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WO2023141856A1 - 靶向cd3的多特异性抗体及其应用 - Google Patents

靶向cd3的多特异性抗体及其应用 Download PDF

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WO2023141856A1
WO2023141856A1 PCT/CN2022/074201 CN2022074201W WO2023141856A1 WO 2023141856 A1 WO2023141856 A1 WO 2023141856A1 CN 2022074201 W CN2022074201 W CN 2022074201W WO 2023141856 A1 WO2023141856 A1 WO 2023141856A1
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Prior art keywords
group
multispecific antibody
antibody according
hetero
galx
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PCT/CN2022/074201
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English (en)
French (fr)
Inventor
杨熠
李劼
陈计
杨洋
Original Assignee
岩唐生物科技(杭州)有限责任公司
南京大学
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Application filed by 岩唐生物科技(杭州)有限责任公司, 南京大学 filed Critical 岩唐生物科技(杭州)有限责任公司
Priority to PCT/CN2022/074201 priority Critical patent/WO2023141856A1/zh
Priority to CN202280089487.8A priority patent/CN118574857A/zh
Publication of WO2023141856A1 publication Critical patent/WO2023141856A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides

Definitions

  • This application relates to the field of biomedicine, in particular to multispecific antibodies and their applications.
  • the structures of common multispecific antibodies include, for example: tetravalent IgG-scFv fusion protein (Coloma M.J. et al., Nat.Biotechnol, 1997,15,159), bispecific antibody body (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 antibody ("DVD-Ig", Wu C. et al., Nat.
  • FIT-Ig tetravalent Fab tandem immunoglobulin
  • bivalent rat/mouse hybrid multispecific IgG Longdhofer H. et al., J. Immunol, 1995, 155, 219
  • multispecific Crossmab binding proteins see, eg, Auer J. et al., WO2013026831A1.
  • multispecific antibodies for treating cancer especially multispecific antibodies capable of retargeting T cells to kill various tumor cells, have attracted widespread attention.
  • the common T cell receptor is a heterodimer (“TCR ⁇ ”) covalently linked by an alpha chain and a beta chain.
  • the CD3 molecule is a T cell co-receptor or T cell co-receptor, which consists of five different polypeptide chains (ie CD3 gamma chain, CD3 delta chain, two CD3 epsilon chains and two zeta chains). These polypeptide chains form a complex of three dimers (ie, ⁇ , ⁇ , and ⁇ complexes) by associating with each other.
  • the CD3 complex binds to the TCR to generate an activation signal in T lymphocytes. In the absence of CD3, the TCR cannot assemble properly and is degraded.
  • TCR complex is very important and contains approximately 10 immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • T cells are key players in various organ-specific autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis.
  • T cells are key players in various organ-specific autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis.
  • T cells are key players in various organ-specific autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis.
  • two signals are required to activate T cells, in order to avoid a single signal causing inappropriate immune responses against self-antigens.
  • the T cell is not activated and no adaptive immune response occurs.
  • T cells can be recruited or activated by binding multispecific antibodies to an activating component (such as CD3) of the T cell receptor (TCR) complex on immature T cells.
  • an activating component such as CD3 of the T cell receptor (TCR) complex on immature T cells.
  • simultaneous binding of multispecific antibodies to two cell types e.g., tumor cells and T cells
  • CTLs cytotoxic T lymphocytes
  • the application provides a multispecific antibody comprising an antibody part A capable of specifically binding to a first target; and 2 antibodies comprising formula (I) The sugar chain part of the structure shown; and the multispecific antibody has the structure shown in formula (II):
  • said A comprises a first antigen-binding portion AB1 capable of specifically binding to said first target and an Fc region
  • GlcNAc is N-acetylglucosamine, and said GlcNAc is directly linked to amino acid N297 of said Fc region
  • Fuc is fucose, the Fuc is connected to the GlcNAc by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX is optionally substituted galactose, the GalX is connected to the GlcNAc by ⁇ -1,4 glycosidic bond connection
  • the Fuc* comprises the structure Fuco-L-AB2, wherein the structure of Fuco is shown in formula (III): (III), AB2 is the second antigen-binding mo
  • said first target is different from said second target.
  • said AB1 and said AB2 are each independently an antigen-binding fragment of an antibody.
  • the antigen-binding fragment is Fab, F(ab) 2 , F(ab'), F(ab') 2 , scFv, affibody and/or single domain antibodies.
  • the first target is a tumor-associated antigen
  • the second target is CD3.
  • the tumor-associated antigen is selected from: Her2 and PD-L1.
  • said A is an IgG antibody.
  • said AB1 comprises an antigen-binding portion of an antibody selected from the group consisting of trastuzumab and durvalumab.
  • the AB1 comprises an antibody heavy chain CDR3 (HCDR3)
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:24 and 32.
  • the AB1 comprises an antibody heavy chain CDR2 (HCDR2)
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:23 and 31.
  • the AB1 comprises an antibody heavy chain CDR1 (HCDR1), and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:22 and 30.
  • HCDR1 antibody heavy chain CDR1
  • the AB1 comprises an antibody light chain CDR3 (LCDR3)
  • the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:21 and 29.
  • the AB1 comprises an antibody light chain CDR2 (LCDR2)
  • the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:20 and 28.
  • the AB1 comprises an antibody light chain CDR1 (LCDR1)
  • the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:19 and 27.
  • the AB1 comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:26 and 34.
  • the AB1 comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in any one of SEQ ID NO:25 and 33.
  • the A is trastuzumab or durvalumab.
  • the AB2 comprises a CD3 antigen-binding portion of an antibody selected from the group consisting of OKT3, M291, YTH12.5, blinatumomab, and catumaxomab .
  • the AB2 comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the first target is CD3, and the second target is a tumor-associated antigen.
  • the tumor-associated antigen is selected from: Her2 and PD-L1.
  • the structure of L is J-(L 1 ) n -X 1 Y 1 -(L 1 ') n' , wherein : the L 1 is the first linker, n is 0 or 1; the L 1 ' is the second linker, n' is 0 or 1; the J is the zygote directly connected to Fuco; X 1 Y1 is the residual group after the connection reaction between the group X1 and the group Y1 , wherein the group X1 contains a functional group that can participate in the bioorthogonal connection reaction, and the Y1 contains a functional group that can be combined with the X 1 Functional groups for bioorthogonal ligation reactions.
  • the X1 comprises a functional group selected from the group consisting of azido, terminal alkynyl, cycloalkynyl, tetrazine, 1,2,4-tri Zinc group, terminal alkenyl group, cycloalkenyl group, keto group, aldehyde group, hydroxylamine group, mercapto group, maleimide group and their functional derivatives.
  • said 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)arylene alkylene, and R 2 is selected from the group consisting of: 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.
  • said X 1 comprises a functional group selected from the group consisting of:
  • the X 1 comprises
  • said Y 1 comprises a functional group selected from the group consisting of azido, terminal alkynyl, cycloalkynyl, tetrazine, 1,2,4-tri Zinc group, terminal alkenyl group, cycloalkenyl group, keto group, aldehyde group, hydroxylamine group, mercapto group, maleimide group and their functional derivatives.
  • the Y1 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)arylene alkylene, and R 2 is selected from the group consisting of: 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.
  • said X1 and said Y1 comprise a group of structures selected from the following groups:
  • the X 1 Y 1 comprises a structure selected from the group consisting of:
  • the J is Wherein said Rf is -CH 2 -, -NH- or -O-, and the left end of the J structure is directly connected to said Fuco.
  • the J is The left end of the structure is directly connected to Fuco.
  • said L 1 and said L 1 ' are each independently selected from: C 3 -C 200 subpolypeptide group, C 1 -C 200 alkylene group, 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)aryl alkylene, C 3 -C 200 alkyl (hetero)arylene, their derivatives and any combination thereof, wherein the Polypeptidelidene, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero)arylene, (hetero)arylalkylene or alkyl ( Hetero)arylene is optionally
  • said L is selected from: Wherein each s1 is independently an integer of 1-50, each s2 is independently an integer of 0-50, 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 connected to the J, and the right end of the structure is connected to the X1 .
  • said L is selected from: And the right end of the structure is connected to the X1 .
  • said L 1 ' is selected from: Wherein each s2 is independently an integer of 0-50, each said -CH 2 - is optionally replaced by -O-, but adjacent -CH 2 - is not simultaneously replaced by -O-, the right end of the structure is connected to the AB2, and the left end of the structure is connected to the Y1 .
  • said L 1 ' is selected from: The right end of the structure is connected to the AB2, and the left end of the structure is connected to the Y1 .
  • the GalX is galactose.
  • the GalX is substituted galactose, and one or more hydroxyl groups at C2, C3, C4 and/or C6 positions in the galactose are substituted .
