CN118574857A

CN118574857A - CD 3-targeting multispecific antibody and application thereof

Info

Publication number: CN118574857A
Application number: CN202280089487.8A
Authority: CN
Inventors: 杨熠; 李劼; 陈计; 杨洋
Original assignee: Yantang Biotechnology Hangzhou Co ltd; Nanjing University
Current assignee: Yantang Biotechnology Hangzhou Co ltd; Nanjing University
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2024-08-30
Also published as: WO2023141856A1

Abstract

The application provides a multi-specific antibody targeting CD3 and application thereof.

Description

CD 3-targeting multispecific antibody and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a multispecific antibody and application thereof.

Background

Efforts in new antibody engineering have made a lot of progress over the last few decades, and thus have initially revealed a broad application prospect for multispecific antibodies.

The structures of currently common multispecific antibodies (e.g., multispecific) include, for example: tetravalent IgG-scFv fusion proteins (Coloma m.j. Et al, nat. Biotechnol,1997,15,159), bispecific antibodies (diabody) (Holliger p. Et al, proc. Natl. Acad. Sci. Usa,1993,90,6444), tandem scFv molecules (see, e.g., bargou R. Et al, science,2008,321,974), tetravalent IgG-like double variable domain antibodies ("DVD-Ig", wu C. Et al, nat. Biotechnol,2007,25,1290), tetravalent Fab tandem immunoglobulins ("FIT-Ig") (Wu C. Et al, WO2015103072A 1), bivalent rat/mouse hybrid multispecific IgG (Lindhofer H. Et al, J. Immunol,1995,155,219), and multispecific Crossmab binding proteins (see, e.g., auer J. Et al, WO2013026831A 1.).

For the treatment of certain diseases or conditions, the complexity and high cost of combination therapies may be avoided by designing and producing engineered multispecific antibodies that can bind to two different epitopes and/or two different target antigens.

Among these, there is a great interest in multispecific antibodies for treating cancer, particularly those capable of re-targeting T cells to kill various tumor cells.

A common T cell receptor ("TCR") is a heterodimer ("tcrαβ") covalently linked by an α chain and a β chain. The CD3 molecule is a T cell co-receptor, alternatively referred to as a T cell co-receptor, which consists of five distinct polypeptide chains (i.e., a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and two zeta chains). These polypeptide chains form a complex of three dimers (i.e., εy, εδ, and ζζ complex) by binding to each other. The CD3 complex binds to the TCR to generate an activation signal in T lymphocytes. In the absence of CD3, TCRs do not assemble properly and are degraded. The TCR complex is very important, comprising about 10 activating motifs of immunoreceptor tyrosine groups (ITAMs). In pathological conditions, T cells are key players in various organ-specific autoimmune diseases, such as type I diabetes, rheumatoid arthritis and multiple sclerosis. Currently, it is thought that activation of T cells requires two signals to avoid a single signal causing an inappropriate immune response against autoantigens. In other words, when contact of a T cell with other cells results in the production of only one of the two necessary signals, the T cell is not activated and no adaptive immune response occurs.

Thus, T cells can be recruited or activated by binding the multispecific antibody to an activating component (e.g., CD 3) in a T Cell Receptor (TCR) complex on a T cell that has not yet matured. The simultaneous binding of a multispecific antibody to two cell types (e.g., tumor cells and T cells) can establish a temporal association between target cells and T cells, resulting in activation of Cytotoxic T Lymphocytes (CTLs) that attack the targeted cancer cells.

However, many multispecific forms of molecules, such as BiTE, diabodies, DARTs, and tandabs, all use single-chain forms, with different variable domains linked by peptide linkers to achieve multispecific. Since these forms do not contain an Fc region, they generally have a very short in vivo half-life and are physically unstable. On the other hand, the molecular configuration types of the currently selected multispecific antibodies are still single, and the design and the application of multispecific molecules are limited to a certain extent. In addition, tumor cells can escape the multi-specific antibody targeting CD3 through antigen loss, and development of the multi-specific antibody targeting various tumor antigens is helpful for improving the drug effect.

Thus, there remains a need to design improved multispecific antibodies to meet the requirements of efficacy, safety, and design flexibility.

Disclosure of Invention

The present application provides a multispecific antibody comprising an antibody moiety a capable of specifically binding to a first target; and 2 containing formulae (I)Sugar chain portions of the structures shown; and the multispecific antibody has a structure represented by formula (II): Wherein: the a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage; the Fuc comprises a structure Fuco-L-AB2, wherein the structure of Fuco is shown as a formula (III): (III) AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc being linked to said GlcNAc by an α -1,3 glycosidic bond; at least one of the first target and the second target is CD3; and the position of the amino acid N297 is determined according to the EU index numbering in Kabat.

In certain embodiments of the multispecific antibodies of the present application, the first target is not the same as the second target.

In certain embodiments of the multispecific antibodies of the present application, each of the AB1 and the AB2 is independently an antigen-binding fragment of an antibody.

In certain embodiments of the multispecific antibodies of the present application, the antigen-binding fragment is a Fab, F (ab) ₂,F(ab'),F(ab') ₂, scFv, affibody (affibody), and/or single domain antibody.

In certain embodiments of the multispecific antibodies of the present application, the first target is a tumor-associated antigen and the second target is CD3. In certain embodiments of the multispecific antibodies of the present application, the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.

In certain embodiments of the multispecific antibodies of the present application, the a is an IgG antibody.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antigen-binding portion of an antibody selected from the group consisting of: trastuzumab and dulcis You Shan antibody.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody heavy chain CDR3 (HCDR 3), and the HCDR3 comprises the amino acid sequence of SEQ ID NO:24 and 32 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody heavy chain CDR2 (HCDR 2), and the HCDR2 comprises the amino acid sequence of SEQ ID NO:23 and 31 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody heavy chain CDR1 (HCDR 1), and the HCDR1 comprises the amino acid sequence of SEQ ID NO:22 and 30 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody light chain CDR3 (LCDR 3), and the LCDR3 comprises the amino acid sequence of SEQ ID NO:21 and 29 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody light chain CDR2 (LCDR 2), and the LCDR2 comprises the amino acid sequence of SEQ ID NO:20 and 28 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody light chain CDR1 (LCDR 1), and the LCDR1 comprises the amino acid sequence of SEQ ID NO:19 and 27 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody heavy chain variable region VH, and the VH comprises SEQ ID NO:26 and 34 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the present application, the AB1 comprises an antibody light chain variable region VL, and the VL comprises SEQ ID NO:25 and 33 the amino acid sequence shown.

In certain embodiments of the multispecific antibodies of the application, the a is trastuzumab or rivarox You Shan antibody.

In certain embodiments of the multispecific antibodies of the present application, the AB2 comprises a CD3 antigen-binding portion of an antibody selected from the group consisting of: OKT3, M291, YTH12.5, bob and Cartuzumab.

In certain embodiments of the multispecific antibodies of the present application, the AB2 comprises SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In certain embodiments of the multispecific antibodies of the present application, the first target is CD3 and the second target is a tumor-associated antigen. In certain embodiments of the multispecific antibodies of the present application, the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.

In certain embodiments of the multispecific antibodies of the present application, L has the structure J- (L ₁) _n-X ₁Y ₁-(L ₁') _n') in Fuco-L-AB2, wherein L ₁ is a first linker, n is 0 or 1, L ₁ 'is a second linker, n' is 0 or 1, J is a linker directly attached to Fuco, X ₁Y ₁ is a residue group after the ligation reaction of group X ₁ with group Y ₁, wherein group X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction, and Y ₁ comprises a functional group capable of undergoing a bioorthogonal ligation reaction with X ₁.

In certain embodiments of the multispecific antibodies of the present application, the X ₁ comprises a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof.

In certain embodiments of the multispecific antibodies of the present application, the X ₁ comprises a functional group selected from the group consisting of: Wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.

In certain embodiments of the multispecific antibodies of the present application, the X ₁ comprises a functional group selected from the group consisting of:

in certain embodiments of the multispecific antibodies of the present application, the X ₁ comprises

In certain embodiments of the multispecific antibodies of the present application, the Y ₁ comprises a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof.

In certain embodiments of the multispecific antibodies of the present application, the Y ₁ comprises a functional group selected from the group consisting of: Wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.

In certain embodiments of the multispecific antibodies of the present application, the X ₁ and the Y ₁ comprise a set of structures selected from the group consisting of:

a) X ₁ includes And Y ₁ includes

B) X ₁ includesAnd Y ₁ includes

C) X ₁ includesAnd Y ₁ includes

D) X ₁ includesAnd Y ₁ includesAnd

E) X ₁ includesAnd Y ₁ includesWherein R ₁ and R ₂ are as described in the foregoing

Defined as follows.

In certain embodiments of the multispecific antibodies of the present application, the X ₁Y ₁ comprises a structure selected from the group consisting of:

In certain embodiments of the multispecific antibodies of the present application, the J is Wherein said Rf is-CH ₂ -, -NH-or-O-, the left end of the J-structure is directly connected to the Fuco.

In certain embodiments of the multispecific antibodies of the present application, the J isThe left end of the structure is directly connected to Fuco.

In certain embodiments of the multispecific antibodies of the present application, each of said L ₁ and said L ₁' is independently selected from the group consisting of: c ₃-C ₂₀₀ subunit, C ₁-C ₂₀₀ alkylene, C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, Derivatives thereof and any combination thereof, wherein said subunit, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene is optionally substituted with one or more Rs ¹ and/or is optionally interrupted by one or more Rs ², wherein each of said Rs ¹ is independently selected from the group consisting of: halogen, halogen, -OH, -NH ₂ and-COOH, each of said Rs ² being independently selected from: -O-, -S-,Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

In certain embodiments of the multispecific antibodies of the present application, the L ₁ is selected from the group consisting of: Wherein each s1 is independently an integer from 1 to 50, each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but consecutive adjacent-CH ₂ -is not simultaneously replaced by-O-, the left end of the structure is attached to said J and the right end of the structure is attached to said X ₁.

In certain embodiments of the multispecific antibodies of the present application, the L ₁ is selected from the group consisting of: And the right end of the structure is connected with the X ₁.

In certain embodiments of the multispecific antibodies of the present application, the L ₁' is selected from: Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure being attached to said AB2 and the left end of the structure being attached to said Y ₁.

In certain embodiments of the multispecific antibodies of the present application, the L ₁' is selected from: The right end of the structure is connected with the AB2, and the left end of the structure is connected with the Y ₁.

In certain embodiments of the multispecific antibodies of the present application, the GalX is galactose.

In certain embodiments of the multispecific antibodies of the present application, the GalX is a substituted galactose and one or more hydroxyl groups at the C2, C3, C4, and/or C6 positions in the galactose are substituted.

In certain embodiments of the multispecific antibodies of the present application, the GalX is a substituted galactose and the hydroxyl group at the C2 position in the galactose is substituted.

In certain embodiments of the multispecific antibodies of the present application, the GalX is a monosaccharide.

In certain embodiments of the multispecific antibodies of the present application, the GalX is substituted with the substituent Rg ¹, and Rg ¹ is a group selected from the group consisting of: hydrogen, halogen, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, C ₂-C ₂₄ alkenyl, C ₅-C ₂₄ cycloalkenyl, C ₂-C ₂₄ alkynyl, C ₆-C ₂₄ cycloalkynyl, C ₃-C ₂₄ (hetero) aryl, C ₃-C ₂₄ alkyl (hetero) aryl, and C ₃-C ₂₄ (hetero) arylalkyl; wherein the alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, (hetero) aryl, alkyl (hetero) aryl, and/or (hetero) arylalkyl are each independently optionally substituted with one or more substituents Rs ⁴, and/or are each independently optionally interrupted by one or more substituents Rs ⁵; wherein each of said Rs ⁴ is independently selected from the group consisting of: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN; each of said Rs ⁵ is independently selected from the group consisting of: -O-, -S-,Wherein, rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

In certain embodiments of the multispecific antibodies of the present application, the GalX is substituted Substitution, wherein: t is 0 or 1; rg ² is a group selected from the group consisting of: c ₁-C ₂₄ Alkylene, C ₃-C ₂₄ cycloalkylene, C ₂-C ₂₄ alkenylene, C ₅-C ₂₄ Cycloalkenylene, C ₂-C ₂₄ alkynylene, C ₆-C ₂₄ Cycloalkynylene, C ₃-C ₂₄ (hetero) arylene, C ₃-C ₂₄ alkyl (hetero) arylene and C ₃-C ₂₄ (hetero) arylalkylene, wherein the alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, alkyl (hetero) arylene and/or (hetero) arylalkylene are each independently optionally substituted with one or more substituents Rs ⁴, And/or each independently optionally interrupted by one or more substituents Rs ⁵, said Rg ³ being selected from: hydrogen, halogen, -OH, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, c ₂-C ₂₄ alkynyl, C ₅-C ₂₄ cycloalkynyl and C ₂-C ₂₄ (hetero) aryl, wherein the alkyl, cycloalkyl, alkynyl, cycloalkynyl and/or (hetero) aryl are each independently optionally substituted with one or more Rs ⁴, Wherein each of said Rs ⁵ is independently selected from: -O-, -S-,The Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl, and each of said Rs ⁴ is independently selected from the group consisting of: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN.

In certain embodiments of the multispecific antibodies of the present application, the GalX is selected from the group consisting of:

in certain embodiments of the multispecific antibodies of the present application, b is 0.

In another aspect, the application provides a multispecific antibody comprising: an antibody moiety a capable of specifically binding to a first target; and 2 comprising formula (IV)Sugar chain portions of the structures shown; and the multispecific antibody has a structure represented by formula (V):

Wherein: the a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; fuc comprises structure Fuco-L-AB2, wherein Fuco has a structure shown in formula (III): AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc is linked to said GlcNAc by an α -1,3 glycosidic bond; galX is substituted galactose, said GalX is linked to said GlcNAc by a β -1,4 glycosidic bond, and said GalX comprises a third antigen binding portion AB3 capable of specifically binding to a third target; at least one of the first target, the second target and the third target is CD3; and the position of the amino acid N297 is determined according to the EU index numbering in Kabat.

In certain embodiments of the multispecific antibodies of the present application, the GalX has the structure GalX ₂Y ₂-(L ₂') _m -AB3 wherein GalX ₂Y ₂ is the residue after the ligation of group GalX ₂ with group Y ₂, wherein the GalX ₂ comprises X _2, the X ₂ comprises a functional group capable of participating in a bioorthogonal ligation reaction, and the Y ₂ comprises a functional group capable of undergoing a bioorthogonal ligation reaction with the X ₂; and L ₂' is a linker, m is 0 or 1.

In certain embodiments of the multispecific antibodies of the present application, the GalX ₂ is galactose with one or more hydroxyl groups at the C2, C3, C4 and/or C6 positions substituted.

In certain embodiments of the multispecific antibodies of the present application, the GalX ₂ is galactose with a hydroxy group at the C2 position substituted.

In certain embodiments of the multispecific antibodies of the present application, the GalX ₂ is a monosaccharide.

In certain embodiments of the multispecific antibodies of the present application, X ₂ in GalX ₂ comprises

In certain embodiments of the multispecific antibodies of the present application, the GalX ₂ has the structure

In certain embodiments of the multispecific antibodies of the present application, the Y ₂ comprises

In certain embodiments of the multispecific antibodies of the present application, the X ₂Y ₂ comprises a structure selected from the group consisting of:

In certain embodiments of the multispecific antibodies of the present application, the L ₂' is selected from: c ₃-C ₂₀₀ polypeptide subunit, C ₁-C ₂₀₀ alkylene, C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenyl, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, derivatives thereof, and any combination thereof, wherein the polypeptide subunit, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene is optionally substituted with one or more Rs ¹ and/or is optionally interrupted with one or more Rs ², wherein each of the Rs ¹ is independently selected from the group consisting of: halogen, -OH, -NH ₂, and-COOH, each of said Rs ² being independently selected from: -O-, -S-, Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

In certain embodiments of the multispecific antibodies of the present application, the L ₂' is selected from:

Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure is attached to said AB3, and the left end of the structure is attached to said Y ₂.

In certain embodiments of the multispecific antibodies of the present application, the L ₂' is

In certain embodiments of the multispecific antibodies of the present application, the X ₁ comprisesWherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.

In certain embodiments of the multispecific antibodies of the present application, the Y ₁ comprises

In certain embodiments of the multispecific antibodies of the present application, the J is Wherein said Rf is-CH ₂ -, -NH-or-O-, wherein the left end of the J-structure is connected to the Fuco.

In certain embodiments of the multispecific antibodies of the present application, the J isWherein the left end of the J-structure is connected with the Fuco.

In certain embodiments of the multispecific antibodies of the present application, the L ₁ is selected from the group consisting of: Wherein s1 is an integer from 1 to 50, each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but consecutive adjacent-CH ₂ -is not simultaneously replaced by-O-, the left end of the structure is attached to said J, and the right end of the structure is attached to said X ₁.

In certain embodiments of the multispecific antibodies of the present application, the L ₁ is selected from the group consisting of: and the right end of the structure is connected with the X ₁, and the left end is connected with the J.

In certain embodiments of the multispecific antibodies of the present application, the L ₁' is selected from:

Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure being attached to said AB2 and the left end of the structure being attached to said Y ₁.

In certain embodiments of the multispecific antibodies of the present application, the L ₁' isThe left end of the structure is connected with the Y ₁, and the right end is connected with the AB 2.

In certain embodiments of the multispecific antibodies of the present application, the first target, the second target, and the third target are each different from one another.

In certain embodiments of the multispecific antibodies of the present application, each of the AB1, the AB2, and the AB3 is independently an antigen-binding fragment of an antibody.

In certain embodiments of the multispecific antibodies of the present application, the first target is a tumor-associated antigen, the second target is CD3, and the third target is a tumor-associated antigen. In certain embodiments of the multispecific antibodies of the present application, the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.

In certain embodiments of the multispecific antibodies of the present application, the first target is Her2, the second target is CD3, and the third target is PD-L1.

In certain embodiments of the multispecific antibodies of the present application, the first target is PD-L1, the second target is CD3, and the third target is Her2.

In certain embodiments of the multispecific antibodies of the application, the a is rivarox You Shan antibody or trastuzumab.

In certain embodiments of the multispecific antibodies of the present application, the AB2 comprises an antigen-binding portion of an antibody selected from the group consisting of: OKT3, M291, YTH12.5, bob and Cartuzumab.

In certain embodiments of the multispecific antibodies of the present application, the AB3 comprises an antigen-binding portion of an antibody selected from the group consisting of: duvalli You Shan antibody, atelizumab, en Wo Lishan antibody, trastuzumab, pertuzumab and ZHer2:342.

In certain embodiments of the multispecific antibodies of the present application, the AB3 comprises SEQ ID NO:10 and 12 the amino acid sequence shown.

In another aspect, the application provides a method of making a multispecific antibody of the application.

In certain embodiments, the method comprises: i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety a in the presence of a catalyst, wherein said sugar chain comprises the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI) to obtain a protein having the structure of formula (VII)Wherein: said a comprises said AB1 and said Fc region; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage; q is a ribonucleotide diphosphate; and Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure of formula (III): The J is an adapter directly connected with Fuco, and the J is connected with the left end of the formula (III); the X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction; the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and ii) reacting the protein of formula (VII) with Y ₁-(L ₁') _n' -AB2 to obtain the multispecific antibody of the application; wherein A, galX, X ₁,Y ₁,J,L ₁,L ₁ ', n, n', AB1 and AB2 are as defined in the foregoing description of the application.

In certain embodiments, the method further comprises the steps of: treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI), the structure of the protein being as shown in formula (VIII): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage.

In certain embodiments, the method further comprises the steps of: treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI), the structure of the protein being as shown in formula (VIII): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage.

In certain embodiments, the method comprises: i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety a in the presence of a catalyst, wherein said sugar chain comprises the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX) to obtain a protein of formula (X)Wherein: said a comprises said AB1 and said Fc region; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX ₂ is substituted galactose and GalX ₂ comprises X ₂,X ₂ comprises a functional group capable of participating in a bioorthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage; q is a ribonucleotide diphosphate; and Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure of formula (III): The J is an adapter directly connected with Fuco, and the J is connected at the left end of the formula (III); the X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction; the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and ii) reacting the protein of formula (X) with Y ₁-(L ₁') _n' -AB2 and Y ₂-(L ₂') _m -AB3 to obtain the multispecific antibody of the application; wherein A,X ₁,Y ₁,J,L ₁,L ₁',n,n',AB1,AB2,X ₂,GalX ₂,AB3,L ₂',Y ₂ and m are as defined in the present application.

In certain embodiments, the method further comprises the steps of: treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein; contacting the treated protein with UDP-GalX ₂ in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and GalX ₂ is a substituted galactose and which comprises X ₂,X ₂ comprising a functional group capable of participating in a bioorthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage.

In certain embodiments, the method further comprises the steps of: treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and GalX ₂ is a substituted galactose and which comprises X ₂,X ₂ comprising a functional group capable of participating in a bioorthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage.

In certain embodiments of the methods of the application, the Q is Guanosine Diphosphate (GDP), uridine Diphosphate (UDP), and/or Cytidine Diphosphate (CDP).

In certain embodiments of the methods of the present application, the Q-Fuc is GDP-Fuc.

In certain embodiments of the methods of the present application, the Q-Fuc' is selected from the following structures:

in certain embodiments of the methods of the application, the catalyst comprises a fucosyltransferase.

