding fragment according to any one of claims 49 to 64, wherein the first antigen is OX40; and the second antigen is NKp46.

96. An antibody or antigen-binding fragment according to claim 93, wherein the CDR of the OX40 antigen-binding fragment is the corresponding VH CDR1, 2, 3 and VL CDR1, 2, 3 of amritelimab, locatinlimab, INBRX-106, BGB-A445, INCAGN-1949, ibuxolimab, MEDI-6469, BAT-6026, IMG-007, ABBV-368, or BMS-986178.

97. An antibody or antigen-binding fragment according to any one of claims 49 to 96, wherein the Fc region has enhanced complement-dependent cell cytotoxicity (CDC), enhanced antibody-dependent cell cytotoxicity (ADCC), or an extended half-life.

98. An antibody or antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment according to any one of claims 49 to 97.

99. A method for treating a subject having cancer, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98.

100. A method according to claim 99, wherein the cancer is a solid tumor or a hematological cancer.

101. A method according to claim 100, wherein the cancer is breast cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, ovarian cancer, colon cancer, esophageal cancer, tracheal cancer, bladder cancer, uterine cancer, rectal cancer, small intestine cancer, pancreatic cancer, and liver cancer.

102. A method according to claim 100, wherein the cancer is multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma, acute B-cell leukemia, chronic lymphocytic leukemia, B-cell prelymphocytic leukemia, splenic lymphoma with hairy lymphocytes, hairy cell leukemia, follicular lymphoma, and mantle cell lymphoma.

103. A method according to any one of claims 99 to 102, wherein the subject is further treated with an effective amount of anti-4-1BB antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-CD40 antibody, or anti-PD-L1 antibody.

104. A method for reducing the rate of tumor growth, comprising the step of contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98.

105. A method for killing tumor cells, comprising the step of contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98.

106. A method for enhancing the immune response in a subject, comprising the step of administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98.

107. A method for treating a subject having an autoimmune disease, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98.

108. A method according to claim 107, wherein the autoimmune disease is selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu's arteritis, cold agglutinin disease, warm autoimmune hemolytic anemia, and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (e.g., granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) or microscopic polyangiitis (MPA)).

109. The method according to claim 108, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis.

110. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 49 to 98 and a pharmaceutically acceptable carrier.

Images