  • the GalX is substituted galactose, and the hydroxyl group at the C2 position in the galactose is substituted.
  • the GalX is a monosaccharide.
  • the GalX is substituted by a 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 radical, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, (hetero)aryl, alkyl(hetero)aryl, alkyl(hetero
  • the GalX is substituted by 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 said alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, ( Hetero)arylene, alkyl(hetero)arylene and/or (hetero)arylalkylene are each independently optionally substituted by one
  • the GalX is selected from the group consisting of:
  • said b is 0.
  • the application provides a multispecific antibody comprising: an antibody part A capable of specifically binding to a first target; and 2 components comprising formula (IV) The sugar chain part of the structure shown; and the multispecific antibody has the structure shown in formula (V):
  • said A comprises a first antigen-binding portion AB1 capable of specifically binding to said first target and an Fc region;
  • 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 by ⁇ -1,6 glycosidic bonds, b is 0 or 1;
  • Fuc* contains the structure Fuco-L-AB2, wherein the structure of Fuco is as in formula (III) Shown: AB2 is the second antigen-binding part capable of specifically binding the second target, L is a linker, and the left end of the formula (III) is connected to L, and the Fuc* and the GlcNAc are connected by ⁇ -1,3 Glycosidic linkage; GalX* is substituted galactose, the GalX* is connected to the GlcNAc through a ⁇ -1,4 glycosidic linkage
  • the GalX* has the following structure GalX 2 Y 2 -(L 2 ') m -AB3, wherein GalX 2 Y 2 is the group GalX 2 and the group Y 2 The residual group after the ligation reaction occurs, wherein the GalX 2 includes X 2 , the X 2 includes a functional group capable of participating in a bioorthogonal ligation reaction, and the Y 2 includes a functional group capable of bioorthogonal with the X 2 A functional group for a linking reaction; and the L 2 ' is a linker, and m is 0 or 1.
  • the GalX 2 is galactose in which one or more hydroxyl groups at positions C2, C3, C4 and/or C6 are substituted.
  • the GalX 2 is galactose in which the hydroxyl group at the C2 position is substituted.
  • the GalX 2 is a monosaccharide.
  • X 2 in the GalX 2 comprises
  • X 2 in the GalX 2 comprises
  • the GalX 2 has the following structure
  • the Y 2 comprises
  • said X2Y2 comprises a structure selected from the group consisting of:
  • the L 2 ' is selected from: C 3 -C 200 subpolypeptide group, C 1 -C 200 alkylene group, C 3 -C 200 cycloalkylene group , 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) aryl alkylene, C 3 -C 200 alkyl (hetero) arylene, their derivatives and any combination thereof, wherein the polypeptide subgroup, alkylene group, Cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero)arylene, (hetero)arylalkylene or alkyl(hetero)arylene optionally Substit
  • said L 2 ' is selected from:
  • each s2 is independently an integer of 0-50, each said -CH 2 - is optionally replaced by -O-, but adjacent -CH 2 - is not simultaneously replaced by -O-, the right end of the structure is connected to the AB3, and the left end of the structure is connected to the Y2 .
  • the L 2 ' is
  • the structure of L is J-(L 1 ) n -X 1 Y 1 -(L 1 ') n' , wherein : the L 1 is the first linker, n is 0 or 1; the L 1 ' is the second linker, n' is 0 or 1; the J is the zygote directly connected to Fuco; X 1 Y1 is the residual group after the connection reaction between the group X1 and the group Y1 , wherein the group X1 contains a functional group that can participate in the bioorthogonal connection reaction, and the Y1 contains a functional group that can be combined with the X 1 Functional groups for bioorthogonal ligation reactions.
  • the X 1 comprises 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)arylene alkylene, and R 2 is selected from the group consisting of: 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 X 1 comprises
  • the Y1 comprises
  • the X 1 Y 1 comprises a structure selected from the group consisting of:
  • the J is Wherein said Rf is -CH 2 -, -NH- or -O-, wherein the left end of said J structure is connected with said Fuco.
  • the J is Wherein the left end of the J structure is connected with the Fuco.
  • said L 1 and said L 1 ' are each independently selected from: C 3 -C 200 subpolypeptide group, C 1 -C 200 alkylene group, 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)aryl alkylene, C 3 -C 200 alkyl (hetero)arylene, their derivatives and any combination thereof, wherein the Polypeptidelidene, alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero)arylene, (hetero)arylalkylene or alkyl ( Hetero)arylene is optionally
  • said L is selected from: Wherein s1 is an integer of 1-50, each s2 is independently an integer of 0-50, each said -CH 2 - is optionally replaced by -O-, but consecutive adjacent -CH 2 - are not simultaneously replaced by -O- Instead, the left end of the structure is connected to the J, and the right end of the structure is connected to the X1 .
  • said L is selected from: And the right end of the structure is connected to the X1 , and the left end is connected to the J.
  • said L 1 ' is selected from:
  • each s2 is independently an integer of 0-50, each said -CH 2 - is optionally replaced by -O-, but adjacent -CH 2 - is not simultaneously replaced by -O-, the right end of the structure is connected to the AB2, and the left end of the structure is connected to the Y1 .
  • said L 1 ' is The left end of this structure is connected to the Y1 , and the right end is connected to the AB2.
  • the first target, the second target and the third target are all different from each other.
  • said AB1, said AB2 and said AB3 are each independently an antigen-binding fragment of an antibody.
  • the antigen-binding fragment is Fab, F(ab) 2 , F(ab'), F(ab') 2 , scFv, affibody and/or single domain antibodies.
  • the first target is a tumor-associated antigen
  • the second target is CD3
  • the third target is a tumor-associated antigen.
  • the tumor-associated antigen is selected from: Her2 and PD-L1.
  • the first target is Her2
  • the second target is CD3
  • the third target is PD-L1.
  • the first target is PD-L1
  • the second target is CD3
  • the third target is Her2.
  • said AB1 comprises an antigen-binding portion of an antibody selected from the group consisting of trastuzumab and durvalumab.
  • the AB1 comprises an antibody heavy chain CDR3 (HCDR3)
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:24 and 32.
  • the AB1 comprises an antibody heavy chain CDR2 (HCDR2)
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:23 and 31.
  • the AB1 comprises an antibody heavy chain CDR1 (HCDR1), and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:22 and 30.
  • HCDR1 antibody heavy chain CDR1
  • the AB1 comprises an antibody light chain CDR3 (LCDR3)
  • the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:21 and 29.
  • the AB1 comprises an antibody light chain CDR2 (LCDR2)
  • the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:20 and 28.
  • the AB1 comprises an antibody light chain CDR1 (LCDR1)
  • the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 19 and 27.
  • the AB1 comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:26 and 34.
  • the AB1 comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in any one of SEQ ID NO:25 and 33.
  • the A is durvalumab or trastuzumab.
  • the AB2 comprises an antigen-binding portion of an antibody selected from the group consisting of OKT3, M291, YTH12.5, blinatumomab, and catutaxomab.
  • the AB2 comprises the amino acid sequence shown in SEQ ID NO: 14.
  • the AB3 comprises an antigen-binding portion of an antibody selected from the group consisting of durvalumab, atezolizumab, envolimumab, Trastuzumab, Pertuzumab and ZHer2:342.
  • the AB3 comprises the amino acid sequence shown in any one of SEQ ID NO: 10 and 12.
  • the present application provides methods for preparing the multispecific antibodies described herein.
  • the method comprises: i) contacting a donor Q-Fuc*' with a protein comprising a sugar chain and said antibody portion A in the presence of a catalyst, wherein said sugar chain comprises the formula Structure-GlcNAc(Fuc) b -GalX(VI) shown in (VI) to obtain the protein with structure as shown in formula (VII)
  • said A comprises said AB1 and said Fc region
  • GlcNAc is N-acetylglucosamine, and said GlcNAc is directly linked to amino acid N297 of said Fc region
  • Fuc is fucose, and said Fuc is connected to said Fc region GlcNAc are connected by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX is optionally substituted galactose, and the GalX and the GlcNAc are connected by ⁇ -1,4 glycosidic bonds
  • Q is Ribonucleotide diphosphate
  • Fuc*' comprises the formula
  • the method further comprises the steps of: treating the protein comprising sugar chains and the antibody part A with an endoglycosidase to obtain a processed protein; making the processed protein in a suitable Contact with UDP-GalX in the presence of a catalyst to obtain a protein with a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX(VI) shown in formula (VI), the structure of the protein is shown in formula (VIII) : Wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond, b is 0 or 1; and GalX is optionally substituted galactose, and the GalX is connected to the GlcNAc through a ⁇ -1,4 glycosidic bond.