In certain embodiments of the methods of the application, the fucosyltransferase is an α1, 3-fucosyltransferase or a functional variant or fragment thereof.

In certain embodiments of the methods of the application, the fucosyltransferase is derived from a bacterium.

In certain embodiments of the methods of the application, the fucosyltransferase is derived from helicobacter pylori Helicobacter pylori.

In certain embodiments of the methods of the application, the fucosyltransferase is derived from helicobacter pylori Helicobacter pylori 26695.

In certain embodiments of the methods of the application, the fucosyltransferase is helicobacter pylori alpha-1, 3 fucosyltransferase from GenBank accession AAD 07710.1.

In certain embodiments of the methods of the application, the fucosyltransferase comprises a catalytically active region comprising the amino acid sequence set forth in SEQ ID NO. 1 and at least one heptad repeat comprising the amino acid sequence set forth in SEQ ID NO. 2.

In certain embodiments of the methods of the application, the fucosyltransferase comprises a catalytically active region comprising the amino acid sequence set forth in SEQ ID NO. 1 and 1-10 heptad repeat fragments comprising the amino acid sequence set forth in SEQ ID NO. 2.

In certain embodiments of the methods of the application, the fucosyltransferase is an alpha-1, 3-fucosyltransferase or a functional variant or fragment thereof, and which comprises the amino acid sequence shown in SEQ ID NO. 3.

In certain embodiments of the methods of the application, the catalyst comprises a fucosyltransferase and a tag sequence of the application.

In certain embodiments of the methods of the application, the catalyst comprises an amino acid sequence set forth in any one of SEQ ID NOs 3 and 4.

In another aspect, the application provides a composition comprising a multispecific antibody according to the application.

In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In another aspect, the application provides a method of preventing, alleviating and/or treating a disease or condition comprising administering to a subject in need thereof a multispecific antibody of the application, and/or a composition of the application.

In a further aspect, the application provides the use of a multispecific antibody according to the application and/or a composition according to the application for the manufacture of a medicament for the prevention, alleviation and/or treatment of a disease or condition.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

FIGS. 1A-1B are schematic diagrams showing a method for preparing a multispecific antibody of the present application, wherein Is N-acetylglucosamine which is prepared from N-acetylglucosamine,Is a compound of alpha 1,6 fucose,For antibody moiety a, fuc' is Fuco-J- (L ₁) _n-X ₁), fuc is J- (L ₁) _n-X ₁Y ₁-(L ₁') _n' -AB2, galX is GalX ₂Y ₂-(L ₂') _n "-AB3, AB2 is an antigen binding moiety targeting CD3, and AB3 is a third antigen binding moiety.

Fig. 2 shows some exemplary structures of Q-Fuc' in the present application.

Figures 3A-3E show the preparation and characterization of exemplary CD 3-targeting multispecific antibodies of the present application.

FIGS. 4A-4C show the binding affinities of exemplary CD 3-targeting multispecific antibodies of the present application.

FIGS. 5A-5B show in vitro killing activity of exemplary CD 3-targeting multispecific antibodies of the present application.

FIGS. 6A-6C show in vitro killing activity of exemplary CD 3-targeting multispecific antibodies of the present application.

Figures 7A-7C show antigen dependent T cell activation characterization of exemplary CD 3-targeting multispecific antibodies of the present application.

FIGS. 8A-8B show concentration-dependent T cell activation profiles of exemplary CD 3-targeting multispecific antibodies of the present application.

FIG. 9 shows a comparison of the efficiency of the reaction of antibody conjugates containing different linkers with Y ₁-(L ₁') _n' -AB2 to produce a multispecific antibody of the present application.

FIG. 10 shows the molecular structure of some of the compounds used in the present application.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "directly linked" generally means that the linking site does not comprise additional compounds (e.g., other sugar groups) or linkers. For example, a direct connection of one molecule or entity to another may mean that no additional molecules or entities exist between the two. For example, direct connection may refer to one portion being connected to another portion without any intervening portions or joints. For example, direct attachment of GlcNAc to an amino acid residue of a protein (e.g., an antibody) generally refers to attachment of GlcNAc to the amino acid residue of the protein via a covalent bond, such as attachment of an atom on the side chain of an amino acid (e.g., an asparagine amino acid) of the protein via an N-glycosidic bond to an amide nitrogen bond. In the present application, when GlcNAc is "indirectly linked" to an amino acid of a protein,

At least one monosaccharide moiety is typically present between GlcNAc and an amino acid of the protein.

In the present application, the term "comprising" is generally meant to include what follows from this definition, but does not exclude other contents not specifically mentioned, i.e. it is generally understood that an open definition is provided in the present application.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The compounds disclosed in the present specification and claims may contain one or more asymmetric centers and different diastereomers and/or enantiomers of the compounds may be present. Unless otherwise indicated, the description of any compound in the present specification and claims is intended to include all diastereomers and mixtures thereof. Furthermore, unless otherwise indicated, the description of any compound in the present specification and claims is intended to include individual enantiomers as well as any mixtures, racemates or other forms of enantiomers. While the structure of a compound is described as a particular enantiomer, it is to be understood that the application is not limited to that particular enantiomer.

The compounds may exist in different tautomeric forms. Unless otherwise indicated, the compounds of the present application are meant to include all tautomeric forms. When the structure of a compound is described as a particular tautomer, it is to be understood that the application is not limited to that particular tautomer.

Unsubstituted alkyl groups have the general formula C _nH _2n+1 and may be linear or branched. Unsubstituted alkyl groups may also contain cyclic moieties and thus have the corresponding general formula C _nH _2n-1. Optionally, the alkyl is substituted with one or more substituents further specified in the present application. Examples of alkyl groups include, for example, methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, and the like.

Aryl groups may contain six to twelve carbon atoms and may contain both monocyclic and bicyclic structures. Optionally, the aryl group may be substituted with one or more substituents further specified herein. Examples of aryl groups are phenyl and naphthyl.

Arylalkyl and alkylaryl groups can contain at least seven carbon atoms and can contain both monocyclic and bicyclic structures. Optionally, the arylalkyl and alkylaryl groups can be substituted with one or more substituents further specified in the present application. Arylalkyl may be, for example, benzyl. Alkylaryl groups can be, for example, 4-tert-butylphenyl.

In the present application, the term "heteroaryl" generally refers to an aromatic monocyclic or polycyclic group of 5 to 12 atoms having at least one aromatic ring containing one, two or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or both ring carbon atoms of the heteroaryl group may be substituted with a carbonyl group.

Heteroarylalkyl and alkylheteroaryl contain at least three carbon atoms (i.e., at least C ₃) and may contain both monocyclic and bicyclic structures. Optionally, heteroaryl groups may be substituted with one or more substituents further specified herein.

When aryl is denoted as (hetero) aryl, the notation is meant to include both aryl and heteroaryl. Similarly, alkyl (hetero) aryl is meant to include alkylaryl and alkylheteroaryl, (hetero) arylalkyl is meant to include arylalkyl and heteroarylalkyl. Thus, C ₂-C ₂₄ (hetero) aryl is understood to include C ₂-C ₂₄ heteroaryl and C ₆-C ₂₄ aryl. Similarly, C ₃-C ₂₄ alkyl (hetero) aryl is meant to include C ₇-C ₂₄ alkylaryl and C ₃-C ₂₄ alkylheteroaryl, and C ₃-C ₂₄ (hetero) arylalkyl is meant to include C ₇-C ₂₄ arylalkyl and C ₃-C ₂₄ heteroarylalkyl.

Unless otherwise indicated, alkyl, alkenyl, alkene, alkyne, (hetero) aryl, (hetero) arylalkyl, and alkyl (hetero) aryl groups may be substituted with one or more substituents selected from the group consisting of: c ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₂-C ₁₂ alkynyl, C ₃-C ₁₂ cycloalkyl, C ₅-C ₁₂ cycloalkenyl, C ₇-C ₁₂ cycloalkynyl, C ₁-C ₁₂ alkoxy, C ₂-C ₁₂ alkenyloxy, C ₂-C ₁₂ alkynyloxy, C ₃-C ₁₂ cycloalkoxy, halogen, amino, oxo, wherein alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted or interrupted by one or more heteroatoms selected from O, N and S.

In the present application, the term "alkynyl" generally includes carbon-carbon triple bonds. Unsubstituted alkynyl groups containing one triple bond have the general formula C _nH _2n-3. Terminal alkynyl is alkynyl in which the triple bond is located at the terminal position of the carbon chain. Optionally, the alkynyl group is substituted with one or more substituents further specified in the application, and/or is interrupted or interrupted by a heteroatom selected from oxygen, nitrogen and sulfur. Examples of alkynyl groups include, for example, ethynyl, propynyl, butynyl, octynyl, and the like.

In the present application, the term "cycloalkynyl" generally refers to an unsaturated, mono-, bi-, or tricyclic hydrocarbon ring having the specified number of carbon atoms and one or more carbon-carbon triple bonds. For example, C ₇-C ₁₂ cycloalkynyl refers to cycloalkynyl groups having 7-12 carbon atoms. In certain embodiments, the cycloalkynyl group has one carbon-carbon triple bond in the ring. In other embodiments, cycloalkynyl groups have more than one carbon-carbon triple bond in the ring. Representative examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, and the like.

In the present application, the term "heterocyclylalkynyl" is generally a cycloalkynyl group interrupted or interrupted by a heteroatom selected from oxygen, nitrogen and sulfur. Optionally, the heterocycloalkynyl is substituted with one or more substituents further specified herein. An example of a heterocycloalkynyl group is azacyclooctynyl (azacyclooctynyl)).

When alkyl, (hetero) aryl, alkyl (hetero) aryl, (hetero) arylalkyl, (hetero) cycloalkynyl are optionally substituted, the groups are independently optionally substituted with one or more substituents independently selected from the group consisting of: c ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₂-C ₁₂ alkynyl, C ₃-C ₁₂ cycloalkyl, C ₁-C ₁₂ alkoxy, C ₂-C ₁₂ alkenyloxy, C ₂-C ₁₂ alkynyloxy, C ₃-C ₁₂ cycloalkoxy, halogen, amino, oxo, and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy groups are optionally substituted, the alkyl, alkoxy, cycloalkyl, and cycloalkoxy groups being optionally interrupted or interrupted by one or more heteroatoms selected from O, N and S.

In the present application, the term "subunit" generally refers to a polypeptide group comprising two or more amino acid residues, which may be formed, for example, by condensing two or more amino acids, which may be linked to other structures by amino or carboxyl groups at both ends. For exampleIs a subunit polypeptide, the left end of which is connected with other structures through carboxyl groups, and the right end of which is connected with other parts through amino groups.

In the present application, the term "sugar" generally refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" refers in the present application generally to a derivative of a monosaccharide, i.e. a monosaccharide comprising substituents and/or functional groups. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcNH ₂), galactosamine (GalNH ₂), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (also known as N-acetylneuraminic acid (NeuNAc)), and N-acetylmuramic acid (murmac), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives also include the compound of the present application denoted GalX, which may be galactose or a galactose derivative. Examples of sugar derivatives also include the compounds of the present application denoted Fuc, which may be fucose derivatives.

In the present application, the term "nucleotide" generally refers to a molecule consisting of a nucleobase, a pentose (ribose or 2-deoxyribose) and one, two or three phosphate groups. In the absence of phosphate groups, nucleobases and sugars constitute nucleosides. Thus, a nucleotide is also referred to as a nucleoside monophosphate, nucleoside diphosphate or nucleoside triphosphate. The nucleobase may be adenine, guanine, cytosine, uracil or thymine. Examples of the nucleotide include ribonucleotides diphosphate, such as Uridine Diphosphate (UDP), guanosine Diphosphate (GDP), thymidine Diphosphate (TDP), cytidine Diphosphate (CDP), and Cytidine Monophosphate (CMP).

In the present application, the term "protein" generally refers to a polypeptide comprising about 10 or more amino acids. Proteins may include natural amino acids, but may also include unnatural amino acids.

In the present application, the term "glycoprotein" is used in its usual scientific sense and refers to a protein containing one or more mono-or oligosaccharide chains ("sugar chains") covalently bonded to the protein. The sugar chain may be linked to a hydroxyl group of the protein (O-linked-sugar chain), for example to a hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline; or to an amido group of a protein (N-glycoprotein), such as asparagine or arginine; or to a carbon of a protein (C-glycoprotein), such as tryptophan. The glycoprotein may comprise more than one sugar chain, may comprise a combination of one or more monosaccharide and one or more oligosaccharide sugar chains, and may comprise a combination of N-linked, O-linked and C-linked sugar chains. It is estimated that more than 50% of all proteins have some glycosylated form and thus can be described as glycoproteins.

In the present application, the term "sugar chain" generally refers to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term sugar chain refers to the carbohydrate portion of a glycoprotein. The sugar chain may be attached to the protein via the C1 carbon of one sugar, which may be free of other substituents (monosaccharides) or may be further substituted on one or more of its hydroxyl groups (oligosaccharides). Naturally occurring glycans generally contain 1 to about 10 sugar moieties. However, when longer glycans are attached to the protein, the glycans are also considered sugar chains in the present application.

The sugar chain of the glycoprotein may be a monosaccharide. Typically, the monosaccharide sugar chain of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently bonded to the protein. The sugar chain may be an oligosaccharide.

The oligosaccharide chains of glycoproteins may be linear or branched. Among oligosaccharides, the sugar directly linked to the protein is called core sugar. Among oligosaccharides, a sugar that is not directly linked to a protein and is linked to at least two other sugars is called an internal sugar. In oligosaccharides, a sugar that is not directly linked to the protein but is linked to a single other sugar, i.e. is not linked to other sugars at one or more of its other hydroxyl groups, is referred to as a terminal sugar. For the avoidance of doubt, there may be multiple terminal saccharides in the oligosaccharide of the glycoprotein, but typically only one core saccharide is present.

The sugar chain may be an O-linked sugar chain, an N-linked sugar chain or a C-linked sugar chain. In the O-linked sugar chains, the monosaccharide or oligosaccharide sugar chains are bonded to O atoms in the amino acids of the proteins, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In the N-linked sugar chains, the monosaccharide or oligosaccharide sugar chains are bonded to the protein via the N atom in the amino acid of the protein, typically via an amide nitrogen on the asparagine (Asn) or arginine (Arg) side chain. In the C-linked sugar chains, the monosaccharide or oligosaccharide sugar chains are bonded to the C atom in the amino acid of the protein, typically to the C atom of tryptophan (Trp).

The end of the oligosaccharide directly attached to the protein is called the reducing end of the sugar chain. The other end of the oligosaccharide is called the non-reducing end of the sugar chain.

For O-linked sugar chains, there are many different chains. Naturally occurring O-linked sugar chains typically have serine or threonine-linked alpha-O-GalNAc moieties, which may also be substituted with galactose, sialic acid and/or fucose. The hydroxylated amino acid carrying a sugar chain substituent may be part of any amino acid sequence in a protein.

For N-linked sugar chains, there are many different chains. Naturally occurring N-linked sugar chains typically have an asparagine-linked β -N-GlcNAc moiety which in turn is further linked to β -GlcNAc at its C4, then to β -Man at its C4, then to α -Man at its C3 and C6, forming pentasaccharide Man ₃GlcNAc ₂. The core GlcNAc moiety can also be linked to α -Fuc at its C6. Pentasaccharide Man ₃GlcNAc ₂ is a common oligosaccharide framework of most N-linked glycoproteins and may be further linked to other saccharides, including but not limited to Man, glcNAc, gal and sialic acid. Asparagine that is modified with a sugar chain in the side chain is typically part of the sequence Asn-X-Ser/Thr, where X is any amino acid other than proline and Ser/Thr is serine or threonine.

In the present application, the term "antibody" generally refers to a protein or antigen-binding fragment thereof produced by the immune system that is capable of recognizing and binding to a specific antigen. Antibodies are one example of glycoproteins. The term antibody is used in its broadest sense in the present application and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., multispecific antibodies), antibody fragments, and double-and single-chain antibodies. Antibodies also include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to a cancer antigen. In the present application. The term "antibody" generally includes intact antibodies, but also antibody fragments, such as antibody Fab fragments, (Fab') ₂, fv fragments or Fc fragments, scFv-Fc fragments, minibodies (minibodies), single domain antibodies (also known as nanobodies), multispecific antibodies (diabodies), affibodies (affibodies) or scFv from cleaved antibodies. Furthermore, the term "antibody" also includes engineered or genetically engineered antibodies and/or derivatives of antibodies. In some embodiments, antibodies are referred to as immunoglobulins and include various classes and isotypes, such as IgA (IgA 1 and IgA 2), igD, igE, igM, and IgG (IgG 1, igG3, and IgG 4), and the like. In the present application, the term "antibody" may include polyclonal antibodies and monoclonal antibodies and functional fragments thereof. Antibodies include modified or derivatized antibody variants that retain the ability to specifically bind an epitope. Antibodies are capable of selectively binding to a target antigen or epitope. In the present application, the antibody may be derived from any source, such as mice or humans, including chimeric antibodies thereof, e.g., the antibody may be humanized.

In the present application, the term "humanized antibody" generally refers to an antibody that contains some or all of the CDRs from a non-human animal antibody, and the framework and constant regions of the antibody contain amino acid residues from the human antibody sequence. Antibodies, antibody fragments, and engineered antibodies can all be obtained by methods known in the art.

In the present application, the term "treatment" generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure of the disease and/or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (particularly a human) and includes preventing the disease from occurring in a subject who may be susceptible to the disease but has not been diagnosed with the disease; inhibiting the disease, i.e., arresting its development; remit the disease, i.e. cause regression of the disease.

In the present application, the term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises 2 constant domains, namely a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain and/or a hinge region. In the present application, the term "Fc region" includes any polypeptide (or nucleic acid encoding such polypeptide), regardless of the manner in which it is produced.

In the present application, the term "GlcNAc" or "N-acetylglucosamine" may be used interchangeably and generally refers to an amide derivative of the monosaccharide glucose.

Glycosylation generally refers to the reaction of a carbohydrate, i.e., a glycosyl donor, with a hydroxyl or other functional group of another molecule (glycosyl acceptor). In some embodiments, glycosylation refers primarily to the enzymatic process of attaching glycans to proteins or other organic molecules. Glycosylation in proteins may be modified in terms of glycosyl linkages, glycosyl structure, glycosyl composition, and/or glycosyl length. Glycosylation can include N-linked glycosylation, O-linked glycosylation, phosphoserine glycosylation, C-mannosylation, GPI anchor formation (glypiation) and/or chemical glycosylation and correspondingly, the glycosylated oligosaccharide of the protein can be an N-linked oligosaccharide, an O-linked oligosaccharide, a phosphoserine oligosaccharide, a C-mannosylated oligosaccharide, a glycosylated oligosaccharide and/or a chemical oligosaccharide.

In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for mutations and/or post-translational modifications (e.g., isomerization, amidation) that may occur in minor amounts, which may occur naturally.

In the present application, the term "IgG" generally refers to an immunoglobulin or a functional derivative thereof. Those skilled in the art will appreciate that immunoglobulin heavy chains are divided into gamma, mu, alpha, delta, and epsilon (gamma, mu, alpha, delta, epsilon) and some subtypes thereof (e.g., gamma 1-gamma 4 or alpha 1-alpha 2). It is the nature of this chain that determines the "isotype" of the antibody, igG, igM, igD, igA or IgE, respectively. Immunoglobulin subclasses (subclasses), e.g., igG1, igG2, igG3, igG4, igA1, igA2, etc., have been well characterized and are known to confer functional specificity. A typical feature of human IgG is the glycosylation at position Asn297 (numbering according to the EU of Kabat) of the heavy chain CH2 region of the Fc region.

In the present application, the terms "Asn297" or "N297" are used interchangeably and generally refer to the asparagine at position 297 (numbered according to EU numbering convention of Kabat) of the Fc region of an antibody. Asn297 may be linked to one or more oligosaccharides.

In the present application, the term "Fuc α1,3GlcNAc linkage" generally refers to a linkage between Fuco and GlcNAc of Fuc, such as C3 linking C1 of Fuco to GlcNAc.

In the present application, the term "GalX β1,4glcnac linkage" generally refers to a linkage between optionally substituted galactose GalX and GlcNAc, such as C4 which links C1 of GalX to GlcNAc.

In the present application, the term "functional group" generally refers to a group capable of reacting with another group. Functional groups may be used to attach reagents (e.g., reagents that are non-reactive or have low reactivity) to the multispecific antibodies of the present application. For example, the functional group may be a chemical group or a residue having chemical and/or enzymatic reactivity. In some embodiments, the functional group may be a group capable of reacting in a ligation reaction.

In the present application, the term "tag sequence" generally refers to another molecular entity (e.g., another stretch of amino acid sequences) that is integrated (e.g., linked) into a protein of interest (e.g., an antibody). For example, the tag sequence may be a detectable label, such as a radiolabeled amino acid or a biotinylated polypeptide, which is detectable by the labeled avidin (e.g., streptavidin with a fluorescent label or enzymatic activity, detectable by optical methods or colorimetry). For example, tag sequences may include, but are not limited to: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent tags (e.g., FITC, rhodamine, lanthanum phosphorescence), enzyme tags (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, preselected epitopes of a polypeptide that are recognized by a second receptor (e.g., leucine zipper complementary sequences, binding sites for a second antibody, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates. In certain embodiments, the tag sequence may be an affinity purification tag, such as a polyhistidine tag (His tag), arg tag, FLAG tag, 3xFlag tag, streptavidin tag, nanotag, SBP tag, c-myc tag, S tag, calmodulin binding peptide, cellulose binding domain, chitin binding domain, GST tag, and/or MBP tag. In certain embodiments, the tag sequence may be fused to the N-terminus and/or C-terminus of the protein of interest.

In the present application, the term "fucosyltransferase" generally refers to an enzyme or a functional fragment or variant thereof that can transfer L-fucose from a fucose donor substrate (e.g., guanosine diphosphate-fucose) to an acceptor substrate. The acceptor substrate may be another sugar, for example a sugar comprising GlcNAc-Gal (LacNAc), such as in the case of N-glycosylation or O-glycosylation. The term "fucosyltransferase" may include any functional fragment thereof, or catalytic domain thereof, as well as functional variants (e.g., mutants, isoforms) having a catalytically active domain. An example of a fucosyltransferase may be an alpha-1, 3 fucosyltransferase. The term "fucosyltransferase" may be derived from various species, such as mammals (e.g., humans), bacteria, nematodes or flukes. In some embodiments, the fucosyltransferase is an alpha-1, 3 fucosyltransferase of bacterial origin. In some embodiments, the fucosyltransferase is an alpha-1, 3 fucosyltransferase derived from helicobacter pylori. In some embodiments, the fucosyltransferase is an alpha-1, 3 fucosyltransferase derived from Helicobacter pylori 26695,26695. In some embodiments, wherein the fucosyltransferase is an a-1, 3 fucosyltransferase derived from GenBank accession No. AAD07710.1, genBank accession No. AAD07447.1, or GenBank accession No. AAB 81031.1. In some embodiments, wherein the fucosyltransferase is a fucosyltransferase having GenBank accession No. AAD07710.1, genBank accession No. AAD07447.1, or GenBank accession No. AAB 81031.1. In some embodiments, wherein the fucosyltransferase is a functional variant or fragment of an a-1, 3 fucosyltransferase having GenBank accession No. AAD07710.1, genBank accession No. AAD07447.1, or GenBank accession No. AAB 81031.1. In some embodiments, the fucosyltransferase comprises an amino acid sequence as set forth in GenBank accession No. AAD07710.1, or a functional variant or fragment thereof. For example, the fucosyltransferase may comprise an amino acid sequence set forth in GenBank accession No. AAD07710.1, or the fucosyltransferase may comprise an amino acid sequence having more than 80% (e.g., more than 83%, more than 88%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more) as set forth in GenBank accession No. AAD07710.1, or a functional variant or fragment thereof. For example, the fucosyltransferase may comprise a catalytically active region comprising the amino acid sequence shown in SEQ ID NO. 1 or comprising an amino acid sequence having at least 80% (e.g., more than 83%, more than 88%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO. 1, and at least one heptad repeat (e.g., 1-10) (SEQ ID NO. 2). For another example, the fucosyltransferase may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 3 and 4, or the fucosyltransferase may comprise an amino acid sequence having at least 80% (e.g., more than 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) homology or identity to an amino acid sequence set forth in SEQ ID NOs 3.

In the present application, the term "bioorthogonal ligation reaction" generally refers to a chemical reaction used to prepare the protein conjugates of the application. The reaction specificity occurs between a first functional group located at a specific position of the protein (e.g., on an oligosaccharide of the protein) and a second functional group corresponding to the functional moiety. The first functional group and the second functional group are commonly referred to as a pair of bio-orthogonal ligation reaction pairs. Typically, the first functional group located at a specific position of a protein is easily distinguishable from other groups of other parts of the protein. Typically, the second functional group will not react with other portions of the protein than the first functional group at a particular location. For example, an azide group is a functional group capable of participating in a bioorthogonal ligation reaction. The DBCO or BCN groups complementary thereto can react specifically with azido groups without cross-reacting with other groups on the protein. Many chemically reactive functional groups with suitable reactivity, chemoselectivity and/or biocompatibility may be used for the bio-orthogonal ligation reaction. The group capable of participating in a bioorthogonal ligation reaction may be selected from, but is not limited to, azido, terminal alkynyl, cyclic alkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cyclic alkenyl, keto, aldehyde, hydroxyamino, thiol, N-maleimido, and functional derivatives thereof (see Bertozzi C.R. et al Angew.Chem.Int.Ed.,2009,48,6974;Chin J.W. Et al ACS Chem.Biol.2014,9,16;van Del F.L. Et al Nat.Commun.,2014,5,5378;Prescher J.A. Et al Acc.Chem.Res.2018,51,1073;Devaraj N.K. Et al ACS Cent.Sci.2018,4,952;Liskamp R.M.J. Et al chem. Sci.,2020,11,9011). Functional derivatives herein may refer to modified functional groups that have similar or higher reactivity than unmodified functional groups.

In the present application, a "functional variant" of a parent polypeptide or protein generally has substantial or significant sequence identity or similarity to the parent polypeptide or protein, which functional variant retains at least a portion of the function of its parent polypeptide or protein. For example, a functional variant of an enzyme retains enzymatic activity to a similar extent, to the same extent, or to a higher extent than the parent enzyme. In the case of a parent polypeptide or protein, a functional variant may be, for example, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity to it over the amino acid sequence. In some cases, a functional variant may be a polypeptide that differs from a parent polypeptide or protein by at least one amino acid. For example, a functional variant may be obtained by adding, deleting or substituting one or more amino acids in the parent polypeptide or protein, e.g. 1-200, 1-100, 1-50, 1-40, 1-30,1-20,1-15,1-14,1-13,1-12,1-11,1-10,1-9,1-8,1-7,1-6,1-5, 1-4, 1-3 or 1-2 amino acids.

In the present application, a "functional fragment" of a parent polypeptide or protein generally refers to a peptide or polypeptide (including, but not limited to, enzymes) comprising at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least 250 consecutive amino acid residues, or at least 350 consecutive amino acid residues of the parent polypeptide or protein, wherein the functional fragment has at least a portion of its parent polypeptide or protein function. For example, a "functional fragment" of a parent enzyme retains enzymatic activity to a similar extent, to the same extent, or to a higher extent than the parent enzyme.

In the present application, the term "CD3" generally refers to a CD3 protein multi-subunit complex (e.g., a human CD3 protein multi-subunit complex). The CD3 protein multi-subunit complex may be composed of 6 distinct polypeptide chains. These polypeptide chains include the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), two CD3 epsilon chains (SwissProt P07766) and one CD3 zeta chain homodimer (SwissProt 20963), and the complex is associated with T cell receptor alpha and beta chains. Unless otherwise indicated, the term "CD3" includes any CD3 variant, isoform and species homolog that is naturally expressed by a cell (including T cells) or that can be expressed on a cell transfected with a gene or cDNA encoding those polypeptides.

In the present application, the term "comprising" also encompasses "being", "consisting of …. Composition", etc. For example, a protein comprising the amino acid sequence shown in SEQ ID NO. 1 is understood to describe not only a protein whose amino acid sequence includes, but is not limited to, the amino acid sequence shown in SEQ ID NO. 1. It is also understood that a protein body is also described, the amino acid sequence of which consists of SEQ ID NO. 1.

Detailed Description

Multispecific antibodies and methods of making same

In one aspect, the application provides a multispecific antibody comprising: an antibody moiety a capable of specifically binding to a first target; and 2 containing formulae (I)Sugar chain portions of the structures shown; and the multispecific antibody has a structure represented by formula (II): Wherein: the antibody moiety a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX is optionally substituted galactose (GalX is galactose or is substituted galactose), said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage; the Fuc comprises a structure Fuco-L-AB2, wherein the structure of Fuco is shown as a formula (III): AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc is linked to said GlcNAc by an α -1,3 glycosidic bond; at least one of the first target and the second target is CD3; and the position of the amino acid N297 is determined according to the EU index numbering in Kabat. For example, in some cases, the first target is CD3. In some cases, the second target may be CD3. In some cases, the first target and the second target may both be CD3. In some cases, b is 0. For example, b is 0, formula (II) Is equivalent to

For example, the GlcNAc is directly linked to amino acid N297 of the Fc region via an N-glycosidic bond. For example, the GlcNAc is linked to amino acid N297 of the Fc region via an N-glycosidic bond at the C1 position.

In another aspect, the application provides a multispecific antibody comprising antibody portion a capable of specifically binding to a first target; and 2 comprising formula (IV)Sugar chain portions of the structures shown; and the multispecific antibody has a structure represented by formula (V): Wherein: the a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; fuc comprises structure Fuco-L-AB2, wherein Fuco has a structure shown in formula (III): AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc is linked to said GlcNAc by an α -1,3 glycosidic bond; galX is substituted galactose, said GalX is linked to said GlcNAc by a β -1,4 glycosidic bond, and said GalX comprises a third antigen binding portion AB3 capable of specifically binding to a third target; at least one of the first target, the second target and the third target is CD3; and the position of the amino acid N297 is determined according to the EU index numbering in Kabat. For example, in some cases, the first target is CD3. In some cases, the second target may be CD3. In some cases, the third target may be CD3. In some cases, the first target, the second target, and the third target may all be CD3. In some cases, b is 0. For example, b is 0, formula (V) Is equivalent to

In some cases, the GalX X has the structure GalX ₂Y ₂-(L ₂') _m -AB3, wherein GalX ₂Y ₂ is a residue after a linking reaction of group GalX ₂ with group Y ₂, wherein the GalX ₂ comprises X ₂,X ₂ comprising a functional group capable of participating in a bioorthogonal linking reaction, and the Y ₂ comprises a functional group capable of bioorthogonal linking reaction with the X ₂, and the L ₂' is a linker, m is 0 or 1.

In another aspect, the application provides a method of making a multispecific antibody of the application. The method may include: i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety a in the presence of a catalyst, wherein said sugar chain comprises the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI) to obtain a protein having the structure of formula (VII)Wherein: said a comprises said AB1 and said Fc region; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage; q is a ribonucleotide diphosphate; and Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure of formula (III): The J is an adapter directly connected with Fuco, and the J is connected with the left end of the formula (III); the X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction; the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and ii) reacting the protein of formula (VII) with Y ₁-(L ₁') _n' -AB2 to obtain a multispecific antibody of the application, wherein Y ₁ comprises a functional group capable of bio-orthogonal ligation with X ₁, AB2 is a second antigen-binding moiety capable of specifically binding to a second target, L ₁ is a first linker, and n is 0 or 1; and L ₁ 'is a second linker, n' is 0 or 1. In some cases, b is 0. For example, b is 0, formula (VII) Is equivalent to

In another aspect, the application provides a method of making a multispecific antibody of the application. The method may include: i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety a in the presence of a catalyst, wherein said sugar chain comprises the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX) to obtain a protein of formula (X)Wherein: said a comprises said AB1 and said Fc region; glcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; galX ₂ is substituted galactose and it comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic bond; q is a ribonucleotide diphosphate; and Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure of formula (III):The J is an adapter directly connected with Fuco, and the J is connected with the left end of the formula (III); the X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction; l ₁ is a first linker, n is 0 or 1; the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and ii) reacting the protein of formula (X) with Y ₁-(L ₁') _n' -AB2 and Y ₂-(L ₂') _m -AB3 to obtain the multispecific antibody of the application; wherein the Y ₁ comprises a functional group capable of bio-orthogonal ligation with the X ₁, AB2 is a second antigen binding moiety capable of specifically binding to a second target, L ₁ 'is a second linker, n' is 0 or 1; the AB3 is a third antigen binding portion capable of specifically binding to the third target, the Y ₂ comprises a functional group capable of bio-orthogonal ligation reaction with the X ₂, the L ₂' is a linker, and m is 0 or 1. In some cases, b is 0. For example, b is 0, formula (X) Is equivalent to

In the present application, the first target and the second target may be the same or different. For example, the first target may be the same as the second target. In some cases, the first target may be different from the second target, e.g., the first target and the second target may be different antigenic proteins, or may be different epitopes (e.g., not identical epitope polypeptides) of the same antigenic protein.

In the present application, the third target and the first target may be the same or different. For example, the third target may be the same as the first target. In some cases, the third target may be different from the first target, e.g., the third target and the first target may be different antigenic proteins, or may be different epitopes (e.g., not identical epitope polypeptides) of the same antigenic protein.

In the present application, the third target and the second target may be the same or different. For example, the third target may be the same as the second target. In some cases, the third target may be different from the second target, e.g., the third target and the second target may be different antigenic proteins, or may be different epitopes (e.g., not identical epitope polypeptides) of the same antigenic protein.

In some cases, the first target, the second target, and the third target are all different from one another. For example, the three may be different antigenic proteins, or may be different epitopes of the same antigenic protein (e.g., not identical epitope polypeptides).

In some cases, the antibody moiety a may be an IgG antibody. For example, it may comprise a first light chain, a first heavy chain, a second heavy chain and a second light chain. The first light chain may comprise a first light chain variable region VL1 and a first light chain constant region CL1. The second light chain may comprise a second light chain variable region VL2 and a second light chain constant region CL2. The first heavy chain may comprise a first heavy chain variable region VH1 and a first heavy chain constant region (which may comprise a first heavy chain Fc domain). The second heavy chain may comprise a second heavy chain variable region VH2 and a second heavy chain constant region (which may comprise a second heavy chain Fc domain). In some cases, the antibody moiety a may comprise an scFv (e.g., as AB1 of the application) and an antibody Fc region (e.g., derived from IgG). In some cases, the antibody portion a can comprise a single domain antibody VHH and an antibody Fc region (e.g., an Fc region derived from IgG). In some cases, the antibody moiety a can be a multi-specific (e.g., bispecific) antibody that can comprise, in addition to an AB1 moiety capable of specifically binding to the first target, an antigen-binding fragment capable of specifically binding to another molecule of interest. For example, the antibody moiety a may comprise a first Fab, which may be the AB1, and a second Fab, which may specifically bind to other target molecules.

For example, the antibody portion a may comprise an Fc region. For example, the Fc region may be an Fc region derived from IgG, e.g., the Fc region may be an Fc region derived from IgG1, igG2, igG3, or IgG4, or a functional variant thereof. For example, the Fc region may be an Fc region derived from human IgG, e.g., it may be an Fc region derived from human IgG1, igG2, igG3, or IgG4, or a functional variant thereof. For example, the Fc region may comprise at least a CH2 domain and a CH3 domain. For example, the Fc region may comprise the amino acid sequence of 99-330 in the human IgG1 heavy chain conserved region (UniProtKB: P01857-1) sequence, or a functional variant or fragment thereof. For example, the Fc region may comprise the amino acid sequence of 99-326 in the human IgG2 heavy chain conserved region (UniProtKB: P01859-1) sequence, or a functional variant or fragment thereof. For example, the Fc region may comprise the amino acid sequence of 99-377 in the human IgG3 heavy chain conserved region (UniProtKB: P01860-1) sequence, or a functional variant or fragment thereof. For example, the Fc region may comprise the amino acid sequence of 99-327 in the human IgG4 heavy chain conserved region (UniProtKB: P01861-1) sequence, or a functional variant or fragment thereof.

In the present application, AB1 may be an antigen binding fragment of an antibody, which is capable of specifically binding to the first target.

In the present application, AB2 may be an antigen binding fragment of an antibody, which is capable of specifically binding to the second target.

In the present application, AB3 may be an antigen binding fragment of an antibody, which is capable of specifically binding to the third target.

In the present application, the antigen binding fragment of the antibody may be Fab, F (ab) ₂,F(ab'),F(ab') ₂, scFv, affibody (affibody) and/or a single domain antibody (in the present application, a single domain antibody is also referred to as nanobody (nanobody) or VHH).

In certain instances, the first target is a tumor-associated antigen (in which case, the AB1 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens), and the second target is CD3 (in which case, the AB2 is an antigen-binding fragment of an antibody that targets CD 3). For example, the first target may be derived from the following target proteins ：Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1 and/or EpCAM. In certain embodiments, the first target is derived from Her2 and the second target is CD3. In certain embodiments, the first target is derived from PD-L1 and the second target is CD3. Thus, the AB1 may be an antigen-binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM; and the AB2 may be an antigen binding fragment of an antibody that targets CD3. In certain cases, the AB1 is an antigen-binding fragment of an antibody that targets Her2, and the AB2 is an antigen-binding fragment of an antibody that targets CD3. In certain instances, the AB1 is an antigen-binding fragment of an antibody that targets PD-L1, and the AB2 is an antigen-binding fragment of an antibody that targets CD3.

In some cases, the first target is CD3 (in which case the AB1 is an antigen binding fragment of an antibody that targets CD 3) and the second target is a tumor-associated antigen (in which case the AB2 is an antigen binding fragment of an antibody that targets these tumor-associated antigens). For example, the second target may be derived from the following target proteins ：Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、 ErbB3、ErbB4、PD-L1 and/or EpCAM. In certain embodiments, the first target is CD3 and the second target is derived from Her2. In certain embodiments, the first target is CD3. And the second target is derived from PD-L1. Thus, the AB1 may be an antigen binding fragment of an antibody that targets CD3, and the AB2 may be an antigen binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM. In certain cases, the AB1 is an antigen-binding fragment of an antibody that targets CD3, and the AB2 is an antigen-binding fragment of an antibody that targets Her2. In certain instances, the AB1 is an antigen-binding fragment of an antibody that targets CD3, and the AB2 is an antigen-binding fragment of an antibody that targets PD-L1.

In some cases, the first target is a tumor-associated antigen (in which case the AB1 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens), the second target is CD3 (in which case the AB2 is an antigen-binding fragment of an antibody that targets CD 3), and the third target is a tumor-associated antigen (in which case the AB3 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens). In some cases, the first target is CD3 (in which case the AB1 is an antigen-binding fragment of an antibody that targets CD 3), the second target is a tumor-associated antigen (in which case the AB2 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens), and the third target is a tumor-associated antigen (in which case the AB3 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens). In some cases, the first target is a tumor-associated antigen (in which case the AB1 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens), the second target is a tumor-associated antigen (in which case the AB2 is an antigen-binding fragment of an antibody that targets these tumor-associated antigens), and the third target is CD3 (in which case the AB3 is an antigen-binding fragment of an antibody that targets CD 3). For example, the tumor-associated antigen may be Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM.

In certain embodiments, the first target is Her2, the second target is CD3, and the third target is PD-L1. In certain embodiments, the first target is PD-L1, the second target is CD3, and the third target is Her2.

For example, the AB1 may be an antigen-binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM; the AB2 may be an antigen binding fragment of an antibody that targets CD3, and the AB3 may be an antigen binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM.

For example, the AB1 may be an antigen-binding fragment of an antibody that targets CD3, the AB2 may be an antigen-binding fragment of an antibody that targets Her2、 Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1 and/or EpCAM, and the AB3 may be an antigen-binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM.

For example, the AB1 may be an antigen-binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM, the AB2 may be an antigen-binding fragment of an antibody that targets Her2, her3, trop2, EGFR, VEGFR, VEGFR2, BCMA, nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, erbB3, erbB4, PD-L1, and/or EpCAM, and the AB3 may be an antigen-binding fragment of an antibody that targets CD 3.

In certain cases, the AB1 is an antigen-binding fragment of an antibody that targets Her2, the AB2 is an antigen-binding fragment of an antibody that targets CD3, and the AB3 is an antigen-binding fragment of an antibody that targets PD-L1.

In certain cases, the AB1 is an antigen-binding fragment of an antibody that targets PD-L1, the AB2 is an antigen-binding fragment of an antibody that targets CD3, and the AB3 is an antigen-binding fragment of an antibody that targets Her 2.

In the present application, the Her 2-targeting antibody may be any antibody or antigen-binding fragment thereof capable of specifically binding to Her2 (e.g., human Her 2). For example, the antibody capable of specifically binding Her2 may be selected from: trastuzumab, pertuzumab (pertuzumab) and ZHer2:342. In certain embodiments, the antibody capable of specifically binding Her2 is trastuzumab. In certain embodiments, the antibody capable of specifically binding Her2 is ZHer2:342.

For example, the Her 2-targeting antibody may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:32, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:31, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:30, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise HCDR3 as shown in SEQ ID No. 32, HCDR2 as shown in SEQ ID No. 31, and HCDR1 as shown in SEQ ID No. 30, or functional variants or fragments thereof.

For example, the Her 2-targeting antibody may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:29, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:28, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:27, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise HCDR3 as shown in SEQ ID No. 32, HCDR2 as shown in SEQ ID No. 31, HCDR1 as shown in SEQ ID No. 30, LCDR3 as shown in SEQ ID No. 29, LCDR2 as shown in SEQ ID No. 28 and LCDR1 as shown in SEQ ID No. 27, or functional variants or fragments thereof.

For example, the Her 2-targeting antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:34, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:33, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a VH as set forth in SEQ ID No. 34 and a VL as set forth in SEQ ID No. 33, or functional variants or fragments thereof.

For example, the Her 2-targeting antibody may comprise a heavy chain H, and the heavy chain H may comprise the amino acid sequence shown in SEQ ID No. 16, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a light chain L, and the light chain L may comprise the amino acid sequence shown in SEQ ID No.15, a functional variant or fragment thereof.

For example, the Her 2-targeting antibody may comprise a heavy chain as set forth in SEQ ID NO. 16 and a light chain as set forth in SEQ ID NO. 15, or functional variants or fragments thereof.

For example, the Her 2-targeting antibody may be an affibody (affibody), which may comprise the amino acid sequence shown in SEQ ID No. 10, a functional variant or fragment thereof.