  • the method further comprises the steps of: treating the protein comprising sugar chains and the antibody part A with endoglycosidase and ⁇ 1,6 fucosidase to obtain the treated protein; making the The treated protein is contacted with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX(VI) shown in formula (VI), the protein The structure is shown in formula (VIII): Wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond, b is 0; and GalX is optionally substituted galactose, and the GalX is connected to the GlcNAc through a ⁇ -1,4 glycosidic bond.
  • the method comprises: i) contacting a donor Q-Fuc*' with a protein comprising a sugar chain and said antibody portion A in the presence of a catalyst, wherein said sugar chain comprises the formula Structure shown in (IX)-GlcNAc(Fuc) b -GalX 2 (IX), to obtain the protein with structure shown in formula (X)
  • said A comprises said AB1 and said Fc region
  • GlcNAc is N-acetylglucosamine, and said GlcNAc is directly linked to amino acid N297 of said Fc region
  • Fuc is fucose, and said Fuc is connected to said Fc region GlcNAc are linked by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX 2 is substituted galactose and GalX 2 contains X 2
  • X 2 contains functional groups that can participate in bioorthogonal linking reactions, and the GalX 2 is connected to the GlcNAc by a
  • the method further comprises the steps of: treating the protein comprising sugar chains and the antibody part A with an endoglycosidase to obtain a processed protein; making the processed protein in a suitable Contact with UDP-GalX 2 in the presence of a catalyst to obtain a protein having a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX 2 (IX) shown in formula (IX), the protein having a structure such as formula (XI) Shown: Wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond, b is 0 or 1; and GalX 2 is substituted galactose and it contains X 2 , X 2 contains a functional group capable of participating in a bioorthogonal connection reaction, and the
  • the method further comprises the steps of: treating the protein comprising sugar chains and the antibody part A with endoglycosidase and ⁇ 1,6 fucosidase to obtain the treated protein; making the The above-mentioned treated protein is contacted with UDP-GalX under the condition that a suitable catalyst exists, so as to obtain a protein having a sugar chain comprising the structure shown in formula (IX)-GlcNAc(Fuc) b -GalX 2 (IX), the protein The structure of is as shown in formula (XI): Wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond, b is 0; and GalX 2 is a substituted galactose containing X 2 , X 2 contains a functional group
  • the Q is guanosine diphosphate (GDP), uridine diphosphate (UDP) and/or cytidine diphosphate (CDP).
  • GDP guanosine diphosphate
  • UDP uridine diphosphate
  • CDP cytidine diphosphate
  • the Q-Fuc*' is GDP-Fuc*'.
  • the Q-Fuc*' is selected from the following structures:
  • the catalyst comprises a fucosyltransferase.
  • the fucosyltransferase is ⁇ 1,3-fucosyltransferase or a functional variant or fragment thereof.
  • the fucosyltransferase is derived from bacteria.
  • the fucosyltransferase is derived from Helicobacter pylori.
  • the fucosyltransferase is derived from Helicobacter pylori 26695.
  • the fucosyltransferase is Helicobacter pylori ⁇ -1,3 fucosyltransferase derived from GenBank accession number AAD07710.1.
  • the fucosyltransferase comprises a catalytically active region and at least one heptad repeat fragment
  • the catalytically active region comprises the amino acid sequence shown in SEQ ID NO: 1
  • the heptapeptide repeating fragment comprises the amino acid sequence shown in SEQ ID NO:2.
  • the fucosyltransferase comprises a catalytically active region and 1-10 heptad repeats, the catalytically active region comprising amino acids shown in SEQ ID NO:1 sequence, and the heptad repeat fragment comprises the amino acid sequence shown in SEQ ID NO:2.
  • the fucosyltransferase is ⁇ -1,3-fucosyltransferase or a functional variant or fragment thereof, and it comprises SEQ ID NO:3 The amino acid sequence shown in.
  • the catalyst comprises a fucosyltransferase described herein and a tag sequence.
  • the catalyst comprises the amino acid sequence shown in any one of SEQ ID NO:3 and 4.
  • the present application provides a composition comprising the multispecific antibody described in the present application.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the present application provides a method for preventing, alleviating and/or treating a disease or disorder, the method comprising administering the multispecific antibody described in the present application to a subject in need, and/or the present application said composition.
  • the present application provides the use of the multispecific antibody described in the present application and/or the composition described in the present application for the preparation of a medicament for preventing, alleviating and/or treating a disease or condition.
  • Figure 1A-1B shows a schematic diagram of the method for preparing the multispecific antibody of the present application, wherein is N-acetylglucosamine, for ⁇ 1,6 fucose,
  • Fuc*' is Fuco-J-(L 1 ) n -X 1
  • Fuc* is J-(L 1 ) n -X 1 Y 1 -(L 1 ') n' -AB2
  • GalX* is GalX 2 Y 2 -(L 2 ') n ”-AB3
  • AB2 is the antigen-binding part targeting CD3
  • AB3 is the third antigen-binding part.
  • FIG. 2 shows some exemplary structures of Q-Fuc*' in this application.
  • Figures 3A-3E show the preparation and characterization of exemplary CD3-targeting multispecific antibodies of the present application.
  • Figures 4A-4C show the binding affinities of exemplary CD3-targeting multispecific antibodies of the present application.
  • 5A-5B show the in vitro killing activity of exemplary CD3-targeting multispecific antibodies of the present application.
  • 6A-6C show the in vitro killing activity of exemplary CD3-targeting multispecific antibodies of the present application.
  • Figures 7A-7C show antigen-dependent T cell activation characterization of exemplary CD3-targeting multispecific antibodies of the present application.
  • Figures 8A-8B show concentration-dependent T cell activation characterization of exemplary CD3-targeting multispecific antibodies of the present application.
  • Figure 9 shows the comparison of the efficiencies of antibody conjugates containing different conjugators reacting with Y 1 -(L 1 ') n' -AB2 to prepare the multispecific antibody described in this application.
  • Figure 10 shows the molecular structures of some of the compounds used in this application.
  • directly linked generally means that the site of attachment does not contain additional compounds (eg, other sugar groups) or linkers.
  • a molecule or entity directly linked to another can mean that there is no other molecule or entity in between.
  • directly linked can mean that one part is joined to another part without any intervening parts or joints.
  • the direct linking of GlcNAc to the amino acid residues of a protein generally means that GlcNAc is linked to the amino acid residues of the protein through a covalent bond, for example, the amino acid of the protein is linked by an N-glycosidic bond and an amide nitrogen bond (such as Tian Atoms in the side chain of paragine amino acid).
  • amide nitrogen bond such as Tian Atoms in the side chain of paragine amino acid
  • the compounds disclosed in the specification and claims of this application may contain one or more asymmetric centers and may exist as different diastereomers and/or enantiomers of said compounds. Unless otherwise stated, the description of any compound in the specification and claims of this application is meant to include all diastereoisomers and mixtures thereof. Furthermore, unless otherwise stated, a description of any compound in the specification and claims is meant to include the individual enantiomers as well as any mixture, racemate or other form of the enantiomers. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to that specific enantiomer.
  • the compounds may exist in different tautomeric forms. Unless otherwise stated, the compounds of the present invention are meant to include all tautomeric forms. When the structure of a compound is described as a specific tautomer, it should be understood that the invention of the present application is not limited to the specific tautomer.
  • Unsubstituted alkyl groups have the general formula C n H 2n+1 and may be linear or branched. Unsubstituted alkyl groups may also contain cyclic moieties and thus have the corresponding general formula C n H 2n-1 .
  • the alkyl group is substituted with one or more substituents as further specified in this application. Examples of alkyl groups include, for example, methyl, ethyl, propyl, 2-propyl, tert-butyl, 1-hexyl, 1-dodecyl, and the like.
  • Aryl groups can contain six to twelve carbon atoms and can contain monocyclic and bicyclic structures.
  • the aryl group may be substituted with one or more substituents as further specified herein. Examples of aryl are phenyl and naphthyl.
  • Arylalkyl and alkylaryl groups can contain at least seven carbon atoms and can contain monocyclic and bicyclic structures.
  • the arylalkyl and alkylaryl groups may be substituted with one or more substituents as further specified in this application.
  • Arylalkyl may be, for example, benzyl.
  • Alkylaryl may be, for example, 4-tert-butylphenyl.
  • heteroaryl generally refers to an aromatic monocyclic or polycyclic group of 5-12 atoms, which has at least one group consisting of one, two or three atoms selected from N, O, and S ring heteroatoms, and the remaining ring atoms are C.
  • One or two ring carbon atoms of a heteroaryl group may be substituted by a carbonyl group.
  • Heteroarylalkyl and alkylheteroaryl groups contain at least three carbon atoms (ie, at least C3 ) and can contain monocyclic and bicyclic structures.
  • heteroaryl may be substituted with one or more substituents as further specified herein.
  • aryl When aryl is indicated as (hetero)aryl, this notation is meant to include both aryl and heteroaryl.