In the present application, the PD-L1-targeting antibody may be any antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 (e.g., human PD-L1). For example, the antibody capable of specifically binding to PD-L1 may be selected from: atilizumab (atezolimumab), simvastatin You Shan antibody (durvalumab) and en Wo Lishan antibody (KN 035). In certain embodiments, the antibody capable of specifically binding to PD-L1 is a rivarotid You Shan antibody. In certain embodiments, the antibody capable of specifically binding to PD-L1 is an en Wo Lishan antibody. For example, the antibody capable of specifically binding to PD-L1 may be a single domain antibody VHH.

For example, the PD-L1-targeting antibody may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:24, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:23, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:22, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise HCDR3 as set forth in SEQ ID NO. 24, HCDR2 as set forth in SEQ ID NO. 23 and HCDR1 as set forth in SEQ ID NO. 22, or functional variants or fragments thereof.

For example, the PD-L1-targeting antibody may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:21, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:20, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:19, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise HCDR3 as set forth in SEQ ID NO. 24, HCDR2 as set forth in SEQ ID NO. 23, HCDR1 as set forth in SEQ ID NO. 22, LCDR3 as set forth in SEQ ID NO. 21, LCDR2 as set forth in SEQ ID NO. 20 and LCDR1 as set forth in SEQ ID NO. 19, or functional variants or fragments thereof.

For example, the PD-L1 targeting antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:26, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:25, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a VH shown in SEQ ID NO:26 and a VL shown in SEQ ID NO:25, or functional variants or fragments thereof.

For example, the PD-L1-targeting antibody may comprise a heavy chain H, and the heavy chain H may comprise the amino acid sequence set forth in SEQ ID NO. 18, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a light chain L, and the light chain L may comprise the amino acid sequence set forth in SEQ ID NO. 17, a functional variant or fragment thereof.

For example, the PD-L1-targeting antibody may comprise a heavy chain as set forth in SEQ ID NO. 18 and a light chain as set forth in SEQ ID NO. 17, or functional variants or fragments thereof.

For example, the PD-L1-targeting antibody may be a single domain antibody VHH, which may comprise the amino acid sequence shown in SEQ ID NO. 12, a functional variant or fragment thereof.

In the present application, the CD 3-targeting antibody may be any antibody or antigen-binding fragment thereof capable of specifically binding to CD3 (e.g., human CD 3). For example, the antibody capable of specifically binding CD3 may be selected from the group consisting of: OKT3, M291, YTH12.5, bonatumomab (blinatumomab) and cetuximab (catumaxomab). For example, the antibody capable of specifically binding CD3 may be a single domain antibody VHH.

For example, the CD 3-targeting antibody may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:42, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:41, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:40, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise HCDR3 as shown in SEQ ID NO. 42, HCDR2 as shown in SEQ ID NO. 41 and HCDR1 as shown in SEQ ID NO.40, or functional variants or fragments thereof.

For example, the CD 3-targeting antibody may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:39, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:38, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:37, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise HCDR3 as shown in SEQ ID NO. 42, HCDR2 as shown in SEQ ID NO. 41, HCDR1 as shown in SEQ ID NO. 40, LCDR3 as shown in SEQ ID NO. 39, LCDR2 as shown in SEQ ID NO. 38 and LCDR1 as shown in SEQ ID NO. 37, or functional variants or fragments thereof.

For example, the CD 3-targeting antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:36, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:35, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a VH as set forth in SEQ ID NO:36 and a VL as set forth in SEQ ID NO:35, or functional variants or fragments thereof.

For example, the CD 3-targeting antibody may comprise a heavy chain H, and the heavy chain H may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 44, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a light chain L, and the light chain L may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 43, a functional variant or fragment thereof.

For example, the CD 3-targeting antibody may comprise a heavy chain as set forth in SEQ ID NO. 44 and a light chain as set forth in SEQ ID NO. 43, or functional variants or fragments thereof.

For example, the CD 3-targeting antibody may be a single domain antibody VHH, which may comprise the amino acid sequence shown in SEQ ID NO. 14, a functional variant or fragment thereof.

In the present application, the AB1 may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:24 and 32, a functional variant or fragment thereof.

In the present application, the AB1 may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:23 and 31, a functional variant or fragment thereof.

In the present application, the AB1 may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:22 and 30, a functional variant or fragment thereof.

For example, the AB1 may comprise HCDR3 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 23 and HCDR1 shown in SEQ ID NO. 22, or functional variants or fragments thereof.

For example, the AB1 may comprise HCDR3 shown in SEQ ID NO. 32, HCDR2 shown in SEQ ID NO. 31 and HCDR1 shown in SEQ ID NO. 30, or functional variants or fragments thereof.

In the present application, the AB1 may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:21 and 29, a functional variant or fragment thereof.

In the present application, the AB1 may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:20 and 28, a functional variant or fragment thereof.

In the present application, the AB1 may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:19 and 27, a functional variant or fragment thereof.

For example, the AB1 may comprise HCDR3 shown in SEQ ID NO. 32, HCDR2 shown in SEQ ID NO. 31, HCDR1 shown in SEQ ID NO. 30, LCDR3 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 28 and LCDR1 shown in SEQ ID NO. 27, or functional variants or fragments thereof.

For example, the AB1 may comprise HCDR3 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 23, HCDR1 shown in SEQ ID NO. 22, LCDR3 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 20 and LCDR1 shown in SEQ ID NO. 19, or functional variants or fragments thereof.

In the present application, the AB1 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:26 and 34, a functional variant or fragment thereof.

In the present application, the AB1 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:25 and 33, a functional variant or fragment thereof.

For example, the AB1 may comprise a VH shown in SEQ ID NO. 26 and a VL shown in SEQ ID NO. 25, or functional variants or fragments thereof.

For example, the AB1 may comprise a VH shown in SEQ ID NO:34 and a VL shown in SEQ ID NO:33, or functional variants or fragments thereof.

In the present application, the AB1 may be a single domain antibody VHH, which may comprise the amino acid sequence shown in SEQ ID NO. 12, a functional variant or fragment thereof.

In the present application, the AB1 may be an affibody, which may comprise the amino acid sequence shown in SEQ ID NO. 10, a functional variant or fragment thereof.

In the present application, the AB2 may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:42, a functional variant or fragment thereof.

In the present application, the AB2 may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:41, a functional variant or fragment thereof.

In the present application, the AB2 may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:40, a functional variant or fragment thereof.

For example, the AB2 may comprise HCDR3 shown in SEQ ID NO. 42, HCDR2 shown in SEQ ID NO. 41 and HCDR1 shown in SEQ ID NO. 40, or functional variants or fragments thereof.

In the present application, the AB2 may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:39, a functional variant or fragment thereof.

In the present application, the AB2 may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:38, a functional variant or fragment thereof.

In the present application, the AB2 may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:37, a functional variant or fragment thereof.

For example, the AB2 may comprise HCDR3 shown in SEQ ID NO. 42, HCDR2 shown in SEQ ID NO. 41, HCDR1 shown in SEQ ID NO. 40, LCDR3 shown in SEQ ID NO. 39, LCDR2 shown in SEQ ID NO. 38 and LCDR1 shown in SEQ ID NO. 37, or functional variants or fragments thereof.

In the present application, the AB2 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:36, a functional variant or fragment thereof.

In the present application, the AB2 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:35, a functional variant or fragment thereof.

For example, the AB2 may comprise a VH shown in SEQ ID NO:36 and a VL shown in SEQ ID NO:35, or functional variants or fragments thereof.

In the present application, the AB2 may be a single domain antibody VHH, which may comprise the amino acid sequence shown in SEQ ID NO. 14, a functional variant or fragment thereof.

In the present application, the AB3 may comprise heavy chain CDR3 (HCDR 3), and the HCDR3 may comprise the amino acid sequence of SEQ ID NO:24 and 32, a functional variant or fragment thereof.

In the present application, the AB3 may comprise heavy chain CDR2 (HCDR 2), and the HCDR2 may comprise the amino acid sequence of SEQ ID NO:23 and 31, a functional variant or fragment thereof.

In the present application, the AB3 may comprise heavy chain CDR1 (HCDR 1), and the HCDR1 may comprise the amino acid sequence of SEQ ID NO:22 and 30, a functional variant or fragment thereof.

For example, the AB3 may comprise HCDR3 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 23 and HCDR1 shown in SEQ ID NO. 22, or functional variants or fragments thereof.

For example, the AB3 may comprise HCDR3 shown in SEQ ID NO. 32, HCDR2 shown in SEQ ID NO. 31 and HCDR1 shown in SEQ ID NO. 30, or functional variants or fragments thereof.

In the present application, the AB3 may comprise a light chain CDR3 (LCDR 3), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO:21 and 29, a functional variant or fragment thereof.

In the present application, the AB3 may comprise a light chain CDR2 (LCDR 2), and the LCDR2 may comprise the amino acid sequence of SEQ ID NO:20 and 28, a functional variant or fragment thereof.

In the present application, the AB3 may comprise a light chain CDR1 (LCDR 1), and the LCDR1 may comprise the amino acid sequence of SEQ ID NO:19 and 27, a functional variant or fragment thereof.

For example, the AB3 may comprise HCDR3 shown in SEQ ID NO. 32, HCDR2 shown in SEQ ID NO. 31, HCDR1 shown in SEQ ID NO. 30, LCDR3 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 28 and LCDR1 shown in SEQ ID NO. 27, or functional variants or fragments thereof.

For example, the AB3 may comprise HCDR3 shown in SEQ ID NO. 24, HCDR2 shown in SEQ ID NO. 23, HCDR1 shown in SEQ ID NO. 22, LCDR3 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 20 and LCDR1 shown in SEQ ID NO. 19, or functional variants or fragments thereof.

In the present application, the AB3 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence of SEQ ID NO:26 and 34, a functional variant or fragment thereof.

In the present application, the AB3 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence of SEQ ID NO:25 and 33, a functional variant or fragment thereof.

For example, the AB3 may comprise a VH shown in SEQ ID NO:26 and a VL shown in SEQ ID NO:25, or functional variants or fragments thereof.

For example, the AB3 may comprise a VH shown in SEQ ID NO:34 and a VL shown in SEQ ID NO:33, or functional variants or fragments thereof.

In the present application, the AB3 may be a single domain antibody VHH, which may comprise the amino acid sequence shown in SEQ ID NO. 12, a functional variant or fragment thereof.

In the present application, the AB3 may be an affibody, which may comprise the amino acid sequence shown in SEQ ID NO. 10, a functional variant or fragment thereof.

In the present application, the Fuc may comprise structure Fuco-L-AB2. In Fuco-L-AB2, the structure of L may be J- (L ₁) _n-X ₁Y ₁-(L ₁') _n').

In the present application, the Fuc may comprise structure Fuco-J- (L ₁) _n-X ₁).

In the present application, the Fuco may have a structure represented by formula (III):

In the present application, the group X ₁ contains a functional group capable of participating in a bio-orthogonal ligation reaction. For example, the X ₁ may comprise a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof. For example, the X ₁ may comprise a functional group selected from the group consisting of: wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl. For example, the X ₁ may comprise a functional group selected from the group consisting of: For example, the X ₁ may include For example, the X ₁ may includeFor example, the X ₁ may beFor example, the X ₁ may be

In the present application, the Y ₁ contains a functional group capable of bio-orthogonal ligation with the X ₁. For example, the Y ₁ may comprise a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof. For example, the Y ₁ may comprise a functional group selected from the group consisting of: Wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl. For example, the Y ₁ may comprise a functional group selected from the group consisting of: For example, the Y ₁ may include For example, Y ₁ may be

For example, the X ₁ and the Y ₁ may comprise a set of structures selected from the group consisting of:

a) X ₁ includes And Y ₁ includes

B) X ₁ includesAnd Y ₁ includes

C) X ₁ includesAnd Y ₁ includes

D) X ₁ includesAnd Y ₁ includesAnd

E) X ₁ includesAnd Y ₁ includes

For example, the X ₁ may includeAnd the Y ₁ may include

In the application, X ₁Y ₁ is a residual group after the connection reaction of the group X ₁ and the group Y ₁. For example, the X ₁Y ₁ may comprise a structure selected from the group consisting of:

In the present application, J is an adapter directly linked to Fuco. The J is connected with the left end of the formula (III). For example, the J may be Wherein said Rf may be-CH ₂ -, -NH-or-O-. In some cases, the J may beWherein the left end of the J-structure is connected to the Fuco.

In the present application, L ₁ is a first linker, and n may be 0 or1. When n is 0, the first linker L ₁ is absent and the J may be directly attached to the X ₁ or X ₁Y ₁. When n is 1, the J is connected with the X ₁ or the X ₁Y ₁ through the L ₁. In some cases, the L ₁ may be a C ₃-C ₂₀₀ subunit, a C ₁-C ₂₀₀ alkylene, a C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, derivatives thereof, or any combination thereof. Wherein the polypeptide-ene, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene may be optionally substituted with one or more Rs ¹ and/or optionally interrupted by one or more Rs ² (e.g., one or more Rs ² groups may be inserted into any of the foregoing groups, For example, a plurality of-O-groups may be inserted into the aforementioned alkylene groups to obtain polyethylene glycol groups).

Each of the Rs ¹ may be independently selected from: halogen, -OH, -NH ₂, and-COOH.

Each of the Rs ² may be independently selected from: -O-, -S-,Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

For example, the L ₁ may be selected from: wherein s1 is an integer from 1 to 50 (e.g., 1 to 45,1 to 40,1 to 35,1 to 30,1 to 25,1 to 20,1 to 15, 14, 13, 12, 11, 10,9,8,7,6,5,4,3,2, or 1); wherein each s2 is independently an integer from 0 to 50 (e.g., 0 to 45,0 to 40,0 to 35,0 to 30,0 to 25,0 to 20,0 to 15, 14, 13, 12, 11, 10,9,8,7,6,5,4,3,2,1, or 0). Each of the-CH ₂ - (CH ₂ in brackets) is optionally replaced with-O-, but adjacent-CH ₂ -is not simultaneously replaced with-O- (e.g., there are no two or more linked-O-); the left end of the structure may be connected to the J, and the right end of the structure may be connected to the X ₁. In certain instances, the L ₁ is a C ₂-C ₂₀ polyethylene glycol (PEG) group. For example, L ₁ can be- (CH ₂OCH ₂) _s1') -where s1' can be an integer from 0 to 20, in some cases, the L ₁ is C ₁-C ₅₀ alkylene.

In the present application, the L ₁ may be selected from the group consisting of: And the right end of the structure is connected with the X ₁.

In the present application, L ₁ 'is a second linker, and n' may be 0 or1. When n 'is 0, the second linker L ₁' is absent, and Y ₁ or X ₁Y ₁ may be directly linked to the AB 2. When n 'is 1, the Y ₁ or X ₁Y ₁ is connected with the AB2 through the L ₁'. In some cases, the L ₁' may be a C ₃-C ₂₀₀ subunit, a C ₁-C ₂₀₀ alkylene, a C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, derivatives thereof, or any combination thereof. Wherein the polypeptide-ene, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene may be optionally substituted with one or more Rs ¹ and/or optionally interrupted by one or more Rs ² (e.g., one or more Rs ² groups may be inserted into any of the foregoing groups, For example, a plurality of-O-groups may be inserted into the aforementioned alkylene groups to obtain polyethylene glycol groups).

For example, the L ₁' may be selected from Wherein each s2 is independently an integer from 0 to 50 (e.g., 0 to 45,0 to 40,0 to 35,0 to 30,0 to 25,0 to 20,0 to 15, 14, 13, 12, 11, 10,9,8,7,6,5,4,3,2,1, or 0). Each of the-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, e.g., there are no two or more connected-O-, the right end of the structure may be attached to the AB2, and the left end of the structure may be attached to the Y ₁ or X ₁Y ₁. In some cases, the right end of the L ₁' structure may be linked (e.g., by a condensation reaction) by an amino group linked to the-COOH forming peptide bond at the C-terminus of the AB2 structure.

For example, the L ₁' may be selected from the following structures:

The left end of the structure is connected with Y ₁, and the right end of the structure can be connected with the AB2 through amino.

In the present application, galX is optionally substituted galactose. In some cases, the GalX is galactose.

In some cases, the GalX is substituted galactose. For example, one or more of the hydroxyl groups at the C2, C3, C4 and C6 positions in the galactose may be substituted. In some cases, the hydroxyl group at the C2 position in the galactose may be substituted. In some cases, the GalX is a monosaccharide. The monosaccharides may be, for example, molecules that can no longer be simply hydrolyzed to smaller sugars. For example, the monosaccharide may be a monosaccharide derivative, but contains only one core monosaccharide backbone. For example GalNAz, galNH ₂ or GalNAc is a monosaccharide. for another example, lacNAc containing both Gal and GlcNAc is not a monosaccharide. In some cases, in the GalX, the galactose may be substituted with a substituent Rg ¹. The Rg ¹ can be selected from the group consisting of: hydrogen, halogen, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, C ₂-C ₂₄ alkenyl, C ₅-C ₂₄ cycloalkenyl, C ₂-C ₂₄ alkynyl, C ₆-C ₂₄ cycloalkynyl, C ₃-C ₂₄ (hetero) aryl, c ₃-C ₂₄ alkyl (hetero) aryl and C ₃-C ₂₄ (hetero) arylalkyl. Wherein the alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, (hetero) aryl, alkyl (hetero) aryl, and/or (hetero) arylalkyl groups each independently may be optionally substituted with one or more substituents Rs ⁴, and/or each independently may be optionally interrupted by one or more substituents Rs ⁵ (e.g., one or more Rs ⁵ groups may be inserted in any of the foregoing groups, for example, a plurality of-O-groups may be inserted into the aforementioned alkyl groups to obtain polyethylene glycol groups).

Each of the Rs ⁴ may be selected from the group consisting of: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN.

Each of the Rs ⁵ may be independently selected from the group consisting of: -O-, -S-,Wherein, the Rs ³ may be selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

In some cases, the GalX may be substitutedAnd a substitution, wherein t is 0 or 1.

The Rg ² can be selected from the group consisting of: c ₁-C ₂₄ alkylene, C ₃-C ₂₄ cycloalkylene, C ₂-C ₂₄ alkenylene, C ₅-C ₂₄ cycloalkenylene, C ₂-C ₂₄ alkynylene, C ₆-C ₂₄ cycloalkynylene, C ₃-C ₂₄ (hetero) arylene, C ₃-C ₂₄ alkyl (hetero) arylene, and C ₃-C ₂₄ (hetero) arylalkylene. Wherein the alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, alkyl (hetero) arylene, and/or (hetero) arylalkylene groups may each independently be optionally substituted with one or more substituents Rs ⁴, and/or each independently be optionally interrupted by one or more substituents Rs ⁵ (e.g., one or more Rs ⁵ groups may be inserted in any of the foregoing groups, e.g., multiple-O-groups may be inserted into the foregoing alkylene groups to obtain polyethylene glycol groups).

The Rg ³ can be selected from the group consisting of: hydrogen, halogen, -OH, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, C ₂-C ₂₄ alkynyl, C ₅-C ₂₄ cycloalkynyl and C ₂-C ₂₄ (heteroaryl), wherein the alkyl, cycloalkyl, alkynyl, cycloalkynyl and/or (heteroaryl) groups may each independently be optionally substituted with one or more Rs ⁴.

Each of the Rs ⁴ may be independently selected from: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN.

Each of the Rs ⁵ may be independently selected from: -O-, -S-,The Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.

In some cases, the GalX may be selected from the group consisting of:

In the present application, galX may have the following structure: galX ₂Y ₂-(L ₂') _m -AB3. Wherein GalX ₂Y ₂ is a residue group after a ligation reaction of GalX ₂ with group Y ₂, wherein the GalX ₂ comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction and the Y ₂ comprises a functional group capable of bio-orthogonal ligation reaction with the X ₂.

According to any aspect of the application, L ₂' is a linker and m may be 0 or1. When m is 0, the linker L ₂' is absent and Y ₂ or X ₂Y ₂ may be directly linked to the AB 3. when m is 1, the Y ₂ or X ₂Y ₂ and the AB3 may be connected by the L ₂'. in some cases, the L ₂' may be a C ₃-C ₂₀₀ subunit, a C ₁-C ₂₀₀ alkylene, a C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, derivatives thereof, or any combination thereof. Wherein the polypeptide-ene, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene may be optionally substituted with one or more Rs ¹ and/or optionally interrupted by one or more Rs ² (e.g., one or more Rs ² groups may be inserted into any of the foregoing groups, For example, a plurality of-O-groups may be inserted into the aforementioned alkylene groups to obtain polyethylene glycol groups).

For example, the L ₂' may be selected from: Wherein each s2 is independently an integer from 0 to 50 (e.g., 0-45,0-40,0-35,0-30,0-25,0-20,0-15, 14, 13, 12, 11, 10,9,8,7,6,5,4,3,2,1, or 0), each of said-CH ₂ -is optionally replaced with-O-, but adjacent-CH ₂ -is not simultaneously replaced with-O-, e.g., there are no two or more connected-O-, the right end of the structure may be connected to said AB3, and the left end of the structure may be connected to said Y ₂ or X ₂Y ₂. In some cases, the right end of the L ₂' structure may be linked by an amino group to the-COOH of the C-terminal end of the AB3 structure to form a peptide bond (e.g., by a condensation reaction).

For example, the L ₂' may have the following structure: The left end of the structure is connected with Y ₂, and the right end of the structure can be connected with the AB2 through amino.

In the present application, galX ₂ may be galactose in which one or more hydroxyl groups at C2, C3, C4 and C6 positions are substituted. In some cases, the GalX ₂ may be galactose with a hydroxyl group at the C2 position substituted. In some cases, the GalX ₂ is a monosaccharide.

For example, X ₂ of GalX ₂ may be azide

In some cases, the GalX ₂ may be

In some cases, the Y ₂ may include

For example, the X ₂ may be azideAnd the Y ₂ may include

In some cases, the X ₂Y ₂ may comprise a structure selected from the group consisting of:

in the present application, in some cases, there are The antibody-Fuc conjugate with the zygotic structure has higher efficiency in preparing the multi-specific antibody disclosed by the application by reacting with Y ₁-(L ₁') _n' -AB 2. For example, haveOr alternatively(Wherein Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁, J is)) The conjugate with the structure has higher efficiency in preparing the multispecific antibody by reacting with Y ₁-(L ₁') _n' -AB 2. For example, haveOr alternatively(Wherein Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁, J is)X ₁ is) The conjugate with the structure has higher efficiency in preparing the multispecific antibody by reacting with Y ₁-(L ₁') _n' -AB 2. For example, the comparison of the efficiency of the multi-specific antibodies of the application prepared by reacting antibody-Fuc' conjugates with different binders with Y ₁-(L ₁') _n' -AB2 is shown in example 26.

In the present application, the Q may be Guanosine Diphosphate (GDP), uridine Diphosphate (UDP), and/or Cytidine Diphosphate (CDP). In some cases, the Q may be Guanosine Diphosphate (GDP).

In the present application, the Q-Fuc may be GDP-Fuc.

In the present application, in some cases, there isQ-fucose derivatives of the zygotic structure (e.g. Q-Fuc') compared to having the structureThe conversion rate of the Q-fucose derivative (e.g., Q-Fuc') of the zygotic structure transferred to the antibody under the catalysis of fucosyltransferase is significantly improved. For example, haveGDP-fucose derivatives (e.g. GDP-Fuc') of the zygotic structure compared to having the structureThe conversion rate of GDP-fucose derivative (such as GDP-Fuc') of the zygotic structure transferred to antibody (such as antibody- (Galβ1, 4) GlcNAc and antibody- (GalNAzβ1, 4) GlcNAc) under the catalysis of helicobacter pylori alpha 1, 3-fucosyltransferase is obviously improved. For example, it is shown in example 27 that there isGDP-FAmP ₄ Biotin having the zygotic structure compared with that having the structureThe GDP-FAzP ₄ Biotin of the zygotic structure has obviously improved conversion rate when being transferred to trastuzumab- (Galβ1, 4) GlcNAc and trastuzumab- (GalNAzβ1, 4) GlcNAc under the catalysis of helicobacter pylori alpha 1, 3-fucosyltransferase.

In the present application, the Q-Fuc' may be selected from the following structures:

In the preparation method of the present application, the catalyst in step i) may comprise a fucosyltransferase. For example, the fucosyltransferase may be an alpha-1, 3-fucosyltransferase or a functional variant or fragment thereof. For example, the fucosyltransferase may be derived from bacteria, e.g., it may be derived from helicobacter pylori Helicobacter pylori (e.g., helicobacter pylori 26695). For example, the fucosyltransferase may be derived from an alpha-1, 3 fucosyltransferase having GenBank accession number AAB81031.1, genBank accession number AAD07447.1, or GenBank accession number AAD07710.1, or a variant or fragment thereof. For example, the fucosyltransferase may be derived from an alpha-1, 3 fucosyltransferase having GenBank accession AAD 07710.1. For example, the fucosyltransferase may be an alpha-1, 3 fucosyltransferase having GenBank accession AAD07710.1, or a functional variant or fragment thereof. For example, a wild-type α -1,3 fucosyltransferase having GenBank accession AAD07710.1 has a catalytically active region, 10 polypeptide repeat fragments and a C-terminal tail structure, and a functional variant or fragment thereof may comprise the catalytically active region and 1-10 polypeptide repeat fragments.

For example, the fucosyltransferase in step i) may comprise a catalytically active region and at least one heptad repeat (HPR), e.g., the fucosyltransferase in step i) may comprise a catalytically active region and 1-10 heptad repeat (HPR) (e.g., 1,2,3,4,5,6,7,8,9, or 10 HPRs). The catalytically active region may be located at the N-terminus of the HRP. For example, the C-terminus of the catalytically active region may be linked to an HPR (e.g., the N-terminus of the HPR).

For example, the catalytically active region may comprise the amino acid sequence shown in SEQ ID NO.1 or a functional variant or fragment thereof.

For example, the catalytically active region may comprise the amino acid sequence set forth in SEQ ID NO. 1 or an amino acid sequence having at least about 80% (e.g., at least about 82%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least about 95%, at least about 98%, at least about 99% or more) sequence identity to SEQ ID NO. 1.

The amino acid sequence of the variant may have at least about 80% (e.g., at least about 82%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least about 95%, at least about 98%, at least about 99% or more) sequence identity to the amino acid sequence of the parent fucosyltransferase (e.g., those fucosyltransferases or catalytically active regions of the application described above).

In the present application, "sequence identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference sequence after aligning the candidate sequence (e.g., the sequence of a variant) with the reference sequence (e.g., the sequence of a parent) and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity. Sequence alignment may be performed in a manner known to those skilled in the art to determine the percent amino acid sequence identity, for example, BLAST-2,Clustal W,ALIGN-2, megalign (DNASTAR) software, or FASTA package, etc. may be used. One skilled in the art can determine appropriate parameters for aligning sequences.

For example, the catalytically active region may comprise the amino acid sequence shown in SEQ ID NO. 1.

For example, the HPR may comprise the amino acid sequence shown in SEQ ID NO. 2.

For example, the fucosyltransferase may comprise the amino acid sequence shown in SEQ ID NO. 3

For example, the catalyst may comprise the fucosyltransferase and tag sequences described above.

For example, the catalyst may comprise the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

In some cases, the catalyst in step i) may comprise the amino acid sequence shown in SEQ ID NO. 3. In some cases, the catalyst in step i) may comprise the amino acid sequence shown in SEQ ID NO. 4.

In some cases, the preparation method of the present application may further comprise the steps of: treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI), the structure of the protein being as shown in formula (VIII): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage. For example, the suitable catalyst may be beta 1,4 galactose transferase or a functional variant thereof. For example, the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the endoglycosidase may be Endo S, endo S2, endo a, endo F, endo M, endo D, endo H and/or functional variants thereof. For example, the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO. 6. In some cases, b is 1 if the protein comprising a sugar chain and the antibody moiety a has a modification with core α -1,6 fucose. In some cases, b is 0 if the protein comprising a sugar chain and the antibody moiety a has no core alpha-1, 6 fucose modification.

In some cases, the preparation method according to the present application may further comprise the steps of: treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI), the structure of the protein being as shown in formula (VIII): (VIII) wherein GlcNAc is N-acetylglucosamine and said GlcNAc is directly linked to amino acid N297 of said Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage. For example, when b is 0, formula (VIII) Is equivalent toFor example, the suitable catalyst may be beta 1,4 galactose transferase or a functional variant thereof. For example, the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the endoglycosidase may be Endo S, endo S2, endo a, endo F, endo M, endo D, endo H and/or functional variants thereof. For example, the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the α1,6 fucosidase may be BfFucH, alfc, BKF, and/or functional variants thereof. For example, the alpha 1,6 fucosidase may comprise the amino acid sequence shown in SEQ ID NO. 7.

In some cases, the preparation method of the present application may further comprise the steps of: treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein; contacting the treated protein with UDP-GalX ₂ in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI): Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and GalX ₂ is a substituted galactose and comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage. For example, the suitable catalyst may be beta 1,4 galactose transferase or a functional variant thereof. For example, the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the endoglycosidase may be Endo S, endo S2, endo a, endo F, endo M, endo D, endo H and/or functional variants thereof. For example, the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO. 6. In some cases, b is 1 if the protein comprising a sugar chain and the antibody moiety a has a modification with core α -1,6 fucose. In some cases, b is 0 if the protein comprising a sugar chain and the antibody moiety a has no core alpha-1, 6 fucose modification.

In some cases, the preparation method of the present application may further comprise the steps of: treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein; contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI): (XI), wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region; fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and GalX ₂ is a substituted galactose and comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage. For example, b is 0, Equivalent toFor example, the suitable catalyst may be beta 1,4 galactose transferase or a functional variant thereof. For example, the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the endoglycosidase may be Endo S, endo S2, endo a, endo F, endo M, endo D, endo H and/or functional variants thereof. For example, the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the α1,6 fucosidase may be BfFucH, alfc, BKF, and/or functional variants thereof. For example, the alpha 1,6 fucosidase may comprise the amino acid sequence shown in SEQ ID NO. 7.

Composition and medical application

In another aspect, the application provides a composition that may comprise one or more of the multispecific antibodies of the application.

The composition of the application may be a pharmaceutical composition. For example, it may comprise one or more pharmaceutically acceptable carriers or excipients.

In another aspect, the application provides the use of a multispecific antibody of the application and/or a composition of the application in the manufacture of a medicament for the prevention, alleviation and/or treatment of a disease or condition.

In the present application, the disease or condition may include, for example, a tumor, cancer, or other proliferative disease. The disease or disorder may also include, for example, an immune system related disease or disorder (e.g., an autoimmune disease).

Without intending to be limited by any theory, the following examples are meant to illustrate the multispecific antibodies, methods of manufacture, uses, and the like of the present application, and are not intended to limit the scope of the application.

Examples

Materials and methods

Protein molecular weight mass spectrometry

Molecular weight analysis of the proteins was performed using a Xex G2-XS QTOF mass spectrometer (Waters Corporation). The mass spectrometer was equipped with an electrospray ionization source (ESI) and Acuqity UPLC I-Class plus system (Waters Corporation). The purified protein is treated by Waters ACQUITY UPLC Protein BEH C to 4 columns1.7 Μm,2.1mm x 100 mm) were separated and desalted. Mobile phase a was 0.1% formic acid/water solution, mobile phase B was acetonitrile containing 0.1% formic acid water, and the flow rate was 0.2mL/min. Data analysis was performed with Waters Unify software (version 1.9.4,Waters Corporation).

BGalT1 (Y289L) (Bovine beta-1, 4-galactosyltransferase I, bovine beta-1, 4-galactosyltransferase carrying the Y289L mutation), endoS (Streptococcus pyogenes endoglycosidase S, endoparasidase S), alfc (Lactobacillus casei alpha-1, 6-fucosidase Alfc, lactobacillus casei alpha-1, 6-fucosidase) and Sortase 5M (Staphylococcus aureus Sortase M, staphylococcus aureus transpeptidase 5M) cloning, expression and purification

Reference Qasba P.K et al (J.biol. Chem.2002,277,20833; prot. Expr. Far. 2003,30,219) cloning, expression and purification of BGalT (Y289L) (SEQ ID NO: 5), reference Collin, M. Et al (EMBO J.2001,20,3046; select. Immun.2001,69, 7187) cloning, expression and purification of EndoS (SEQ ID NO: 6), reference Wang L., et al (Methods mol. Biol.2018,19,367) cloning, expression and purification of Alfc (SEQ ID NO: 7), and reference Liu D et al (Proc. Natl. Acad. Sci. USA 2011,108,11399) cloning, expression and purification of Sortase 5M (SEQ ID NO: 8).

HpFT-2HR cloning, expression and purification

The nucleic acid sequence encoding HpFT-2HR (which comprises the catalytically active region, 2 heptad repeats, and a C-terminal fused His tag with the amino acid sequence shown in SEQ ID NO: 4) was synthesized by gene synthesis and inserted into the pET24b vector (Nanjin Style) via NdeI and BamHI cleavage sites. E.coli BL21 (DE 3) was transformed with the constructed recombinant plasmid. The transformed recombinant bacteria were cultured in LB medium containing 50. Mu.g/mL kanamycin, and when OD ₆₀₀ reached 0.6-0.8 at 37℃IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to a final concentration of 0.2mM, followed by further incubation at 25℃and 200rpm for 16 hours for protein-induced expression. After centrifugation, the induced cells were suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 500mM sodium chloride, 20mM imidazole, 1mM PMSF (phenylmethylsulfonyl fluoride)). The suspended cells were sonicated and then purified with Ni-NTA packing (GE Health). The main fraction with a purity of more than 90% was collected and then dialyzed into a preservation buffer (25 mM Tris-HCl, pH 7.5, 150mM sodium chloride, 5% glycerol).

ZHer _2:342 affinity, cloning, expression and purification of aPDL1 and aCD3

The nucleic acid sequence was inserted into the pET24b vector (Nanjinspire) by gene synthesis encoding ZHer _2:342 affibody (SEQ ID NO: 9) comprising a C-terminal fusion LPETGG and a His tag (SEQ ID NO: 2010,107,15039), and using NdeI and BamHI cleavage sites (SEQ ID NO: eigenbrot, C.et al, proc.Natl. Acad. Sci. USA). Nucleic acid sequences encoding PDL1 nanobody (aPDL) (sequence reference Zhang f. Et al, cell discovery, 2017,3,17004.) (which comprises a C-terminal fusion LPETGG and His-tag, the amino acid sequence of which is shown in SEQ ID No. 11) and CD3 nanobody (aCD 3) (sequence reference Annelies r. Et al, US 2019/0382485 A1) (which comprises a C-terminal fusion LPETGG and His-tag, the amino acid sequence of which is shown in SEQ ID No. 13) were synthesized by gene synthesis and inserted into pET26b vector (nanjing prestrel) using NcoI and BamHI cleavage sites. E.coli BL21 (DE 3) was transformed with the constructed recombinant plasmid. The transformed recombinant bacteria were cultured in LB medium containing 50. Mu.g/mL kanamycin, and when OD ₆₀₀ reached 0.6-0.8 at 37℃IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to a final concentration of 0.2mM, followed by further incubation at 25℃and 200rpm for 16 hours for protein-induced expression. After centrifugation, the induced cells were suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 500mM sodium chloride, 20mM imidazole, 1mM PMSF (phenylmethylsulfonyl fluoride)). The suspended cells were sonicated and then purified with Ni-NTA (GE Health) packing. The main fraction with a purity of more than 90% was collected and then dialyzed into a preservation buffer (25 mM Tris-HCl, pH 7.5, 150mM sodium chloride).

Synthesis of examples 1 GDP-FAz

GDP-FAz was synthesized by the method reported by Wu P et al (Proc. Natl. Acad. Sci. USA 2009,106,16096). And passed through a Bio-Gel P-2Gel column using 50mM ammonium bicarbonate solution as eluent to complete the purification.

HRMS (ESI-): C ₁₆H ₂₄N ₈O ₁₅P ₂(M-H ⁺) calculated 629.0764, found 629.0785.

Examples 2 GDP-FAm Synthesis

100Mg of GDP-FAz was dissolved in 8.75mL of a methanol/water (volume ratio 1:1.5) solution, then 5mg of palladium on carbon was added to the system, the hydrogen was replaced by evacuation, and the pressure of the system was maintained at 0.28MPa. The reaction was stirred at room temperature for 4h, filtered, concentrated and lyophilized to give a white solid (84.8 mg, 88%). HRMS (ESI-): C ₁₆H ₂₆N ₆O ₁₅P ₂(M-H ⁺) calculated 603.0859, found 603.0874. ¹H NMR(400MHz,D ₂O)δ8.10(s,1H),5.92(d,J＝6.1Hz,1H),4.97(t,J＝7.6Hz,1H),4.76-4.73(m,1H),4.51(dd,J＝5.2,3.4Hz,1H),4.37-4.34(m,1H),4.23-4.21(m,2H),3.96(dd,J＝9.6,2.4Hz,1H),3.92-3.91(m,1H),3.70(dd,J＝10.0,3.3Hz,1H),3.63(dd,J＝10.0,7.6Hz,1H),3.31(dd,J＝13.4,9.6Hz,1H),3.24(dd,J＝13.4,3.1Hz,1H).

EXAMPLE 3 Synthesis of GDP-FAmAz

To 600. Mu.L GDP-FAm (100 mM aqueous solution) were added 200. Mu.L NaHCO ₃ (200 mM aqueous solution), 780. Mu.L THF (tetrahydrofuran) and 220. Mu.L NHS-azide (Simultaneous technology) (100 mM THF solution) in this order. The reaction was stirred at room temperature overnight and monitored by thin layer chromatography. Purification by preparative high performance liquid chromatography (Pre-HPLC) afforded the product as a white solid (8.7 mg, 63%). HRMS (ESI-): C ₁₈H ₂₇N ₉O ₁₆P ₂(M-H ⁺) calculated 686.0978, found 686.1002. ¹H NMR(400MHz,D ₂O):δ8.10(s,1H),5.92(d,J＝6.0Hz,1H),4.92(t,J＝7.9Hz,1H),4.78-4.76(m,1H),4.52(dd,J＝5.2,3.4Hz,1H),4.35-4.34(m,1H),4.23-4.21(m,2H),4.00(s,2H),3.87(d,J＝3.2Hz,1H),3.71(dd,J＝8.8,3.9Hz,1H),3.66(dd,J＝10.0,3.3Hz,1H),3.62-3.57(m,2H),3.32(dd,J＝14.0,8.6Hz,1H).

EXAMPLE 4 Synthesis of GDP-FAmP ₄ Az

200. Mu.L of NaHCO ₃ (200 mM aqueous solution), 780. Mu.L of THF, 220. Mu.L of NHS-PEG ₄ -azide (Simultaneous technology) (100 mM THF solution), and 600. Mu. L H ₂ O were added sequentially to 200. Mu.L of GDP-FAm (100 mM aqueous solution). The reaction was stirred at room temperature overnight and monitored by thin layer chromatography. Purification by preparative high performance liquid chromatography (Pre-HPLC) afforded the product as a white solid (9 mg, 51%). HRMS (ESI-): C ₂₇H ₄₅N ₉O ₂₀P ₂(M-H ⁺) calculated 876.2183, found 876.2187. ¹H NMR(400MHz,D ₂O)δ8.14(s,1H),5.90(d,J＝6.0Hz,1H),4.90(t,J＝7.8Hz,1H),4.75(t,J＝5.5Hz,1H),4.50(dd,J＝5.0,3.5Hz,1H),4.33-4.32(m,1H),4.21-4.19(m, 2H),3.84(d,J＝3.2Hz,1H),3.73(t,J＝6.2Hz,2H),3.70-3.61(m,16H),3.60-3.53(m,2H),3.47-3.45(m,2H),3.26(dd,J＝14.0,8.6Hz,1H),2.53(t,J＝6.2Hz,2H).

EXAMPLE 5 Synthesis of GDP-FAmP ₈ Tz

To 200. Mu.L GDP-FAm (100 mM in water) were added sequentially 200. Mu.L NaHCO ₃ (200 mM in water), 780. Mu.L THF, and 220. Mu.L NHS-PEG ₈ -Tz (Simultaneous technology) (100 mM in THF). The reaction was stirred at room temperature for 4h and monitored by thin layer chromatography. Purification by Pre-HPLC gave the product as a pink solid (9.2 mg, 38%). HRMS (ESI-): C ₄₄H ₆₈N ₁₀O ₂₅P ₂(M-2H ⁺)/2 calculated 598.1844, found 598.1880. ¹H NMR(400MHz,D ₂O)δ8.28-8.24(m,2H),8.02(s,1H),7.16-7.12(m,2H),5.81(d,J＝6.0Hz,1H),4.92(t,J＝7.8Hz,1H),4.72(t,J＝5.6Hz,1H),4.50(dd,J＝5.2Hz,3.5,1H),4.32-4.29(m,3H),4.21-4.19(m,2H),3.97-3.95(m,2H),3.85(d,J＝3.0Hz,1H),3.81-3.78(m,2H),3.75-3.59(m,32H),3.27(dd,J＝14.1,8.7Hz,1H),3.02(s,3H),2.53(t,J＝6.2Hz,2H).

EXAMPLE 6 Synthesis of GDP-FAmP ₄ Tz

To 200. Mu.L GDP-FAm (100 mM in water) were added sequentially 200. Mu.L NaHCO ₃ (200 mM in water), 780. Mu.L THF, and 220. Mu.L NHS-PEG ₄ -Tz (CLICK CHEMISTRY Tools) (100 mM in THF). The reaction was stirred at room temperature for 4h and monitored by thin layer chromatography. Purification by Pre-HPLC afforded the product as a pink solid (9.9 mg, 48%). HRMS (ESI-): C ₃₆H ₅₂N ₁₀O ₂₁P ₂(M-H ⁺) calculated 1021.2711, found 1021.2725. ¹H NMR(400MHz,D ₂O)δ8.17-8.13(m,2H),8.00(s,1H),7.06-7.02(m,2H),5.76(d,J＝5.6Hz,1H),4.91(t,J＝7.7Hz,1H),4.67(t,J＝5.4Hz,1H),4.49(dd,J＝5.1,3.7Hz,1H),4.30-4.28(m,1H),4.27-4.25(m,2H),4.21-4.19(m,2H),3.95-3.93(m,2H),3.84-3.83(m,1H),3.80-3.78(m,2H),3.74-3.71(m,2H),3.70-3.57(m,13H),3.51(dd,J＝14.1,4.2Hz,1H),3.24(dd,J＝14.0,8.6Hz,1H),3.00(s,3H),2.49(t,J＝6.3Hz,2H).