  • alkyl(hetero)aryl is meant to include both alkylaryl and alkylheteroaryl
  • (hetero)arylalkyl is meant to include both arylalkyl and heteroarylalkyl.
  • C 2 -C 24 (hetero)aryl is therefore understood to include C 2 -C 24 heteroaryl and C 6 -C 24 aryl.
  • C 3 -C 24 alkyl(hetero)aryl is meant to include C 7 -C 24 alkylaryl and C 3 -C 24 alkylheteroaryl
  • C 3 -C 24 (hetero)aryl Alkyl is meant to include C 7 -C 24 arylalkyl and C 3 -C 24 heteroarylalkyl.
  • alkyl, alkenyl, alkene, alkyne, (hetero)aryl, (hetero)arylalkyl and alkyl(hetero)aryl may be substituted by one or more substituents selected from Substitution: 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 ring Alkynyl, 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,
  • alkynyl generally contains a carbon-carbon triple bond.
  • Unsubstituted alkynyl groups containing one triple bond have the general formula C n H 2n-3 .
  • a terminal alkynyl group is an alkynyl group in which the triple bond is at the terminal position of the carbon chain.
  • the alkynyl group is substituted with one or more substituents as further specified in this application, and/or 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.
  • cycloalkynyl generally refers to an unsaturated monocyclic, bicyclic or tricyclic hydrocarbon ring having the specified number of carbon atoms and one or more carbon-carbon triple bonds.
  • C 7 -C 12 cycloalkynyl refers to a cycloalkynyl group having 7-12 carbon atoms.
  • cycloalkynyl groups have one carbon-carbon triple bond in the ring.
  • cycloalkynyl groups have more than one carbon-carbon triple bond in the ring.
  • Representative examples of cycloalkynyl include, but are not limited to, cycloheptynyl, cyclooctynyl, and the like.
  • heterocycloalkynyl is generally a cycloalkynyl interrupted or interrupted by a heteroatom selected from oxygen, nitrogen and sulfur.
  • heterocycloalkynyl is substituted with one or more substituents as further specified herein.
  • An example of a heterocycloalkynyl is azacyclooctynyl.
  • alkyl, (hetero)aryl, alkyl(hetero)aryl, (hetero)arylalkyl, (hetero)cycloalkynyl are optionally substituted
  • said groups are independently optionally replaced by one or a plurality of substituents independently selected from the following substituents: 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 alkane Base, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, said alkyl, alkoxy,
  • subpolypeptide group generally refers to a polypeptide group containing two or more amino acid residues, for example, it can be formed by condensation of two or more amino acids, and it can be formed by the amino acid residues at both ends.
  • carboxyl group is linked to other structures.
  • sucrose generally refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc).
  • saccharide derivative in the application generally refers to derivatives of monosaccharides, ie monosaccharides comprising substituents and/or functional groups.
  • sugar derivatives include amino sugars and sugar acids such as glucosamine ( GlcNH2 ), galactosamine ( GalNH2 ), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), also known as N-acetylneuramic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA).
  • sugar derivatives also include compounds denoted GalX in this application, which may be galactose or a galactose derivative.
  • sugar derivatives also include compounds denoted Fuc*' in this application, which may be fucose derivatives.
  • nucleotide generally refers to a molecule consisting of a nucleobase, a five-carbon sugar (ribose or 2-deoxyribose) and one, two or three phosphate groups. In the absence of a phosphate group, nucleobases and sugars make up nucleosides. Therefore, nucleotides are also called nucleoside monophosphates, nucleoside diphosphates or nucleoside triphosphates.
  • the nucleobase can be adenine, guanine, cytosine, uracil or thymine.
  • nucleotides examples include diphosphate ribonucleotides such as uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).
  • UDP uridine diphosphate
  • GDP guanosine diphosphate
  • TDP thymidine diphosphate
  • CDP cytidine diphosphate
  • CMP cytidine monophosphate
  • protein generally refers to a polypeptide comprising about 10 or more amino acids. Proteins can include natural amino acids, but can also include unnatural amino acids.
  • glycoprotein is used in its ordinary scientific sense and refers to a protein containing one or more chains of monosaccharides or oligosaccharides ("sugar chains") covalently bonded to the protein .
  • the sugar chain can be attached to a hydroxyl group of the protein (O-linkage-sugar chain), for example to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline; or to the acyl group of the protein Amino groups (N-glycoproteins), such as asparagine or arginine; or attached to protein carbons (C-glycoproteins), such as tryptophan.
  • O-linkage-sugar chain for example to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline
  • Amino groups N-glycoproteins
  • C-glycoproteins protein carbons
  • Glycoproteins may contain more than one sugar chain, may contain a combination of one or more monosaccharide and one or more oligosaccharide sugar chains, and may contain a combination of N-linked, O-linked, and C-linked sugar chains . It is estimated that more than 50% of all proteins have some form of glycosylation and can therefore be described as glycoproteins.
  • sugar chain generally refers to a monosaccharide or oligosaccharide chain attached to a protein.
  • sugar chain refers to the carbohydrate portion of a glycoprotein.
  • Sugar chains which can be attached to proteins through the C1 carbon of a sugar, can contain no other substituents (monosaccharides) or can be further substituted on one or more of their hydroxyl groups (oligosaccharides).
  • Naturally occurring glycans typically contain from 1 to about 10 sugar moieties. However, longer glycans are also considered sugar chains in this application when they are attached to proteins.
  • the sugar chains of glycoproteins may be monosaccharides.
  • 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.
  • Sugar chains may also be oligosaccharides.
  • the oligosaccharide chains of glycoproteins can be linear or branched.
  • the sugar directly attached to the protein is called core sugar.
  • sugars that are not directly attached to a protein but are attached to at least two other sugars are called endosaccharides.
  • multiple terminal sugars may be present in an oligosaccharide of a glycoprotein, but usually only one core sugar is present.
  • the sugar chains may be O-linked sugar chains, N-linked sugar chains or C-linked sugar chains.
  • O-linked sugar chains monosaccharide or oligosaccharide sugar chains are bonded to O atoms in the amino acids of the protein, usually via the hydroxyl groups of serine (Ser) or threonine (Thr).
  • N-linked sugar chains monosaccharide or oligosaccharide sugar chains are bonded to the protein via the N atom in the amino acid of the protein, usually via the asparagine (Asn) or arginine (Arg) side chain on the amide nitrogen.
  • C-linked sugar chains monosaccharide or oligosaccharide sugar chains are bonded to C atoms in amino acids of the protein, usually 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.
  • O-linked sugar chains there are many different chains.
  • Naturally occurring O-linked sugar chains usually have a serine- or threonine-linked ⁇ -O-GalNAc moiety, which may also be substituted with galactose, sialic acid and/or fucose.
  • Hydroxylated amino acids carrying sugar chain substituents can be part of any amino acid sequence in proteins.
  • N-linked sugar chains there are many different chains.
  • Naturally occurring N-linked sugar chains usually have an asparagine-linked ⁇ -N-GlcNAc moiety, which in turn is further linked to ⁇ -GlcNAc at its C4, which is then further linked to ⁇ -Man at its C4, followed by It is further connected with ⁇ -Man on its C3 and C6 to form a pentasaccharide Man 3 GlcNAc 2 .
  • the core GlcNAc portion can also be linked to ⁇ -Fuc at its C6.
  • the pentasaccharide Man 3 GlcNAc 2 is the common oligosaccharide framework of most N-linked glycoproteins and can be further linked to other sugars, including but not limited to Man, GlcNAc, Gal and sialic acid.
  • Asparagine modified with a sugar chain on the side chain is usually part of the sequence Asn-X-Ser/Thr, where X is any amino acid except proline and Ser/Thr is serine or threonine.
  • antibody generally refers to a protein or an antigen-binding fragment thereof produced by the immune system that can recognize and bind to a specific antigen.
  • An antibody is an example of a glycoprotein.
  • the term antibody is used in this application in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g. multispecific antibodies), antibody fragments as well as double chain and single chain antibody.
  • Antibodies also include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind cancer antigens. in this application.
  • antibody generally includes whole antibodies, but also antibody fragments, such as antibody Fab fragments, (Fab') 2 , Fv fragments or Fc fragments, scFv-Fc fragments, minibodies, from cleaved antibodies, Single domain antibody (also known as nanobody, nanobody), multispecific antibody (diabody), affibody (affibody) or scFv. Furthermore, the term “antibody” also includes engineered or genetically modified antibodies and/or antibody derivatives.
  • antibodies are referred to as immunoglobulins and include various classes and isotypes, such as IgA (IgAl and IgA2), IgD, IgE, IgM, and IgG (IgGl, IgG3, and IgG4), among others.
  • 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 a target antigen or epitope.