EXAMPLE 7 Synthesis of GDP-FAmP ₄ BCN

200. Mu.L of NaHCO ₃ (200 mM aqueous solution), 560. Mu.L of THF and 440. Mu.L of NHS-PEG ₄ -BCN (Simultaneous technology) (50 mM THF solution) were added sequentially to 200. Mu.L of GDP-FAm (100 mM aqueous solution). The reaction was stirred at room temperature for 4h and monitored by thin layer chromatography. Purification by Pre-HPLC gave the product as a white solid (7.4 mg, 36%). HRMS (ESI-): C ₃₈H ₅₉N ₇O ₂₂P ₂(M-2H ⁺)/2 calculated 512.6522, found 512.6532. ¹H NMR(400MHz,D ₂O)δ8.17(s,1H),5.92(d,J＝5.9Hz,1H),4.93(t,J＝7.8Hz,1H),4.78-4.75(m,1H),4.52(dd,J＝5.1,3.5Hz,1H),4.36-4.32(m,1H),4.22(dd,J＝5.4,3.4Hz,2H),4.14(d,J＝8.2Hz,2H),3.86(d,J＝2.3Hz,1H),3.75(t,J＝6.3Hz,2H),3.69-3.64(m,14H),3.62-3.58(m,4H),3.31(t,J＝5.3Hz,2H),3.29-3.26(m,1H),2.55(t,J＝6.2Hz,2H),2.29-2.15(m,6H),1.54-1.51(m,2H), 1.39-1.31(m,1H),0.92(t,J＝9.8Hz,2H).

EXAMPLE 8 Synthesis of GDP-FAmP ₄ TCO

To 400. Mu.L GDP-FAm (100 mM in water) was added sequentially 400. Mu.L NaHCO ₃ (200 mM in water), 1.36mL THF, and 440. Mu.L NHS-PEG ₄ -TCO (CLICK CHEMISTRY Tools) (50 mM in THF). The reaction was stirred at room temperature for 4h and monitored by thin layer chromatography. Purification by Pre-HPLC gave the product as a white solid (8.2 mg, 20%). HRMS (ESI-): C ₃₆H ₅₉N ₇O ₂₂P ₂(M-H ⁺)/2 calculated 1002.3116, found 1002.3134.

EXAMPLE 9 Synthesis of GDP-FAmP ₄ Biotin

To 500. Mu.L GDP-FAm (100 mM in water) were added sequentially 500. Mu.L NaHCO ₃ (200 mM in water), 1.95mL THF, and 550. Mu.L NHS-PEG ₄ -Biotin (CLICK CHEMISTRY Tools) (100 mM in THF). The reaction was stirred at room temperature for 4h and monitored by thin layer chromatography. Purification by Pre-HPLC afforded the product as a white solid (20.5 mg, 38%). HRMS (ESI-): C ₃₇H ₆₁N ₉O ₂₂P ₂S(M-H ⁺) calculated 1076.3054, found 1076.3068. ¹H NMR(400MHz,D ₂O)δ8.12(s,1H),5.92(d,J＝6.1Hz,1H),4.93(t,J＝7.8Hz,1H),4.78-4.77(m,1H),4.59(dd,J＝7.9,4.6Hz,1H),4.53(dd,J＝5.2,3.4Hz,1H),4.39(dd,J＝7.9,4.4Hz,1H),4.35-4.34(m,1H),4.22(dd,J＝5.4,3.4Hz,2H),3.87(d,J＝3.1Hz,1H),3.76(t,J＝6.3Hz,2H),3.69-3.66(m,14H),3.63-3.60(m,3H),3.59-3.56(m,1H),3.38(t,J＝5.3Hz,2H),3.32-3.26(m,2H),2.97(dd,J＝13.1,5.0Hz,1H),2.77(d,J＝13.0Hz,1H),2.56(t,J＝6.2Hz,2H),2.26(t,J＝7.3Hz,2H),1.74-1.51(m,4H),1.42-1.34(m,2H).

EXAMPLE 10 Synthesis of GDP-FAzP ₄ Biotin

To 400. Mu.L of GDP-FAz (50 mM aqueous solution) were added 400. Mu.L of CuSO ₄/BTTP (5 mM/10mM aqueous solution), 210. Mu. L propargyl-PEG ₄ -Biotin (CLICK CHEMISTRY Tools) (100 mM methanol solution) and 40. Mu.L of sodium ascorbate (250 mM aqueous solution) in this order, and the reaction was stirred at room temperature for 5 hours and monitored by thin layer chromatography. Then, 2mM BCS (disodium salt of 2, 9-dimethyl-4, 7-diphenyl-1, 10-phenanthroline disulfonic acid) was added to quench the reaction. Purification by Pre-HPLC gave the product as a white solid (14.4 mg, 60%). HRMS (ESI-): C ₃₇H ₅₉N ₁₁O ₂₁P ₂S(M-H ⁺) calculated 1086.3010, found 1086.3040. ¹H NMR(400MHz,D ₂O)δ8.15(s,1H),8.11(s,1H),5.89(d,J＝6.0Hz,1H),4.94-4.90(m,1H),4.77-4.76(m,1H),4.73-4.68(m,1H),4.65(d, J＝3.1Hz,2H),4.62-4.59(m,1H),4.58-4.56(m,1H),4.53-4.51(m,1H),4.38(dd,J＝8.0,4.4Hz,1H),4.34-4.31(m,1H),4.25-4.16(m,2H),4.05-4.02(m,1H),3.82(s,1H),3.72-3.65(m,14H),3.61(t,J＝5.3Hz,2H),3.37(t,J＝5.2Hz,2H),3.30-3.25(m,1H),2.96(dd,J＝13.1,5.0Hz,1H),2.77-2.72(m,1H),2.24(t,J＝7.3Hz,2H),1.74-1.49(m,4H),1.40-1.32(m,2H).

Example 11 preparation of Trastuzumab- (Galβ1, 4) GlcNAc

First, trastuzumab (final concentration 5 mg/mL), endoS (final concentration 0.05 mg/mL), and Alfc (final concentration 1.5 mg/mL) were sequentially added to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 37℃for 24 hours. Then, UDP-galactose (uridine diphosphate galactose) (final concentration 5 mM), manganese chloride (final concentration 5 mM), and BGalT (Y289L) (final concentration 0.5 mg/mL) were sequentially added to the above reaction solution, and the above mixture was reacted at 30℃for 24 hours. Subsequently, the reaction solution was purified by protein A resin (gold Style) to obtain a modified target antibody. The main peak was identified by mass spectrometry as trastuzumab- (Galβ1, 4) GlcNAc (145909 Da, > 90%).

The amino acid sequence of the light chain of Trastuzumab is shown as SEQ ID NO. 15, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 16.

Example 12 preparation of Trastuzumab- (GalNAzβ1, 4) GlcNAc

First, trastuzumab (final concentration 5 mg/mL), endoS (final concentration 0.05 mg/mL), and Alfc (final concentration 1.5 mg/mL) were added sequentially to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 37℃for 24 hours. Then, UDP-GalNAz (uridine diphosphate-N-acetylazidoaminogalactose) (final concentration 5 mM), manganese chloride (final concentration 5 mM), and BGalT (Y289L) (final concentration 0.5 mg/mL) were sequentially added to the above reaction solution, and the above mixture was reacted at 30℃for 16 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry as trastuzumab- (GalNAzβ1, 4) GlcNAc (146077 Da, > 90%).

EXAMPLE 13 preparation Durvalumab- (GalNAzβ1, 4) GlcNAc

First, in 50mM Tris-HCl (pH 7.0) buffer, dulcis You Shan antibody (Durvalumab) (final concentration 5 mg/mL), endoS (final concentration 0.05 mg/mL), and Alfc (final concentration 1.5 mg/mL) were added in this order, and the above mixture was reacted at 37℃for 24 hours. Then, UDP-GalNAz (uridine diphosphate-N-acetylazidoaminogalactose) (final concentration 5 mM), manganese chloride (final concentration 5 mM), and BGalT (Y289L) (final concentration 0.5 mg/mL) were sequentially added to the above reaction solution, and the above mixture was reacted at 30℃for 16 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry as durvalumab- (GalNAzβ1, 4) GlcNAc (146963 Da, > 90%).

Durvalumab has the amino acid sequence shown in SEQ ID NO. 17, and has the amino acid sequence shown in SEQ ID NO. 18.

Example 14 preparation of Trastuzumab- (Galβ1, 4) GlcNAc-FAz

First, trastuzumab- (Galβ1, 4) GlcNAc (final concentration 5 mg/mL), GDP-FAz (final concentration 5 mM), magnesium chloride (final concentration 10 mM) and HpFT-2HR (final concentration 0.5 mg/mL) were added in this order to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 30℃for 16 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry as trastuzumab- (Galβ1, 4) GlcNAc-FAz (146268 Da, > 90%).

Example 15 preparation of Trastuzumab- (Galβ1, 4) GlcNAc-FAmAz

First, trastuzumab- (Galβ1, 4) GlcNAc (final concentration 5 mg/mL), GDP-FAmAz (final concentration 5 mM), magnesium chloride (final concentration 10 mM) and HpFT-2HR (final concentration 0.5 mg/mL) were added in this order to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 30℃for 16 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry as trastuzumab- (Galβ1, 4) GlcNAc-FAmAz (146383 Da, > 90%).

Example 16 preparation of Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az

First, trastuzumab- (Galβ1, 4) GlcNAc (final concentration 5 mg/mL), GDP-FAmP ₄ Az (final concentration 5 mM), magnesium chloride (final concentration 10 mM) and HpFT-2HR (final concentration 0.5 mg/mL) were added in this order to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 30℃for 16 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry to be trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az (146773 Da, > 90%).

EXAMPLE 17 preparation of Trastuzumab- (GalNAzβ1, 4) GlcNAc-FAmP ₈ Tz

First, trastuzumab- (GalNAz. Beta.1, 4) GlcNAc (final concentration 5 mg/mL), GDP-FAmP ₈ Tz (final concentration 5 mM), magnesium chloride (final concentration 10 mM), and HpFT-2HR (final concentration 0.5 mg/mL) were added in this order to 50mM Tris-HCl buffer, and the above mixture was reacted at 30℃for 24 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry to be trastuzumab- (GalNAzβ1, 4) GlcNAc-FAmP ₈ Tz (147578 Da, > 90%).

EXAMPLE 18 preparation Durvalumab- (GalNAzβ1, 4) GlcNAc-FAmP ₈ Tz

First, durvalumab- (GalNAzβ1, 4) GlcNAc (final concentration 5 mg/mL), GDP-FAmP ₈ Tz (final concentration 5 mM), magnesium chloride (final concentration 10 mM) and HpFT-2HR (final concentration 0.5 mg/mL) were added in this order to 50mM Tris-HCl (pH 7.0) buffer, and the above mixture was reacted at 30℃for 24 hours. And then purifying the reaction solution through protein A resin to obtain the modified target antibody. The main peak was identified by mass spectrometry to be durvalumab- (GalNAzβ1, 4) GlcNAc-FAmP ₈ Tz (148475 Da, > 90%).

EXAMPLE 19 preparation of DBCO-ZHer _2:342

To 50mM Tris-HCl (pH 7.0) buffer, ZHer _2:342 affinity (final concentration 1.5 mg/mL), DBCO-PEG ₅ -GGG (final concentration 2 mM) (Simultaneous technology), sodium chloride (final concentration 150 mM), calcium chloride (final concentration 5 mM) and Sortase 5M (final concentration 0.2 mg/mL) were added in this order, and the above mixture was reacted at 25℃for 3 hours. After the reaction time point is reached, a proper amount of Ni-NTA filler is added into the reaction solution, and the mixture is evenly mixed at room temperature in a reverse way for 1h of incubation, so that the Sortase 5M and unreacted ZHer _2:342 affibody are removed. After the incubation, the reaction solution containing the Ni-NTA filler is centrifuged at 4000rpm for 1min, the filler is removed, and the reaction solution is purified by an SP column (cation exchange chromatography column, suzhou Nami), thereby obtaining the modified target product. The main peak is shown to be DBCO-ZHer _2:342 (7923 Da) by mass spectrum identification.

EXAMPLE 20 preparation of DBCO-aPDL1

To 50mM Tris-HCl (pH 7.0) buffer, aPDL (final concentration 1.5 mg/mL), DBCO-PEG ₅ -GGG (final concentration 2 mM) (Simultaneous technology), sodium chloride (final concentration 150 mM), calcium chloride (final concentration 5 mM) and Sortase5M (final concentration 0.2 mg/mL) were added in this order, and the above mixture was reacted at 25℃for 3 hours. After the reaction time point is reached, a proper amount of Ni-NTA filler is added into the reaction solution, and the mixture is evenly mixed at room temperature in a reverse way for 1h of incubation, and the Sortase5M and unreacted aPDL1 are removed. After the incubation, the reaction solution containing the Ni-NTA filler is centrifuged at 4000rpm for 1min, the filler is removed, and the reaction solution is purified by an SP column, so that a modified target product is obtained. The main peak is DBCO-aPDL1 (15213 Da) by mass spectrum identification.

EXAMPLE 21 preparation of DBCO-aCD3

To 50mM Tris-HCl (pH 7.0) buffer, aCD3 (final concentration 1.5 mg/mL), DBCO-PEG ₅ -GGG (final concentration 2 mM) (Simultaneous technology), sodium chloride (final concentration 150 mM), calcium chloride (final concentration 5 mM) and Sortase 5M (final concentration 0.2 mg/mL) were added in this order, and the above mixture was reacted at 25℃for 3 hours. After reaching the reaction time point, adding a proper amount of Ni-NTA filler into the reaction solution, and uniformly mixing at room temperature in a reverse way for incubation for 1h to remove the Sortase 5M and unreacted aCD3 nano antibody. After the incubation, the reaction solution containing the Ni-NTA filler is centrifuged at 4000rpm for 1min, the filler is removed, and the reaction solution is purified by an SP column, so that a modified target product is obtained. The main peak was identified by mass spectrometry as DBCO-aCD3 (15099 Da).

EXAMPLE 22 preparation of BCN-aCD3

To 50mM Tris-HCl (pH 7.0) buffer was added aCD3 (final concentration 1.5 mg/mL), BCN-PEG ₅ -GGG (final concentration 2 mM) (Simultaneous technology), sodium chloride (final concentration 150 mM), calcium chloride (final concentration 5 mM) and Sortase5M (final concentration 0.2 mg/mL) in this order, and the above mixture was reacted at 25℃for 3 hours. After the reaction time point was reached, a proper amount of Ni-NTA filler was added to the reaction solution, and the mixture was incubated for 1 hour at room temperature with upside down mixing, to remove Sortase5M and unreacted aCD3. After the incubation, the reaction solution containing the Ni-NTA filler is centrifuged at 4000rpm for 1min, the filler is removed, and the reaction solution is purified by an SP column, so that a modified target product is obtained. The main peak was identified by mass spectrometry to be BCN-aCD3 (14990 Da).

EXAMPLE 23 preparation of Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az-DBCO-aCD3

Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az (final concentration 1 mg/mL), DBCO-aCD3 (final concentration 3 mg/mL) was added sequentially to 1 XPBS (pH 7.0) buffer, and the mixture was reacted at room temperature for 8 hours. And purifying the reaction solution through an SP column to obtain the modified target antibody. The main peak was identified by mass spectrometry and was Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az-DBCO-aCD3 (abbreviated as Tras-aCD 3) (176973 Da, MAR 2). MAR (molecule of interest to antibody ratio, ratio of molecules of interest to antibodies). The results are shown in fig. 3E, which shows that the obtained bispecific antibodies have good homogeneity.

EXAMPLE 24 preparation of Trastuzumab- (GalNAz-DBCO-aPDL 1) GlcNAc-FAmP ₈ Tz-BCN-aCD3

Trastuzumab- (GalNAz) GlcNAc-FAmP ₈ Tz (final concentration 1 mg/mL), DBCO-aPDL1 (final concentration 3 mg/mL) was added sequentially to 1 XPBS (pH 7.0) buffer, and the mixture was reacted at room temperature for 8h. BCN-aCD3 (final concentration 2 mg/mL) was then added to the reaction solution, and the reaction was continued at room temperature for 16 hours. And purifying the reaction solution through an SP column to obtain the modified target antibody. The main peak was identified by mass spectrometry and was Trastuzumab- (GalNAz-DBCO-aPDL 1) GlcNAc-FAmP ₈ Tz-BCN-aCD3 (abbreviated as Tras-aPDL-aCD 3) (207930Da, MAR2+MAR2). The results are shown in fig. 3E, which shows that the obtained trispecific antibodies have good homogeneity.

EXAMPLE 25 preparation Durvalumab- (GalNAz-DBCO-ZHer _2:342)GlcNAc-FAmP ₈ Tz-BCN-aCD3

To 1 XPBS (pH 7.0) buffer solution was added Durvalumab- (GalNAz) GlcNAc-FAmP ₈ Tz (final concentration 1 mg/mL), DBCO-ZHer _2:342 (final concentration 1.5 mg/mL) in this order, and the above mixture was reacted at room temperature for 8 hours. BCN-aCD3 (final concentration 2 mg/mL) was then added to the reaction solution, and the reaction was continued at room temperature for 16 hours. And purifying the reaction solution through an SP column to obtain the modified target antibody. The main peak was Durvalumab- (GalNAz-DBCO-ZHer _2:342)GlcNAc-FAmP ₈ Tz-BCN-aCD3 (Dur-ZHer-aCD 3 for short) (194249 Da, MAR2+MAR2) by mass spectrometry, and the results are shown in FIG. 3E, which shows that the obtained trispecific antibody has excellent uniformity.

Example 26 comparison of the efficiency of multispecific antibodies of the application was made by reacting antibody-Fuc x' conjugates containing different linkers with Y ₁-(L ₁') _n' -AB 2.

DBCO-aCD3 (final concentration 1 mg/mL) was added to 1 XPBS (pH 7.0) buffer, followed by Trastuzumab- (Galβ1, 4) GlcNAc-FAz, trastuzumab- (Galβ1, 4) GlcNAc-FAmAz or Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az (final concentration 0.5 mg/mL), and the above mixture was reacted at room temperature for 2 hours. After the reaction time point was reached, 20. Mu.L of each sample was taken, 5. Mu.L of loading buffer was added, and the sample was treated at 90℃for 5 minutes, and then the same volume of sample was taken for SDS-PAGE detection. The results are shown in fig. 9. The results show that the device hasThe antibody-Fuc' conjugate of the zygote structure has higher efficiency in preparing the multi-specific antibody disclosed by the application through reaction with Y ₁-(L ₁') _n' -AB 2. By comparing the amounts of the product bands (HC (heavy chain) +aCD3) after the reaction, trastuzumab- (Galβ1, 4) GlcNAc-FAmAz and Trastuzumab- (Galβ1, 4) GlcNAc-FAmP ₄ Az (withThe adaptor structure) and DBCO-aCD3 are significantly more efficient than Trastuzumab- (Galβ1, 4) GlcNAc-FAz and DBCO-aCD 3.

Example 27 comparison of conversion efficiency of GDP-fucose derivatives containing different zygotes transferred to antibodies under the catalysis of fucose transferase

Trastuzumab- (Galβ1, 4) GlcNAc (final concentration 2 mg/mL), GDP-FAmP ₄ Biotin or GDP-FAzP ₄ Biotin (final concentration 1 mM), magnesium chloride (final concentration 5 mM), and HpFT-2HR (final concentration 0.5 mg/mL) were added sequentially to 50mM Tris-HCl buffer, and the above mixture was reacted at 30℃for 10min, with three groups being placed in parallel. After reaching the reaction time point, 10mM LacNAc was added to the reaction solution and the reaction was stopped by a desalting column (Zeba ^TM SPIN DESALTING Columns/2ml/40K, siemens technology), and finally detected by mass spectrometry, and the conversion was calculated.

Trastuzumab- (GalNAz. Beta.1, 4) GlcNAc (final concentration 2 mg/mL), GDP-FAmP ₄ Biotin or GDP-FAzP ₄ Biotin (final concentration 1 mM), magnesium chloride (final concentration 5 mM), and HpFT-2HR (final concentration 0.5 mg/mL) were sequentially added to 50mM Tris-HCl buffer, and the above mixture was reacted at 30℃for 4 hours, with three groups being placed in parallel. After reaching the reaction time point, 10mM LacNAc was added to the reaction solution and the reaction was stopped by a desalting column (Zeba ^TM SPIN DESALTING Columns/2ml/40K, siemens technology), and finally detected by mass spectrometry, and the conversion was calculated. Conversion = (MAR 0 peak intensity x 0+mar1 peak intensity x 1+mar2 peak intensity x 2)/2 (MAR 0 peak intensity+mar1 peak intensity+mar2 peak intensity) ×100%.

The results are shown in the table below. The result shows that hasGDP-fucose derivatives of the zygotic structure (e.g., GDP-FAmP ₄ Biotin) are compared to those having the structureGDP-fucose derivatives of the zygotic structure (e.g., GDP-FAzP ₄ Biotin) have significantly improved conversion rates on antibodies (e.g., trastuzumab- (Galβ1, 4) GlcNAc or Trastuzumab- (GalNAzβ1, 4) GlcNAc).