  • antibodies can be from any source, such as mouse or human, including chimeric antibodies thereof, for example, antibodies can be humanized.
  • humanized antibody generally refers to an antibody that contains part 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 human antibody sequences.
  • Antibodies, antibody fragments and genetically engineered antibodies can all be obtained by methods known in the art.
  • treatment generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or may be therapeutic in terms of partial or complete cure of the disease and/or side effects caused by the disease.
  • Treatment encompasses any treatment of a disease in a mammal, especially a human, and includes preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed To suffer from the disease; to inhibit the disease, that is, to prevent its development; to alleviate the disease, that is, to cause the regression of the disease.
  • the term “Fc region” generally refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of intact antibodies.
  • the Fc region can be a native sequence Fc region or a variant Fc region.
  • the Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain and/or a hinge region.
  • the term “Fc region” includes any polypeptide (or nucleic acid encoding such a polypeptide), regardless of its mode of production.
  • GlcNAc or “N-acetylglucosamine” are used interchangeably and generally refer to the amide derivative of the monosaccharide glucose.
  • glycosylation generally refers to the reaction in which a carbohydrate, the sugar donor, attaches to the hydroxyl or other functional group of another molecule (the sugar acceptor).
  • glycosylation refers primarily to, inter alia, the enzymatic process of attaching glycans to proteins or other organic molecules.
  • Glycosylation in proteins can 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, formation of GPI anchors (glypiation) and/or chemical glycosylation
  • protein glycosylation Glycosylated oligosaccharides may be N-linked oligosaccharides, O-linked oligosaccharides, phosphoserine oligosaccharides, C-mannosylated oligosaccharides, glycosylated oligosaccharides and/or chemical oligosaccharides.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and/or post-translational modifications that may be present in minor amounts (e.g., iso Apart from constitutiveization, amidation), the individual antibodies making up the population are identical.
  • immunoglobulin heavy chains are classified as gamma, mu, alpha, delta, and epsilon (gamma, mu, alpha, delta, epsilon) and some of these subtypes (e.g. gamma1-gamma4 or alpha1-alpha2) . It is the nature of this chain that determines the antibody's "isotype,” IgG, IgM, IgD, IgA, or IgE, respectively.
  • Immunoglobulin subclasses such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc., are well characterized and known to confer functional specificity.
  • a typical feature of human IgG is glycosylation at position Asn297 (EU numbering according to Kabat) of the heavy chain CH2 region of the Fc region.
  • Asn297 or “N297” is used interchangeably and generally refers to the asparagine at position 297 (numbering according to the EU numbering convention of Kabat) of the Fc region of an antibody. Asn297 can be linked to one or more oligosaccharides.
  • Fuc* ⁇ 1,3 GlcNAc linkage generally refers to the linkage between Fuco of Fuc* and GlcNAc, such as linking C1 of Fuco to C3 of GlcNAc.
  • GalX ⁇ 1,4GlcNAc linkage generally refers to the linkage between optionally substituted galactose GalX and GlcNAc, such as linking C1 of GalX to C4 of GlcNAc.
  • a functional group generally refers to a group capable of reacting with another group.
  • Functional groups can be used to incorporate reagents (eg, non-reactive or low-reactive reagents) into the multispecific antibodies of the present application.
  • a functional group may be a chemical group or a chemically and/or enzymatically reactive residue.
  • a functional group may be a group capable of reacting in a ligation reaction.
  • tag sequence generally refers to another molecular entity (eg, another amino acid sequence) that is integrated (eg, linked) into a protein of interest (eg, antibody).
  • the tag sequence can be a detectable label, such as a radiolabeled amino acid or a biotinylated polypeptide, which can be detected by labeled avidin (such as an antibiotic with a fluorescent label or enzymatic activity). streptavidin, which can be detected optically or colorimetrically).
  • tag sequences may include, but are not limited to: radioisotopes or radionuclides (e.g.
  • fluorescent labels e.g. FITC, rhodamine, lanthanum phosphors
  • enzyme label e.g. horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase
  • chemiluminescent label biotin group, pre-selected by a second acceptor Recognized polypeptide epitopes (e.g. leucine zipper complementary sequence, binding site for secondary antibody, metal binding domain, epitope tag), magnetic reagents such as gadolinium chelate.
  • the tag sequence can be an affinity purification tag, such as polyhistidine tag (His tag), Arg tag, FLAG tag, 3xFlag tag, streptavidin tag, nano tag, SBP tag, c-myc tag, S tag, calmodulin binding peptide, cellulose binding domain, chitin binding domain, GST tag and/or MBP tag.
  • a tag sequence can be fused to the N-terminus and/or C-terminus of the protein of interest.
  • the term “fucosyltransferase” generally refers to an enzyme that can transfer L-fucose from a fucose donor substrate (such as guanosine diphosphate-fucose) to an acceptor substrate. Enzymes or functional fragments or variants thereof.
  • the acceptor substrate may be another sugar, for example a sugar comprising GlcNAc-Gal (LacNAc), as in the case of N-glycosylation or in the case of O-glycosylation.
  • the term “fucosyltransferase” may include any functional fragment thereof, or its catalytic domain, as well as functional variants (eg, mutants, isoforms) having a catalytically active domain.
  • fucosyltransferase may be alpha-1,3 fucosyltransferase.
  • the term "fucosyltransferase" may be derived from various species, such as mammals (eg humans), bacteria, nematodes or trematodes.
  • the fucosyltransferase is an alpha-1,3 fucosyltransferase of bacterial origin.
  • the fucosyltransferase is alpha-1,3 fucosyltransferase derived from Helicobacter pylori.
  • the fucosyltransferase is an alpha-1,3 fucosyltransferase derived from Helicobacter pylori 26695.
  • the fucosyltransferase is derived from ⁇ -1,3 fucose with GenBank accession number AAD07710.1, GenBank accession number AAD07447.1 or GenBank accession number AAB81031.1 base transferase. In some embodiments, wherein the fucosyltransferase is a fucosyltransferase with GenBank accession number AAD07710.1, GenBank accession number AAD07447.1 or GenBank accession number AAB81031.1.
  • the fucosyltransferase is ⁇ -1,3 fucosyltransferase with GenBank accession number AAD07710.1, GenBank accession number AAD07447.1 or GenBank accession number AAB81031.1 Functional variants or fragments of enzymes.
  • the fucosyltransferase comprises an amino acid sequence as set forth in GenBank Accession No. AAD07710.1, or a functional variant or fragment thereof.
  • the fucosyltransferase may comprise the amino acid sequence listed in GenBank Accession No.
  • the fucosyltransferase may comprise a 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) amino acid sequence as described in GenBank Accession No. AAD07710.1, or a functional variant or fragment thereof.
  • a fucosyltransferase may comprise a catalytically active region and at least one heptad repeat (eg, 1-10) (SEQ ID NO:2), the catalytically active region comprising the amino acid sequence shown in SEQ ID NO:1 Or comprising 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 98% of the amino acid sequence shown in SEQ ID NO: 1) 99% or more) sequence homology of amino acid sequences.
  • SEQ ID NO:2 heptad repeat
  • the fucosyltransferase may comprise the amino acid sequence shown in any one of SEQ ID NO: 3 and 4, or the fucosyltransferase may comprise the amino acid sequence shown in the item of SEQ ID NO: 3 Amino acid sequences that have at least 80% (eg, more than 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) homology or identity.
  • biorthogonal ligation reaction generally refers to the chemical reaction used to prepare the protein conjugates described herein. This reaction occurs specifically between a first functional group located at a specific position on the protein, for example on an oligosaccharide of the protein, and a second functional group corresponding to said functional group part.
  • the first functional group and the second functional group are generally referred to as a pair of bioorthogonal linkage reactions.
  • the first functional group located at a specific position in a protein is easily distinguished from other groups in other parts of the protein.
  • the second functional group will not react with other parts of the protein other than the first functional group at a specific location.
  • the azido group is a functional functional group capable of participating in bioorthogonal ligation reactions.
  • the complementary DBCO or BCN group can react specifically with the azide group without cross-reacting with other groups on the protein.
  • Many chemically reactive functional groups with suitable reactivity, chemoselectivity and/or biocompatibility are available for bioorthogonal ligation reactions.
  • Groups capable of participating in bioorthogonal ligation reactions can be selected from, but not limited to, azido, terminal alkynyl, cyclic alkynyl, tetrazinyl, 1,2,4-triazinyl, terminal alkenyl, cyclic alkenyl group, ketone group, aldehyde group, hydroxyl amino group, mercapto group, N-maleimide group and their functional derivatives (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.
  • the functional derivative here may refer to a modified functional group, which has a reactivity similar to or higher than that of an unmodified functional group.
  • a "functional variant" of a parent polypeptide or protein usually has substantial or significant sequence identity or similarity with the parent polypeptide or protein, and the functional variant retains at least a part of the function of its parent polypeptide or protein.
  • a functional variant of an enzyme retains enzymatic activity to a similar extent, to the same extent, or to a greater extent than the parent enzyme.
  • functional variants can be, for example, those having 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.
  • a functional variant may be a polypeptide that differs from a parent polypeptide or protein by at least one amino acid.
  • functional variants can be obtained by adding, deleting or substituting one or more amino acids, such as 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.
  • a "functional fragment" of a parent polypeptide or protein generally refers to a peptide or polypeptide (including but not limited to an enzyme) that comprises at least 5 consecutive amino acid residues, at least 10 consecutive Amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues group, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, At least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250 contiguous amino acid residues, or at least 350 contiguous amino acid residues, wherein the functional fragment has at least a portion of
  • CD3 generally refers to a CD3 protein multi-subunit complex (eg, a human CD3 protein multi-subunit complex).
  • CD3 protein multi-subunit complex can be composed of 6 different polypeptide chains. These polypeptide chains include CD3 ⁇ chain (SwissProt P09693), CD3 ⁇ chain (SwissProt P04234), two CD3 ⁇ chains (SwissProt P07766) and a CD3 ⁇ chain homodimer (SwissProt 20963), and the complex binds to T cell receptor ⁇ associated with the ⁇ chain.
  • CD3 includes any CD3 variant, isoform, and species homologue that is naturally expressed by cells, including T cells, or that can be expressed on cells transfected with genes or cDNAs encoding those polypeptides things.
  • a protein comprising the amino acid sequence shown in SEQ ID NO: 1 should be understood as not only describing a protein, but its amino acid sequence includes but is not limited to the amino acid sequence shown in SEQ ID NO: 1. It should also be understood that a protein body is also described, the amino acid sequence of which consists of SEQ ID NO:1.
  • the application provides a multispecific antibody comprising: an antibody part A capable of specifically binding to a first target; and 2 antibodies comprising formula (I) The sugar chain part of the structure shown; and the multispecific antibody has the structure shown in formula (II):
  • the antibody part A comprises a first antigen-binding part AB1 capable of specifically binding the first target and an Fc region
  • GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region
  • Fuc is fucose, the Fuc and the GlcNAc are connected by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX is optionally substituted galactose (GalX is galactose or is substituted galactose), the GalX and the GlcNAc are linked by a ⁇ -1,4 glycosidic bond
  • the Fuc* comprises the structure Fuco-L-AB2, wherein the structure of Fuco-
  • the GlcNAc is directly linked to amino acid N297 of the Fc region through an N-glycosidic bond.
  • the GlcNAc is connected to amino acid N297 of the Fc region through an N-glycosidic bond at the C1 position.
  • the application provides a multispecific antibody comprising an antibody part A capable of specifically binding to a first target; and 2 antibodies comprising formula (IV) The sugar chain part of the structure shown; and the multispecific antibody has the structure shown in formula (V):
  • the A comprises the first antigen-binding part AB1 and an Fc region capable of specifically binding the first target
  • GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region
  • Fuc is fucose, the Fuc and the GlcNAc are connected by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • Fuc* contains the structure Fuco-L-AB2, wherein the structure of Fuco is as in formula (III) Shown: AB2 is the second antigen-binding part capable of specifically binding the second target, L is a linker, and the left end of the formula (III) is connected to L, and the Fuc* and the GlcNAc are
  • the first target is CD3.
  • the second target may be CD3.
  • the third target may be CD3.
  • the first target, the second target, and the third target can all be CD3.
  • b is 0.
  • b is 0, formula (V) Equivalent to
  • the GalX* has the following structure GalX 2 Y 2 -(L 2 ') m -AB3, wherein GalX 2 Y 2 is the residual group after the coupling reaction of the group GalX 2 with the group Y 2 , wherein the GalX 2 includes X 2 , X 2 includes a functional group capable of participating in a bioorthogonal linkage reaction, and the Y 2 includes a functional group capable of undergoing a bioorthogonal linkage reaction with the X 2 , and the L 2 ' is a linker, m is 0 or 1.
  • the present application provides methods for preparing the multispecific antibodies described herein.
  • the method may include: i) contacting the donor Q-Fuc*' with a protein comprising a sugar chain and the antibody part A in the presence of a catalyst, wherein the sugar chain comprises a structure represented by formula (VI) -GlcNAc(Fuc) b -GalX(VI), to obtain the protein with structure as shown in formula (VII)
  • said A comprises said AB1 and said Fc region
  • GlcNAc is N-acetylglucosamine, and said GlcNAc is directly linked to amino acid N297 of said Fc region
  • Fuc is fucose, and said Fuc is connected to said Fc region GlcNAc are connected by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX is optionally substituted galactose, and the GalX and the GlcNAc are connected by ⁇ -1,4 glycosidic bonds
  • Q
  • the present application provides methods for preparing the multispecific antibodies described herein.
  • the method may include: i) contacting the donor Q-Fuc*' with a protein comprising a sugar chain and the antibody part A in the presence of a catalyst, wherein the sugar chain comprises a structure represented by formula (IX) -GlcNAc(Fuc) b -GalX 2 (IX), to obtain a protein with a structure as shown in formula (X)
  • said A comprises said AB1 and said Fc region
  • GlcNAc is N-acetylglucosamine, and said GlcNAc is directly linked to amino acid N297 of said Fc region
  • Fuc is fucose, and said Fuc and said
  • the GlcNAc are connected by ⁇ -1,6 glycosidic bonds, b is 0 or 1
  • GalX 2 is substituted galactose and it contains X 2
  • X 2 contains a functional group that can participate in a bio-ort
  • the first target and the second target may be the same or different.
  • the first target may be the same as the second target.
  • the first target may be different from the second target, for example, the first target and the second target may be different antigenic proteins, or may be different epitopes of the same antigenic protein (eg, not identical epitope polypeptides).
  • the third target may be the same as or different from the first target.
  • the third target may be the same as the first target.
  • the third target may be different from the first target, for example, the third target and the first target may be different antigenic proteins, or may be different epitopes of the same antigenic protein (eg, not identical epitope polypeptides).
  • the third target may be the same or different from the second target.
  • the third target may be the same as the second target.
  • the third target may be different from the second target, for example, the third target and the second target may be different antigenic proteins, or may be different epitopes of the same antigenic protein (eg, not identical epitope polypeptides).
  • the first target, the second target, and the third target are all different from each other.
  • the three can be different antigenic proteins, or can be different epitopes of the same antigenic protein (eg, not completely identical epitope polypeptides).
  • the antibody portion A may be an IgG antibody.
  • 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).
  • the antibody portion A may comprise a scFv (eg, as AB1 of the present application) and an antibody Fc region (eg, an IgG-derived Fc region).
  • the antibody portion A may comprise a single domain antibody VHH and an antibody Fc region (eg, an IgG-derived Fc region).
  • the antibody portion A may be a multispecific (eg, bispecific) antibody that, in addition to the AB1 portion that specifically binds the first target, may also contain other Antigen-binding fragments of target molecules.
  • the antibody portion A may comprise a first Fab and a second Fab, the first Fab of which may be the AB1, and the second Fab of which may specifically bind other target molecules.
  • said antibody portion A may comprise an Fc region.
  • the Fc region may be an IgG-derived Fc region, for example, the Fc region may be an IgG1, IgG2, IgG3 or IgG4-derived Fc region, or a functional variant thereof.
  • the Fc region may be an Fc region derived from human IgG, eg, it may be an Fc region derived from human IgG1, IgG2, IgG3 or IgG4, or a functional variant thereof.
  • the Fc region may comprise at least a CH2 domain and a CH3 domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • AB1 may be an antigen-binding fragment of an antibody capable of specifically binding the first target.
  • AB2 may be an antigen-binding fragment of an antibody capable of specifically binding to said second target.
  • AB3 may be an antigen-binding fragment of an antibody capable of specifically binding to the third target.
  • the antigen-binding fragment of an antibody can be Fab, F(ab) 2 , F(ab'), F(ab') 2 , scFv, affibody and/or single domain antibody (in this application
  • a single domain antibody is also referred to as a nanobody (nanobody or VHH).
  • the first target is a tumor-associated antigen (in this case, the AB1 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens), and the second target is CD3 (in this case, the AB2 is an antigen-binding fragment of an antibody targeting CD3).
  • the first target may be derived from the following target proteins: Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUCl, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM.
  • the first target is derived from Her2 and the second target is CD3.
  • the first target is derived from PD-L1 and the second target is CD3. Therefore, the AB1 can be targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or an antigen-binding fragment of an antibody to EpCAM; and the AB2 may be an antigen-binding fragment of an antibody targeting CD3. In certain instances, the AB1 is an antigen-binding fragment of an antibody targeting Her2 and the AB2 is an antigen-binding fragment of an antibody targeting 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.