Example 28 Tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 antigen binding assay

Affinity detection for Her2 antigen: tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 were added at various concentrations to Her2 positive SKOV3 (Her2+/PDL 1-) cells, and the cells were resuspended in FACs buffer (DPBS containing 2% bovine serum) in duplicate, 60000 cells/well. After incubation at 4℃for 30min, unbound multispecific antibodies on the cells were washed off with FACs buffer (DPBS containing 2% fetal bovine serum), after which the cells were resuspended in 30. Mu.L FACs buffer. Then, a streaming antibody targeting human IgG Fc-PE (Biolegend, clone HP6017,409304) was added to the cells, and after incubation at 4℃for 30 minutes, the streaming antibody not bound to the multispecific antibody was washed off with FACs buffer, and then the cells were resuspended in 100. Mu.L of FACs buffer. Samples were then collected using a flow cytometer (Angilent Novocyte quanteon) and analyzed using a PE channel, and finally analyzed by FlowJo 10.5.3 and GRAPHPAD PRISM 8. As a result, as shown in FIG. 4A, all three multispecific antibodies were able to bind to Her2 antigen, with Tras-aCD3 and Tras-aPDL1-aCD3 having similar binding affinities for Her2 antigen, and Dur-ZHer-aCD3 being slightly weaker than the former two.

Affinity detection for CD3 antigen: various concentrations of Tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 were added to CD3+ Jurkat cells, and the cells resuspended in FACs buffer for 60000 cells/well in duplicate. After incubation at 4 ℃ for 30 minutes, unbound multispecific antibodies on the cells were washed away with FACs buffer, after which the cells were resuspended in 30 μl of FACs buffer. The flow-through antibodies targeting human IgG Fc-PE were added to the cells, and after incubation at 4 ℃ for 30min, the flow-through antibodies that did not bind to the multispecific antibodies were washed away with FACs buffer, and then the cells were resuspended in 100 μl of FACs buffer. Samples were then collected using a flow cytometer (Angilent Novocyte quanteon) and analyzed using a PE channel, and finally data processed through FlowJo 10.5.3 and GRAPHPAD PRISM 8. As shown in FIG. 4C, all three multispecific antibodies can bind to the CD3 antigen, with Tras-aPDL1-aCD3 having similar binding affinity to Dur-ZHer-aCD3 for the CD3 antigen, and Tras-aCD3 being slightly better than the former two.

Affinity detection for PDL1 antigen: recombinant PDL-1 antigen (PDL-1, near shore protein) was diluted to a final concentration of 250ng/mL with coating buffer (50 mM Na ₂CO ₃/NaHCO ₃, pH 9.6), inoculated into 96-well plates at a volume of 100. Mu.L per well and incubated overnight at 4℃in a refrigerator. The following day after removal of the coating solution, the wells were first blocked with a blocking solution (PBS solution containing 3% (v/v) fetal bovine serum albumin) for 2 hours at 30℃in an incubator with 200. Mu.L per well. After washing 3 times with plate wash buffer PBST (0.03% tween-20 in PBS), the samples to be tested (Durvalumab, dur-ZHer-aCD3, tras-aPDL1-aCD 3) were diluted to a series of final concentrations (9000ng/mL,3000ng/mL,1000ng/mL,333.33ng/mL,111.11ng/mL,37.04ng/mL,12.35ng/mL,4.12ng/mL,1.37ng/mL,0.46ng/mL,0.15ng/mL,0ng/mL) and added to the plates, 100. Mu.L per well, respectively, using sample dilution buffer (1% (v/v) in PBS containing fetal bovine serum albumin). After the addition of the sample, the plate was washed with PBST buffer 3 times after the addition of the sample was continued for 1 hour in a 30℃incubator, and horseradish peroxidase (HRP) -conjugated goat anti-human IgG antibody (Biyun) was added to each well and incubated for half an hour at 30 ℃. After the incubation, each well was washed 5 times with PBST buffer, and then 100. Mu.L of 3,3', 5' -tetramethylbenzidine substrate was added for co-processing color development. After 5-10 minutes incubation, the reaction was stopped by adding 100. Mu.L of 3M HCl. Finally, the absorbance was read at 450nm on a Synergy ^TM LX plate reader. As a result, as shown in FIG. 4B, dur-ZHer-aCD3 and Tras-aPDL1-aCD3 can be bound to PDL-1, wherein Dur-ZHer-aCD3 and Durvalumab have similar binding force and are slightly stronger than Tras-aPDL1-aCD 3.

EXAMPLE 29 Tras-aCD3 cell killing Activity assay

Cytotoxicity test of SKOV3

The killing of effector cells to target cells (using adherent cells to generate impedance on a metal electrode plate, the impedance decreases due to the deterioration of the adherent effect after killing) was tested using an RTCA-DP instrument, which detects the number of living cells based on microelectronic impedance technology. First, after 50. Mu.L of DMEM (Gibco) was added to each well of an E-Plate 16PET Plate (RTCA-DP instrument kit), the base line was measured in the RTCA-DP instrument. Then, the E-Plate 16PET Plate was taken out, 10000 SKOV3 cells were inoculated in each well, and after 30 minutes of standing at room temperature, the E-Plate 16PET Plate was put into an RTCA-DP instrument, and the growth curve of the cells was started to be tested. After 25h, when the cell curve grew to late in the logarithmic growth phase of the cells, the test was suspended, the E-Plate 16PET Plate was removed from the instrument, tras-aCD3 and 50000 hBMCs (human peripheral blood mononuclear cells) were added to the experimental wells at a final concentration of 15nM, and control wells were set up to separately add 50000 hPBMC,30nM aCD3 and 50000 hBMCs, or 15nM Trastuzumab and 50000 hBMCs, in duplicate. Finally, the test was continued for 30 hours, data were derived by the instrument and analyzed by map with GRAPHPAD PRISM. As shown in FIG. 5A, the addition of hBMC, hBMC and aCD3 to SKOV3 did not significantly inhibit the growth of SKOV3 cells. The experimental group with both hPBMC and trastuzumab inhibited SKOV3 cell growth slightly. In contrast, the simultaneous addition of hBMC and Tras-aCD3 significantly and efficiently inhibited the growth of SKOV3 cells, indicating that only coupling Trastuzumab and aCD3 together caused T-cells to kill SKOV3.

Cytotoxicity test of MDA-MB-468

Effector cells were tested for killing of target cells using an RTCA-DP instrument. First, after 50. Mu.L of DMEM was added to each well of the E-Plate 16PET Plate, the baseline was measured by placing in an RTCA-DP instrument. Then, the E-Plate 16PET Plate was taken out, 10000 MDA-MB-468 (Her 2-) cells were inoculated in each well, and after 30 minutes of standing at room temperature, the E-Plate 16PET Plate was put into an RTCA-DP instrument, and the growth curve of the cells was started to be tested. After 42h, when the cell curve grew to late in the logarithmic growth phase of the cells, the test was suspended, the E-Plate 16PET Plate was removed from the instrument, tras-aCD3 and 50000 hBMCs were added to the experimental wells at a final concentration of 15nM, and control wells were set up, separately adding 50000 hPBMC,30nM aCD3 and 50000 hBMCs, or 15nM Trastuzumab and 50000 hBMCs, in duplicate. Finally, the test was continued for 38 hours, data were derived by the instrument and plotted using GRAPHPAD PRISM a for analysis. The results are shown in FIG. 5B, and all experimental groups did not significantly affect the growth of MDA-MB-468 cells. Meanwhile, compared with the experimental result of Her2 positive SKOV3 cells, the double antibody cannot kill Her2 negative cells in the environment with the hBMC, so that the double antibody has antigen lazy.

Example 30 Tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 and T cytotoxicity test

Cytotoxicity test of SKOV3

Effector cells were tested for killing of target cells using an RTCA-DP instrument. First, after 50. Mu.L of DMEM was added to each well of the E-Plate 16PET Plate, the baseline was measured by placing in an RTCA-DP instrument. Then, the E-Plate 16PET Plate was taken out, 10000 SKOV3 (Her2+/PDL 1-) cells were inoculated in each well, and after 30 minutes of standing at room temperature, the E-Plate 16PET Plate was put into an RTCA-DP instrument, and the growth curve of the cells was started to be tested. After 25h, when the cell curve grew to the late phase of the logarithmic cell phase, the test was suspended, the E-Plate 16PET Plate was removed from the instrument, and 50000 hBMCs and Tras-aCD3, tras-aPDL1-aCD3 or Dur-ZHer-aCD3, each at a final concentration of 1.5pM, were added to the experimental wells in quadruplicate. Finally, the test was continued for 157h, data was derived by the instrument and analyzed by GRAPHPAD PRISM plot. The results are shown in FIG. 6A, where three multispecific antibodies all significantly killed SKOV3 cells in the presence of hBMC. Wherein Tras-aCD3 and Tras-aPDL1-aCD3 kill SKOV3 more than Dur-ZHer-aCD3.

Cytotoxicity test of JIMT1

Effector cells were tested for killing of target cells using an RTCA-DP instrument. First, after 50. Mu.L of DMEM was added to each well of the E-Plate 16PET Plate, the baseline was measured by placing in an RTCA-DP instrument. Then, the E-Plate 16PET Plate was taken out, 20000 JIMT1 (Her2+/PDL1+) cells were inoculated in each well, and after 30 minutes of standing at room temperature, the E-Plate 16PET Plate was put into an RTCA-DP instrument, and the growth curve of the cells was started to be tested. After 25h, when the cell curve grew to the late phase of the logarithmic cell phase, the test was suspended, the E-Plate 16PET Plate was removed from the instrument, 100000 hBMCs and Tras-aCD3, tras-aPDL1-aCD3 or Dur-ZHer-aCD3, each at a final concentration of 1.5pM, were added to the experimental wells in quadruplicate. Finally, the test is continued for 160 hours, data are exported through an instrument, and are plotted and analyzed by GRAPHPAD PRISM. The results are shown in fig. 6B, where three multispecific antibodies all had significant killing of JIMT 1in the presence of hPBMC. It is worth mentioning that Dur-ZHer-aCD3 kills significantly more than SKOV3 (Her2+/PDL1-) on JIMT1 (Her2+/PDL1+) compared to Tras-aCD3 and Tras-aPDL1-aCD 3. This is probably due to the enhancement of PDL1 targeting in Dur-ZHer-aCD 3.

Cytotoxicity test of MDA-MB-468

Effector cells were tested for killing of target cells using an RTCA-DP instrument. First, after 50. Mu.L of DMEM was added to each well of the E-Plate 16PET Plate, the baseline was measured by placing in an RTCA-DP instrument. Then, the E-Plate 16PET Plate was taken out, 20000 MDA-MB-468 (Her 2-/PDL 1-) cells were inoculated in each well, and after 30 minutes of standing at room temperature, the E-Plate 16PET Plate was put into an RTCA-DP instrument, and the growth curve of the cells was started to be tested. After 42h, when the cell curve grew to the late phase of the logarithmic cell phase, the test was suspended, the E-Plate 16PET Plate was removed from the instrument, 100000 hBMCs and Tras-aCD3, tras-aPDL1-aCD3 or Dur-ZHer-aCD3, each at a final concentration of 1.5pM, were added to the experimental wells in quadruplicate. Finally, the test is continued for 38h, data are exported by an instrument, and plotted and analyzed by GRAPHPAD PRISM. The results are shown in FIG. 6C, which shows that none of the experimental groups significantly inhibited MDA-MB-468 cell growth.

Example 31 Tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 immunoactivation assay

T cell activation assay

Hpbmcs were obtained by isolation of human peripheral blood, 100000 hPBMC cells were seeded in flat bottom 96-well plates, and control groups were designed, respectively, untreated group (non-plus 20000 SKOV 3), aCD3 group with a final concentration of 30nM (non-plus 20000 SKOV 3), commercial anti-human CD3 antibody OKT3 (BioXCell, BE 0001-2) group with a final concentration of 15nM (non-plus 20000 SKOV 3), and Tras-aCD3, tras-aPDL-aCD 3 and Dur-ZHer-aCD3 group with a final concentration of 15nM (non-plus 20000 SKOV 3), all experimental well volumes were 100 μl in triplicate. After 36 hours, 1000g centrifugation for 10min, cell supernatants were collected and frozen in-80 ℃ refrigerator for subsequent analysis. At the same time, the centrifuged cells were resuspended in 30. Mu.L of FACs buffer (DPBS with 2% fetal bovine serum) and stained with anti- CD4-PB(Biolegend,Clone OKT4,317424),CD8a-PE(Biolegend,Clone HIT8a,300908),CD69-APC(Biolegend,Clone FN50,310910),CD25-Percp/Cy5.5(Biolegend,Clone BC96,302626),PD-1-FITC(Biolegend,Clone EH12.2H7,329904) flow antibody, wherein CD4-PB, CD8a-PE is the T cell used to differentiate between CD4+ and CD8+, CD69-APC is used to assess up-regulation of CD69, CD25-Percp/Cy5.5 is used to assess up-regulation of CD25, and PD-1-FITC is used to assess up-regulation of PD-1. Samples were collected using a flow cytometer (Angilent Novocyte quanteon) and analyzed by FlowJo 10.5.3 and GRAPHPAD PRISM 8. As shown in FIGS. 7A-B, tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3, both caused significant up-regulation of CD69, CD25 and PD-1 in CD4+ and CD8+ T cells upon addition of Her2 positive SKOV3 cells. This suggests that T cell activation by these three multispecific antibodies is antigen-lazy.

2. Cytokine measurement

The supernatant collected in the previous step was removed from the-80 ℃ refrigerator, thawed at room temperature, centrifuged at 1000 x g for 10min to remove particles and polymer and the supernatant diluted 5-fold. Human IFN-gamma and human TNF-alpha were measured in samples using an Elisa kit (Beijing four cypress). First, 100. Mu.L of supernatants from different control groups were incubated with coated plates with human IFN-gamma and human TNF-alpha antibodies for 90min at 37℃in an incubator. After the incubation was completed, the plate was washed 3 times with a washing solution, 100. Mu.L of biotin solution was added, and incubated in an incubator at 37℃for 60 minutes. After the incubation was completed, the plate was washed 3 times with a washing solution, 100. Mu.L of the enzyme conjugate was added, and incubated in an incubator at 37℃for 30 minutes. After the incubation, the plate was washed 3 times with washing solution, finally the chromogenic agent was added for 15min incubation in the dark, and finally the stop solution was added to immediately test the OD450 value. A standard curve was fitted by origin and analyzed with GRAPHPAD PRISM a. As shown in FIG. 7C, tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 all caused significant up-regulation of cytokine release upon addition of Her2 positive SKOV3 cells, indicating that cytokine release by these three multispecific antibodies was antigen-lazy.

Example 32 Tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 concentration-dependent T cell activation assay

Human peripheral blood mononuclear cells (hpbmcs) were obtained by isolation of human peripheral blood, 100000 hPBMC cells and 20000 SKOV3 (E/t=5) were plated in flat bottom 96-well plates and Tras-aCD3, tras-aPDL1-aCD3 or Dur-ZHer-aCD3 at different concentrations (1.5 fm,15fm,150fm,1500fm,15000fm,150000fm,1500000 fm) were added to the cell solution in triplicate for all experimental well volumes of 100 μl. After 16 hours, cells were collected and washed with FACs buffer. Finally, cells were resuspended in 30. Mu.L FACs buffer and stained with anti- CD4-PB(Biolegend,Clone OKT4,317424),CD8a-PE(Biolegend,Clone HIT8a,300908),CD69-APC(Biolegend,Clone FN50,310910),CD25-Percp/Cy5.5(Biolegend,Clone BC96,302626),PD-1-FITC(Biolegend,Clone EH12.2H7,329904) flow antibody, where CD4-PB, CD8a-PE is the T cell used to differentiate between CD4+ and CD8+, CD69-APC is used to assess up-regulation of CD69, CD25-Percp/Cy5.5 is used to assess up-regulation of CD25, and PD-1-FITC is used to assess up-regulation of PD-1. Samples were collected using a flow cytometer (Angilent Novocyte quanteon) and analyzed by FlowJo 10.5.3 and GRAPHPAD PRISM 8. As shown in FIG. 8, as the drug concentration increases, tras-aCD3, tras-aPDL1-aCD3 and Dur-ZHer-aCD3 resulted in an increase in the extent of up-regulation of CD69, CD25 and PD-1 in CD4+ and CD8+ T cells, with the ability to activate T cells in a concentration-dependent manner.

Claims

A multispecific antibody comprising:

An antibody moiety a capable of specifically binding to a first target; and

2 Containing formulae (I)Sugar chain portions of the structures shown;

and the multispecific antibody has a structure represented by formula (II):

wherein:

the a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target;

GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1;

GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage;

the Fuc comprises a structure Fuco-L-AB2, wherein the structure of Fuco is shown as a formula (III): AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc is linked to said GlcNAc by an α -1,3 glycosidic bond;

at least one of the first target and the second target is CD3; and

The position of the amino acid N297 is determined according to the EU index numbering in Kabat.
The multispecific antibody of claim 1, wherein the first target is not the same as the second target.
The multispecific antibody of any one of claims 1-2, wherein the AB1 and the AB2 are each independently an antigen-binding fragment of an antibody.
The multi-specific antibody of claim 3, wherein the antigen-binding fragment is a Fab, F (ab) ₂,F(ab'),F(ab') ₂, scFv, affibody (affibody) and/or single domain antibody.
The multispecific antibody of any one of claims 1-4, wherein the first target is a tumor-associated antigen and the second target is CD3.
The multispecific antibody of claim 5, wherein the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.
The multispecific antibody of any one of claims 1-6, wherein the a is an IgG antibody.
The multispecific antibody of any one of claims 1-7, wherein the AB1 comprises an antigen-binding portion of an antibody selected from the group consisting of: trastuzumab and dulcis You Shan antibody.
The multispecific antibody of any one of claims 1-8, wherein the AB1 comprises an antibody heavy chain CDR3 (HCDR 3), and the HCDR3 comprises the amino acid sequence of SEQ ID NO:24 and 32 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-9, wherein the AB1 comprises an antibody heavy chain CDR2 (HCDR 2), and the HCDR2 comprises the amino acid sequence of SEQ ID NO:23 and 31 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-10, wherein the AB1 comprises an antibody heavy chain CDR1 (HCDR 1), and the HCDR1 comprises the amino acid sequence of SEQ ID NO:22 and 30 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-11, wherein the AB1 comprises an antibody light chain CDR3 (LCDR 3), and the LCDR3 comprises the amino acid sequence of SEQ ID NO:21 and 29 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-12, wherein the AB1 comprises an antibody light chain CDR2 (LCDR 2), and the LCDR2 comprises the amino acid sequence of SEQ ID NO:20 and 28 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-13, wherein the AB1 comprises an antibody light chain CDR1 (LCDR 1), and the LCDR1 comprises the amino acid sequence of SEQ ID NO:19 and 27 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-14, wherein the AB1 comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence of SEQ ID NO:26 and 34 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-15, wherein the AB1 comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence of SEQ ID NO:25 and 33 the amino acid sequence shown.
The multispecific antibody of any one of claims 1-16, wherein the a is trastuzumab or rivarox You Shan antibody.
The multispecific antibody of any one of claims 1-17, wherein the AB2 comprises a CD3 antigen-binding portion of an antibody selected from the group consisting of: OKT3, M291, YTH12.5, bob and Cartuzumab.
The multispecific antibody of any one of claims 1-18, wherein the AB2 comprises the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.
The multispecific antibody of any one of claims 1-4 and 7, wherein the first target is CD3 and the second target is a tumor-associated antigen.
The multispecific antibody of claim 20, wherein the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.
The multispecific antibody of any one of claims 1-21, wherein in the Fuco-L-AB2, L has the structure J- (L ₁) _n-X ₁Y ₁-(L ₁') _n'), wherein:

L ₁ is a first linker, n is 0 or 1;

The L ₁ 'is a second linker, and n' is 0 or 1;

j is an adapter directly connected with Fuco;

X ₁Y ₁ is the residue after the ligation of group X ₁ with group Y ₁, wherein the group X ₁ comprises a functional group capable of undergoing a bio-orthogonal ligation reaction and the Y ₁ comprises a functional group capable of undergoing a bio-orthogonal ligation reaction with the X ₁.
The multispecific antibody of claim 22, wherein the X ₁ comprises a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof.
The multispecific antibody of any one of claims 22-23, wherein the X ₁ comprises a functional group selected from the group consisting of: Wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.
The multispecific antibody of any one of claims 22-24, wherein the X ₁ comprises a functional group selected from the group consisting of:
The multispecific antibody of any one of claims 22-25, wherein the X ₁ comprises
The multispecific antibody of any one of claims 22-26, wherein the Y ₁ comprises a functional group selected from the group consisting of: azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2, 4-triazinyl, terminal alkenyl, cycloalkenyl, keto, aldehyde, hydroxylamine, mercapto, maleimide, and functional derivatives thereof.
The multispecific antibody of any one of claims 22-27, wherein the Y ₁ comprises a functional group selected from the group consisting of: Wherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.
The multispecific antibody of any one of claims 22-28, wherein the X ₁ and the Y ₁ comprise a set of structures selected from the group consisting of:

a) X ₁ includes And Y ₁ includes

B) X ₁ includesAnd Y ₁ includes

C) X ₁ includesAnd Y ₁ includes

D) X ₁ includesAnd Y ₁ includesAnd

E) X ₁ includesAnd Y ₁ includes
The multispecific antibody of any one of claims 22-29, wherein the X ₁Y ₁ comprises a structure selected from the group consisting of:
the multispecific antibody of any one of claims 22-30, wherein the J is Wherein said Rf is-CH ₂ -, -NH-or-O-, the left end of the J-structure is directly connected to the Fuco.
The multispecific antibody of any one of claims 22-31, wherein the J isThe left end of the structure is directly connected with the Fuco.
The multispecific antibody of any one of claims 22-32, wherein the L ₁ and the L ₁' are each independently selected from: c ₃-C ₂₀₀ subunit, C ₁-C ₂₀₀ alkylene, C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, Derivatives thereof and any combination thereof, wherein said subunit, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene is optionally substituted with one or more Rs ¹ and/or is optionally interrupted by one or more Rs ², wherein each of said Rs ¹ is independently selected from the group consisting of: halogen, halogen, -OH, -NH ₂ and-COOH, each of said Rs ² being independently selected from: -O-, -S-,Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.
The multispecific antibody of any one of claims 22-33, wherein the L ₁ is selected from: Wherein each s1 is independently an integer from 1 to 50, each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but consecutive adjacent-CH ₂ -is not simultaneously replaced by-O-, the left end of the structure is attached to said J and the right end of the structure is attached to said X ₁.
The multispecific antibody of any one of claims 22-34, wherein the L ₁ is selected from:

And the right end of the structure is connected with the X ₁.
The multispecific antibody of any one of claims 22-35, wherein the L ₁' is selected from:

Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure being attached to said AB2 and the left end of the structure being attached to said Y ₁.
The multispecific antibody of any one of claims 22-36, wherein the L ₁' is selected from:

The right end of the structure is connected with the AB2, and the left end of the structure is connected with the Y ₁.
The multispecific antibody of any one of claims 1-37, wherein the GalX is galactose.
The multispecific antibody of any one of claims 1-37, wherein the GalX is a substituted galactose and one or more hydroxyl groups in the galactose at C2, C3, C4 and/or C6 positions are substituted.
The multispecific antibody of any one of claims 1-37 and 39, wherein the GalX is a substituted galactose and the hydroxyl group at the C2 position in the galactose is substituted.
The multispecific antibody of any one of claims 1-40, wherein the GalX is a monosaccharide.
The multispecific antibody of any one of claims 1-37 and 39-41, wherein the GalX is substituted with a substituent Rg ¹, rg ¹ is selected from the group consisting of: hydrogen, halogen, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, C ₂-C ₂₄ alkenyl, C ₅-C ₂₄ cycloalkenyl, C ₂-C ₂₄ alkynyl, C ₆-C ₂₄ cycloalkynyl, C ₃-C ₂₄ (hetero) aryl, C ₃-C ₂₄ alkyl (hetero) aryl, and C ₃-C ₂₄ (hetero) arylalkyl;

Wherein the alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, (hetero) aryl, alkyl (hetero) aryl, and/or (hetero) arylalkyl are each independently optionally substituted with one or more substituents Rs ⁴, and/or are each independently optionally interrupted by one or more substituents Rs ⁵;

Wherein each of said Rs ⁴ is independently selected from: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN;

Each of said Rs ⁵ is independently selected from: -O-, -S-, Wherein the Rs ³ comprises hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.
The multispecific antibody of any one of claims 1-37 and 39-42, wherein GalX is substitutedInstead of the above-mentioned,

Wherein:

t is 0 or 1;

the Rg ² is selected from the group consisting of: c ₁-C ₂₄ alkylene, C ₃-C ₂₄ cycloalkylene, C ₂-C ₂₄ alkenylene, C ₅-C ₂₄ cycloalkenyl, C ₂-C ₂₄ alkynylene, C ₆-C ₂₄ cycloalkynylene, C ₃-C ₂₄ (hetero) arylene, C ₃-C ₂₄ alkyl (hetero) arylene and C ₃-C ₂₄ (hetero) arylalkylene, wherein the alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, alkyl (hetero) arylene and/or (hetero) arylalkylene are each independently optionally substituted with one or more substituents Rs ⁴ and/or are each independently optionally interrupted with one or more substituents Rs ⁵,

The Rg ³ is selected from the group consisting of: hydrogen, halogen, -OH, -NH ₂、-SH、-N ₃、-COOH、-CN、C ₁-C ₂₄ alkyl, C ₃-C ₂₄ cycloalkyl, C ₂-C ₂₄ alkynyl, C ₅-C ₂₄ cycloalkynyl and C ₂-C ₂₄ (hetero) aryl, wherein each of said alkyl, cycloalkyl, alkynyl, cycloalkynyl and/or (hetero) aryl is independently optionally substituted with one or more Rs ⁴,

Wherein,

Each of said Rs ⁵ is independently selected from: -O-, -S-,The Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl, and

Each of said Rs ⁴ is independently selected from: halogen, -OH, -NH ₂、-SH、-N ₃, -COOH and-CN.
The multispecific antibody of any one of claims 1-43, wherein the GalX is selected from the group consisting of:
the multispecific antibody of any one of claims 1-44, wherein b is 0.
A multispecific antibody comprising:

An antibody moiety a capable of specifically binding to a first target; and

2 Containing formula (IV)Sugar chain portions of the structures shown;

and the multispecific antibody has a structure represented by formula (V):

wherein:

the a comprises a first antigen binding portion AB1 and an Fc region capable of specifically binding to the first target;

GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1;

fuc comprises structure Fuco-L-AB2, wherein Fuco has a structure shown in formula (III): AB2 is a second antigen binding moiety capable of specifically binding to a second target, L is a linker, and the left end of formula (III) is linked to L, said Fuc is linked to said GlcNAc by an α -1,3 glycosidic bond;

GalX is substituted galactose, said GalX is linked to said GlcNAc by a β -1,4 glycosidic bond, and said GalX comprises a third antigen binding portion AB3 capable of specifically binding to a third target;

at least one of the first target, the second target and the third target is CD3; and

The position of the amino acid N297 is determined according to the EU index numbering in Kabat.
The multispecific antibody according to claim 46, wherein the GalX has the structure GalX ₂Y ₂-(L ₂') _m -AB3 wherein GalX ₂Y ₂ is the residue after a ligation of group GalX ₂ with group Y ₂, wherein the GalX ₂ comprises X _2, the X ₂ comprises a functional group capable of participating in a bioorthogonal ligation reaction, and the Y ₂ comprises a functional group capable of undergoing a bioorthogonal ligation reaction with the X ₂; and is also provided with

L ₂' is a linker, and m is 0 or 1.
The multispecific antibody of claim 47, wherein the GalX ₂ is galactose with one or more hydroxyl groups at the C2, C3, C4 and/or C6 positions substituted.
The multispecific antibody of any one of claims 47-48, wherein the GalX ₂ is galactose with a hydroxyl group at the C2 position substituted.
The multispecific antibody of any one of claims 47-49, wherein the GalX ₂ is a monosaccharide.
The multispecific antibody of any one of claims 47-50, wherein X ₂ in the GalX ₂ comprises
The multispecific antibody of any one of claims 47-51, wherein X ₂ in the GalX ₂ comprises
The multispecific antibody of any one of claims 47-52, wherein the GalX ₂ has the structure
The multispecific antibody of any one of claims 47-53, wherein the Y ₂ comprises
The multispecific antibody of any one of claims 47-54, wherein the X ₂Y ₂ comprises a structure selected from the group consisting of:
the multispecific antibody of any one of claims 47-55, wherein the L ₂' is selected from: c ₃-C ₂₀₀ polypeptide subunit, C ₁-C ₂₀₀ alkylene, C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenyl, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, derivatives thereof, and any combination thereof, wherein the polypeptide subunit, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene is optionally substituted with one or more Rs ¹ and/or is optionally interrupted with one or more Rs ², wherein each of the Rs ¹ is independently selected from the group consisting of: halogen, -OH, -NH ₂, and-COOH, each of said Rs ² being independently selected from: -O-, -S-, Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.
The multispecific antibody of any one of claims 47-56, wherein the L ₂' is selected from:

Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure is attached to said AB3, and the left end of the structure is attached to said Y ₂.
The multispecific antibody of any one of claims 47-57, wherein the L ₂' is
The multispecific antibody of any one of claims 46-58, wherein in the Fuco-L-AB2, L has the structure J- (L ₁) _n-X ₁Y ₁-(L ₁') _n'), wherein:

L ₁ is a first linker, n is 0 or 1;

The L ₁ 'is a second linker, and n' is 0 or 1;

j is an adapter directly connected with Fuco;

x ₁Y ₁ is the residue after the ligation of group X ₁ with group Y ₁, wherein the group X ₁ comprises a functional group capable of participating in a bio-orthogonal ligation reaction and the Y ₁ comprises a functional group capable of bio-orthogonal ligation reaction with the X ₁.
The multispecific antibody of claim 59, wherein the X ₁ comprisesWherein R ₁ is selected from: c ₁-C ₁₀ alkylene, C ₅-C ₁₀ (hetero) arylene, C ₆-C ₁₀ alkyl (hetero) arylene and C ₆-C ₁₀ (hetero) arylalkylene, and R ₂ is selected from: hydrogen, C ₁-C ₁₀ alkyl, C ₅-C ₁₀ (hetero) aryl, C ₆-C ₁₀ alkyl (hetero) aryl and C ₆-C ₁₀ (hetero) arylalkyl.
The multispecific antibody of any one of claims 59-60, wherein the X ₁ comprises
The multispecific antibody of any one of claims 59-61, wherein the Y ₁ comprises
The multispecific antibody of any one of claims 59-62, wherein the X ₁Y ₁ comprises a structure selected from the group consisting of:
The multispecific antibody of any one of claims 59-63, wherein the J is Wherein said Rf is-CH ₂ -, -NH-or-O-, wherein the left end of the J-structure is connected to the Fuco.
The multispecific antibody of any one of claims 59-64, wherein the J isWherein the left end of the J-structure is connected with the Fuco.
The multispecific antibody of any one of claims 59-65, wherein the L ₁ and the L ₁' are each independently selected from: c ₃-C ₂₀₀ subunit, C ₁-C ₂₀₀ alkylene, C ₃-C ₂₀₀ cycloalkylene, C ₂-C ₂₀₀ alkenylene, C ₅-C ₂₀₀ cycloalkenylene, C ₂-C ₂₀₀ alkynylene, C ₆-C ₂₀₀ cycloalkynylene, C ₂-C ₂₀₀ (hetero) arylene, C ₃-C ₂₀₀ (hetero) arylalkylene, C ₃-C ₂₀₀ alkyl (hetero) arylene, Derivatives thereof and any combination thereof, wherein said subunit, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero) arylene, (hetero) arylalkylene, or alkyl (hetero) arylene is optionally substituted with one or more Rs ¹ and/or is optionally interrupted by one or more Rs ², wherein each of said Rs ¹ is independently selected from the group consisting of: halogen, halogen, -OH, -NH ₂ and-COOH, each of said Rs ² being independently selected from: -O-, -S-,Wherein Rs ³ is selected from: hydrogen, optionally substituted C ₁-C ₂₄ alkyl, optionally substituted C ₂-C ₂₄ alkenyl, optionally substituted C ₂-C ₂₄ alkynyl and optionally substituted C ₃-C ₂₄ cycloalkyl.
The multispecific antibody of any one of claims 59-66, wherein the L ₁ is selected from: Wherein s1 is an integer from 1 to 50, each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but consecutive adjacent-CH ₂ -is not simultaneously replaced by-O-, the left end of the structure is attached to said J, and the right end of the structure is attached to said X ₁.
The multispecific antibody of any one of claims 59-67, wherein the L ₁ is selected from:

and the right end of the structure is connected with the X ₁, and the left end is connected with the J.
The multispecific antibody of any one of claims 59-68, wherein the L ₁' is selected from:

Wherein each s2 is independently an integer from 0 to 50, each of said-CH ₂ -is optionally replaced by-O-, but the adjacent-CH ₂ -is not simultaneously replaced by-O-, the right end of the structure being attached to said AB2 and the left end of the structure being attached to said Y ₁.
The multispecific antibody of any one of claims 59-69, wherein the L ₁' isThe left end of the structure is connected with the Y ₁, and the right end is connected with the AB 2.
The multispecific antibody of any one of claims 46-70, wherein the first target, second target, and third target are each different from one another.
The multispecific antibody of any one of claims 46-71, wherein each of the AB1, the AB2, and the AB3 is independently an antigen-binding fragment of an antibody.
The multispecific antibody of claim 72, wherein the antigen-binding fragment is a Fab, F (ab) ₂,F(ab'),F(ab') ₂, scFv, affibody (affibody) and/or single domain antibody.
The multispecific antibody of any one of claims 46-73, wherein the first target is a tumor-associated antigen, the second target is CD3, and the third target is a tumor-associated antigen.
The multispecific antibody of claim 74, wherein the tumor-associated antigen is selected from the group consisting of: her2 and PD-L1.
The multispecific antibody of any one of claims 46-75, wherein the first target is Her2, the second target is CD3, and the third target is PD-L1.
The multispecific antibody of any one of claims 46-75, wherein the first target is PD-L1, the second target is CD3, and the third target is Her2.
The multispecific antibody of any one of claims 46-77, wherein the AB1 comprises an antigen-binding portion of an antibody selected from the group consisting of: trastuzumab and dulcis You Shan antibody.
The multispecific antibody of any one of claims 46-78, wherein the AB1 comprises an antibody heavy chain CDR3 (HCDR 3), and the HCDR3 comprises the amino acid sequence of SEQ ID NO:24 and 32 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-79, wherein the AB1 comprises an antibody heavy chain CDR2 (HCDR 2), and the HCDR2 comprises the amino acid sequence of SEQ ID NO:23 and 31 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-80, wherein the AB1 comprises an antibody heavy chain CDR1 (HCDR 1), and the HCDR1 comprises the amino acid sequence of SEQ ID NO:22 and 30 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-81, wherein the AB1 comprises an antibody light chain CDR3 (LCDR 3), and the LCDR3 comprises the amino acid sequence of SEQ ID NO:21 and 29 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-82, wherein the AB1 comprises an antibody light chain CDR2 (LCDR 2), and the LCDR2 comprises the amino acid sequence of SEQ ID NO:20 and 28 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-83, wherein the AB1 comprises an antibody light chain CDR1 (LCDR 1), and the LCDR1 comprises the amino acid sequence of SEQ ID NO:19 and 27 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-84, wherein the AB1 comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence of SEQ ID NO:26 and 34 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-85, wherein the AB1 comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence of SEQ ID NO:25 and 33 the amino acid sequence shown.
The multispecific antibody of any one of claims 46-86, wherein the a is rivarox You Shan antibody or trastuzumab.
The multispecific antibody of any one of claims 46-87, wherein the AB2 comprises an antigen-binding portion of an antibody selected from the group consisting of: OKT3, M291, YTH12.5, bob and Cartuzumab.
The multispecific antibody of any one of claims 46-88, wherein the AB2 comprises the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.
The multispecific antibody of any one of claims 46-89, wherein the AB3 comprises an antigen-binding portion of an antibody selected from the group consisting of: duvalli You Shan antibody, atelizumab, en Wo Lishan antibody, trastuzumab, pertuzumab and ZHer2:342.
The multispecific antibody of any one of claims 46-90, wherein the AB3 comprises the amino acid sequence of SEQ ID NO:10 and 12 the amino acid sequence shown.
A method of making the multispecific antibody of any one of claims 1-91.
The method of claim 92, the method comprising:

i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety A in the presence of a catalyst,

Wherein the sugar chain comprises the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI) to obtain a protein of formula (VII)Wherein:

said a comprises said AB1 and said Fc region;

GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1;

GalX is optionally substituted galactose, said GalX and said GlcNAc being linked by a β -1,4 glycosidic linkage;

q is a ribonucleotide diphosphate; and is also provided with

Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure shown in formula (III): The J is an adapter directly connected with Fuco, and the J is connected with the left end of the formula (III);

The X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction;

the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and

Ii) reacting the protein of formula (VII) with Y ₁-(L ₁') _n' -AB2 to obtain the multispecific antibody of any one of claims 1-45;

Wherein a, galX, X ₁,Y ₁,J,L ₁,L ₁ ', n, n', AB1 and AB2 are as defined in any one of claims 1 to 45.
The method of any of claims 92-93, further comprising the steps of:

treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein;

Contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a polypeptide having a structure comprising formula (VI): -GlcNAc (Fuc) _b -GalX (VI) sugar chain protein having a structure represented by formula (VIII):

Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

Fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and is also provided with

GalX is optionally substituted galactose, said GalX being linked to said GlcNAc by a β -1,4 glycosidic linkage.
The method of any of claims 92-93, further comprising the steps of:

Treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein;

Contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (VI) -GlcNAc (Fuc) _b -GalX (VI), the structure of the protein being as shown in formula (VIII):

Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

Fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and is also provided with

GalX is optionally substituted galactose, said GalX being linked to said GlcNAc by a β -1,4 glycosidic linkage.
The method of claim 92, the method comprising:

i) Contacting a donor Q-Fuc with a protein comprising a sugar chain and said antibody moiety a in the presence of a catalyst, wherein said sugar chain comprises the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX) to obtain a protein of formula (X) Wherein:

said a comprises said AB1 and said Fc region;

GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1;

GalX ₂ is substituted galactose and GalX ₂ comprises X ₂,X ₂ comprises a functional group capable of participating in a bioorthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage;

q is a ribonucleotide diphosphate; and is also provided with

Fuc' comprises structure Fuco-J- (L ₁) _n-X ₁), wherein Fuco has the structure shown in formula (III): the J is an adapter directly connected with Fuco, and the J is connected with the left end of the formula (III);

The X ₁ comprises a functional group capable of participating in a bioorthogonal ligation reaction;

the position of the amino acid N297 is determined according to the EU index numbering in Kabat; and

Ii) reacting the protein of formula (X) with Y ₁-(L ₁') _n' -AB2 and Y ₂-(L ₂') _m -AB3 to obtain the multispecific antibody of any one of claims 46-91;

Wherein A,X ₁,Y ₁,J,L ₁,L ₁',n,n',AB1,AB2,X ₂,GalX ₂,AB3,L ₂',Y ₂ and m are as defined in any one of claims 46 to 91.
The method of claim 96, further comprising the step of:

treating a protein comprising a sugar chain and the antibody moiety a with an endoglycosidase to obtain a treated protein;

Contacting the treated protein with UDP-GalX ₂ in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI):

Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

Fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0 or 1; and is also provided with

GalX ₂ is substituted galactose and it comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage.
The method of claim 96, further comprising the step of:

Treating a protein comprising a sugar chain and said antibody moiety a with an endoglycosidase and an alpha 1,6 fucosidase to obtain a treated protein;

Contacting the treated protein with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure of formula (IX) -GlcNAc (Fuc) _b-GalX ₂ (IX), the structure of which is shown in formula (XI):

Wherein GlcNAc is N-acetylglucosamine and is directly linked to amino acid N297 of the Fc region;

Fuc is fucose, the Fuc and the GlcNAc are connected through alpha-1, 6 glycosidic bond, and b is 0; and is also provided with

GalX ₂ is substituted galactose and it comprises X ₂,X ₂ comprising a functional group capable of participating in a bio-orthogonal ligation reaction, and the GalX ₂ and GlcNAc are linked by a β -1,4 glycosidic linkage.
The method of any one of claims 93-98, wherein the Q is Guanosine Diphosphate (GDP), uridine Diphosphate (UDP), and/or Cytidine Diphosphate (CDP).
The method according to any one of claims 93-99, wherein said Q-Fuc is GDP-Fuc.
The method according to any one of claims 93-100, wherein said Q-Fuc is selected from the following structures:
the method of any one of claims 93-101, wherein the catalyst comprises a fucosyltransferase.
The method of claim 102, wherein the fucosyltransferase is an alpha 1, 3-fucosyltransferase or a functional variant or fragment thereof.
The method of any one of claims 102-103, wherein the fucosyltransferase is derived from a bacterium.
The method according to any one of claims 102-104, wherein the fucosyltransferase is derived from helicobacter pylori Helicobacter pylori.
The method of any one of claims 102-105, wherein the fucosyltransferase is derived from helicobacter pylori Helicobacter pylori 26695,26695.
The method according to any one of claims 102-106, wherein the fucosyltransferase is helicobacter pylori a-1, 3 fucosyltransferase from GenBank accession AAD 07710.1.
The method of any one of claims 102-107, wherein the fucosyltransferase comprises a catalytically active region comprising the amino acid sequence set forth in SEQ ID No. 1 and at least one heptad repeat comprising the amino acid sequence set forth in SEQ ID No. 2.
The method of any one of claims 102-108, wherein the fucosyltransferase comprises a catalytically active region comprising the amino acid sequence set forth in SEQ ID No. 1 and 1-10 heptad repeat fragments comprising the amino acid sequence set forth in SEQ ID No. 2.
The method of any one of claims 102-109, wherein the fucosyltransferase is an a-1, 3-fucosyltransferase or a functional variant or fragment thereof, and comprising the amino acid sequence set forth in SEQ ID No. 3.
The method of any one of claims 93-110, wherein the catalyst comprises the fucosyltransferase and tag sequence of any one of claims 102-110.
The method of any one of claims 93-111, wherein the catalyst comprises an amino acid sequence set forth in any one of SEQ ID NOs 3 and 4.
A composition comprising the multispecific antibody of any one of claims 1-91.
The composition of claim 113, further comprising a pharmaceutically acceptable carrier.
A method of preventing, alleviating and/or treating a disease or disorder, the method comprising administering to a subject in need thereof the multispecific antibody of any one of claims 1-91, and/or the composition of any one of claims 113-114.
Use of the multispecific antibody of any one of claims 1-91 and/or the composition of any one of claims 113-114 for the manufacture of a medicament for preventing, alleviating and/or treating a disease or disorder.