  • the first target is CD3 (in this case, the AB1 is an antigen-binding fragment of an antibody targeting CD3)
  • the second target is a tumor-associated antigen (in this case, the AB2 is Antigen-binding fragments of antibodies targeting these tumor-associated antigens).
  • the second target may be derived from the following target proteins: Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUCl, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM.
  • the first target is CD3 and the second target is derived from Her2.
  • the first target is CD3.
  • the AB1 may be an antigen-binding fragment of an antibody targeting CD3, and the AB2 may be an antibody targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA Antigen-binding fragments of antibodies to , GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM.
  • 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.
  • 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.
  • the first target is a tumor-associated antigen (in this case, the AB1 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens)
  • the second target is CD3 (in this case, the AB2 is an antigen-binding fragment of an antibody targeting CD3)
  • the third target is a tumor-associated antigen (in this case, the AB3 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens).
  • the first target is CD3 (in this case, the AB1 is an antigen-binding fragment of an antibody targeting CD3)
  • the second target is a tumor-associated antigen
  • the AB2 is the target Antigen-binding fragments of antibodies targeting these tumor-associated antigens
  • the third target is a tumor-associated antigen (in this case, the AB3 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens).
  • the first target is a tumor-associated antigen (in this case, the AB1 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens), and the second target is a tumor-associated antigen (in this case, The AB2 is an antigen-binding fragment of an antibody targeting these tumor-associated antigens), and the third target is CD3 (in this case, the AB3 is an antigen-binding fragment of an antibody targeting CD3).
  • the tumor-associated antigen can 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.
  • 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.
  • the AB1 can target Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or an antigen-binding fragment of an antibody to EpCAM;
  • the AB2 may be an antigen-binding fragment of an antibody targeting CD3, and the AB3 may be targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin -4, Antigen-binding fragments of antibodies to MUCl, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM.
  • the AB1 can be an antigen-binding fragment of an antibody targeting CD3, and the AB2 can be an antibody targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA, Antigen-binding fragments of antibodies to GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM, and the AB3 may be targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin -4, Antigen-binding fragments of antibodies to MUCl, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 and/or EpCAM.
  • the AB1 can target Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA, CD20, ErbB3, ErbB4, PD-L1 And/or the antigen-binding fragment of the antibody of EpCAM
  • the AB2 can be targeting Her2, Her3, Trop2, EGFR, VEGFR, VEGFR2, BCMA, Nectin-4, MUC1, c-Met, PSMA, GD2, GPC3, CEA , CD20, ErbB3, ErbB4, PD-L1 and/or an antigen-binding fragment of an antibody to EpCAM
  • the AB3 may be an antigen-binding fragment of an antibody targeting CD3.
  • the AB1 is an antigen-binding fragment of an antibody targeting Her2
  • the AB2 is an antigen-binding fragment of an antibody targeting CD3
  • the AB3 is an antigen-binding fragment of an antibody targeting PD-L1 .
  • the AB1 is an antigen-binding fragment of an antibody targeting PD-L1
  • the AB2 is an antigen-binding fragment of an antibody targeting CD3
  • the AB3 is an antigen-binding fragment of an antibody targeting Her2 .
  • the Her2-targeting antibody can be any antibody or antigen-binding fragment thereof that can specifically bind to Her2 (eg, human Her2).
  • the antibody capable of specifically binding to Her2 may be selected from: trastuzumab, pertuzumab and ZHer2:342.
  • the antibody capable of specifically binding to Her2 is trastuzumab.
  • the antibody capable of specifically binding to Her2 is ZHer2:342.
  • the antibody targeting Her2 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 32, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the antibody targeting Her2 may comprise heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 31, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the antibody targeting Her2 may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • the antibody targeting Her2 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 their functional variants or fragment.
  • the antibody targeting Her2 may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the antibody targeting Her2 may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, a functional variant or fragment thereof.
  • LCDR2 light chain CDR2
  • the antibody targeting Her2 may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • the antibody targeting Her2 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 their functional variants or fragments.
  • the antibody targeting Her2 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 34, a functional variant or fragment thereof.
  • the antibody targeting Her2 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 33, a functional variant or fragment thereof.
  • the antibody targeting Her2 may comprise the VH shown in SEQ ID NO: 34 and the VL shown in SEQ ID NO: 33, or their functional variants or fragments.
  • the antibody targeting Her2 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.
  • the antibody targeting Her2 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.
  • the antibody targeting Her2 may comprise the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 15, or their functional variants or fragments.
  • the antibody targeting Her2 may be an affibody, which may comprise the amino acid sequence shown in SEQ ID NO: 10, a functional variant or fragment thereof.
  • the antibody targeting PD-L1 may be any antibody or antigen-binding fragment thereof that can specifically bind to PD-L1 (eg, human PD-L1).
  • the antibody capable of specifically binding to PD-L1 may be selected from: atezolizumab (atezolimumab), durvalumab (durvalumab) and envolimumab (KN035).
  • the antibody capable of specifically binding to PD-L1 is durvalumab.
  • the antibody capable of specifically binding to PD-L1 is envolimumab.
  • the antibody capable of specifically binding to PD-L1 may be a single domain antibody VHH.
  • the antibody targeting PD-L1 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 24, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the antibody targeting PD-L1 may comprise a heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 23, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the antibody targeting PD-L1 may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • the antibody targeting PD-L1 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 their functional variants or fragment.
  • the antibody targeting PD-L1 may comprise light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 21, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the antibody targeting PD-L1 may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 20, a functional variant or fragment thereof.
  • LCDR2 light chain CDR2
  • the antibody targeting PD-L1 may comprise light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • the antibody targeting PD-L1 may comprise HCDR3 shown in SEQ ID NO:24, HCDR2 shown in SEQ ID NO:23, HCDR1 shown in SEQ ID NO:22, and HCDR1 shown in SEQ ID NO:21 LCDR3, LCDR2 shown in SEQ ID NO:20 and LCDR1 shown in SEQ ID NO:19, or functional variants or fragments thereof.
  • the antibody targeting PD-L1 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 26, a functional variant or fragment thereof.
  • the antibody targeting PD-L1 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 25, a functional variant or fragment thereof.
  • the antibody targeting PD-L1 may comprise the VH shown in SEQ ID NO: 26 and the VL shown in SEQ ID NO: 25, or their functional variants or fragments.
  • the antibody targeting PD-L1 may comprise a heavy chain H, and the heavy chain H may comprise the amino acid sequence shown in SEQ ID NO: 18, a functional variant or fragment thereof.
  • the antibody targeting PD-L1 may comprise a light chain L, and the light chain L may comprise the amino acid sequence shown in SEQ ID NO: 17, a functional variant or fragment thereof.
  • the antibody targeting PD-L1 may comprise the heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 17, or their functional variants or fragments.
  • the antibody targeting PD-L1 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.
  • the antibody targeting CD3 may be any antibody or antigen-binding fragment thereof capable of specifically binding to CD3 (eg, human CD3).
  • the antibody capable of specifically binding to CD3 may be selected from: OKT3, M291, YTH12.5, blinatumomab and catumaxomab.
  • the antibody capable of specifically binding to CD3 may be a single domain antibody VHH.
  • the antibody targeting CD3 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 42, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the antibody targeting CD3 may comprise heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 41, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the CD3-targeting antibody may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 40, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • the antibody targeting CD3 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 their functional variants or fragments .
  • the CD3-targeting antibody may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the CD3-targeting antibody may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 38, a functional variant or fragment thereof.
  • LCDR2 light chain CDR2
  • the CD3-targeting antibody may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 37, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • the antibody targeting CD3 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.
  • the antibody targeting CD3 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 36, a functional variant or fragment thereof.
  • the antibody targeting CD3 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 35, a functional variant or fragment thereof.
  • the antibody targeting CD3 may comprise the VH shown in SEQ ID NO: 36 and the VL shown in SEQ ID NO: 35, or their functional variants or fragments.
  • the CD3-targeting antibody may comprise a heavy chain H, and the heavy chain H may comprise the amino acid sequence shown in any one of SEQ ID NO: 44, a functional variant or fragment thereof.
  • the CD3-targeting antibody may comprise a light chain L, and the light chain L may comprise the amino acid sequence shown in any one of SEQ ID NO: 43, a functional variant or fragment thereof.
  • the CD3-targeting antibody may comprise a heavy chain shown in SEQ ID NO:44 and a light chain shown in SEQ ID NO:43, or functional variants or fragments thereof.
  • the antibody targeting CD3 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.
  • the AB1 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 24 and 32, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the AB1 may comprise heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 23 and 31, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the AB1 may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 22 and 30, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • 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.
  • 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.
  • the AB1 may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 21 and 29, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the AB1 may comprise a light chain CDR2 (LCDR2)
  • the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 20 and 28, a functional variant or fragment thereof.
  • the AB1 may comprise a light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 19 and 27, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • 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, SEQ ID NO LCDR2 shown in :28 and LCDR1 shown in SEQ ID NO:27, or their functional variants or fragments.
  • 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, SEQ ID NO LCDR2 shown in :20 and LCDR1 shown in SEQ ID NO: 19, or their functional variants or fragments.
  • the AB1 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 26 and 34, a functional variant or fragment thereof.
  • the AB1 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 25 and 33, a functional variant or fragment thereof.
  • the AB1 may comprise the VH shown in SEQ ID NO: 26 and the VL shown in SEQ ID NO: 25, or functional variants or fragments thereof.
  • the AB1 may comprise the VH shown in SEQ ID NO: 34 and the VL shown in SEQ ID NO: 33, or functional variants or fragments thereof.
  • 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.
  • the AB1 may be an affimer, which may comprise the amino acid sequence shown in SEQ ID NO: 10, a functional variant or fragment thereof.
  • the AB2 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 42, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the AB2 may comprise heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 41, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the AB2 may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 40, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • 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.
  • the AB2 may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 39, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the AB2 may comprise light chain CDR2 (LCDR2), and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 38, a functional variant or fragment thereof.
  • LCDR2 light chain CDR2
  • the AB2 may comprise light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 37, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • 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, SEQ ID NO LCDR2 shown in :38 and LCDR1 shown in SEQ ID NO:37, or their functional variants or fragments.
  • the AB2 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 36, a functional variant or fragment thereof.
  • the AB2 may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 35, a functional variant or fragment thereof.
  • the AB2 may comprise the VH shown in SEQ ID NO: 36 and the VL shown in SEQ ID NO: 35, or functional variants or fragments thereof.
  • 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.
  • the AB3 may comprise heavy chain CDR3 (HCDR3), and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 24 and 32, a functional variant or fragment thereof.
  • HCDR3 heavy chain CDR3
  • the AB3 may comprise heavy chain CDR2 (HCDR2), and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 23 and 31, a functional variant or fragment thereof.
  • HCDR2 heavy chain CDR2
  • the AB3 may comprise heavy chain CDR1 (HCDR1), and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 22 and 30, a functional variant or fragment thereof.
  • HCDR1 heavy chain CDR1
  • 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.
  • 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.
  • the AB3 may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 21 and 29, a functional variant or fragment thereof.
  • LCDR3 light chain CDR3
  • the AB3 may comprise a light chain CDR2 (LCDR2), and the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 20 and 28, a functional variant or fragment thereof.
  • LCDR2 light chain CDR2
  • the AB3 may comprise light chain CDR1 (LCDR1), and the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 19 and 27, a functional variant or fragment thereof.
  • LCDR1 light chain CDR1
  • 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, SEQ ID NO LCDR2 shown in :28 and LCDR1 shown in SEQ ID NO:27, or their functional variants or fragments.
  • 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, SEQ ID NO LCDR2 shown in :20 and LCDR1 shown in SEQ ID NO: 19, or their functional variants or fragments.
  • the AB3 may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 26 and 34, a functional variant or fragment thereof.
  • the AB3 may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 25 and 33, a functional variant or fragment thereof.
  • the AB3 may comprise the VH shown in SEQ ID NO: 26 and the VL shown in SEQ ID NO: 25, or functional variants or fragments thereof.
  • the AB3 may comprise the VH shown in SEQ ID NO: 34 and the VL shown in SEQ ID NO: 33, or functional variants or fragments thereof.
  • 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.
  • the AB3 may be an affimer, which may comprise the amino acid sequence shown in SEQ ID NO: 10, a functional variant or fragment thereof.
  • said Fuc* may comprise the structure Fuco-L-AB2.
  • the structure of L may be J-(L 1 ) n -X 1 Y 1 -(L 1 ') n' .
  • the Fuc*' may comprise the structure Fuco-J-(L 1 ) n -X 1 .
  • the group X 1 includes a functional group capable of participating in a bioorthogonal linking reaction.
  • said X may comprise a functional group selected from the group consisting of azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2,4-triazinyl, terminal alkenyl, cycloalkenyl, ketone group, aldehyde group, hydroxylamine group, mercapto group, maleimide group and their functional derivatives.
  • the X 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)arylene alkylene, and R 2 is selected from the group consisting of: 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 X may comprise a functional group selected from the group consisting of:
  • the X1 may contain
  • the X1 may contain
  • the X1 could be
  • the Y 1 includes a functional group capable of bioorthogonal linkage reaction with the X 1 .
  • said Y may comprise a functional group selected from the group consisting of azido, terminal alkynyl, cycloalkynyl, tetrazinyl, 1,2,4-triazinyl, terminal alkenyl, cycloalkenyl, ketone group, aldehyde group, hydroxylamine group, mercapto group, maleimide group and their functional derivatives.
  • said Y 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)arylene alkylene, and R 2 is selected from the group consisting of: 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.
  • said Y may comprise a functional group selected from the group consisting of:
  • the Y1 may contain
  • Y1 could be
  • said X and said Y may comprise a set of structures selected from the following groups:
  • the X1 may contain and the Y1 may contain
  • the X1 may contain and the Y1 may contain
  • the X1 may contain and the Y1 may contain
  • the X 1 Y 1 is the residual group after the connection reaction between the group X 1 and the group Y 1 occurs.
  • said X 1 Y 1 may comprise a structure selected from the group consisting of:
  • said J is a zygote directly connected to Fuco.
  • Said J is connected to the left end of formula (III).
  • the J can be Wherein said Rf can be -CH 2 -, -NH- or -O-.
  • the J can be Wherein the left end of the J structure is connected with the Fuco.
  • the L 1 is the first linker, and n can be 0 or 1.
  • the first linker L 1 does not exist, and the J can be directly connected to the X 1 or X 1 Y 1 .
  • n is 1, said J is connected to said X 1 or X 1 Y 1 through said L 1 .
  • the L 1 can be C 3 -C 200 polypeptidylene, 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)aryl Alkylene, C 3 -C 200 alkyl (hetero)arylene, their derivatives, or any combination thereof.
  • the subpolypeptidyl group, alkylene group, cycloalkylene group, alkenylene group, cycloalkenylene group, alkynylene group, cycloalkynylene group, (hetero)arylene group, (hetero)aryl alkylene group Or an alkyl(hetero)arylene group may be optionally substituted by one or more Rs 1 and/or optionally interrupted by one or more Rs 2 (e.g., one or more Rs may be inserted in any of the preceding groups Rs 2 group, for example, a plurality of -O- can be inserted into the aforementioned alkylene to obtain a polyethylene glycol group).
  • Each of said Rs 1 can be independently selected from: halogen, -OH, -NH 2 and -COOH.
  • Each of said Rs 2 can be independently selected from: -O-, -S-, wherein Rs 3 is selected from the group consisting of 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 Substituted C 3 -C 24 cycloalkyl.
  • said L 1 can be selected from: Where s1 is 1-50 (eg 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1); wherein each s2 is independently 0-50 (such as 0-45, 0-40, 0-35, 0-30, 0-25, 0 An integer of -20, 0-15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0).
  • s1 is 1-50 (eg 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1);
  • each of said -CH 2 - (-CH 2 - in brackets) is optionally replaced by -O-, but adjacent -CH 2 - are not simultaneously replaced by -O- (for example, there are no two or more a plurality of connected -O-); the left end of the structure can be connected to the J, and the right end of the structure can be connected to the X1 .
  • the L 1 is a C 2 -C 20 polyethylene glycol (PEG) group.
  • L 1 may be -(CH 2 OCH 2 ) s1' -, wherein s1' may be an integer of 0-20.
  • said L 1 is C 1 -C 50 alkylene.
  • the L can be selected from the following group: And the right end of the structure is connected to the X1 .
  • the L 1 ' is the second linker, and n' can be 0 or 1.
  • the second linker L 1 ' does not exist, and Y 1 or X 1 Y 1 can be directly connected to the AB2.
  • n' is 1, said Y 1 or X 1 and said AB2 are connected through said L 1 '.
  • the L 1 ' can be a C 3 -C 200 polypeptide group, a C 1 -C 200 alkylene group, a C 3 -C 200 cycloalkylene group, a C 2 -C 200 alkenylene group, 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)arylene alkylene, C 3 -C 200 alkyl(hetero)arylene, their derivatives, or any combination thereof.
  • the subpolypeptidyl group, alkylene group, cycloalkylene group, alkenylene group, cycloalkenylene group, alkynylene group, cycloalkynylene group, (hetero)arylene group, (hetero)aryl alkylene group Or an alkyl(hetero)arylene group may be optionally substituted by one or more Rs 1 and/or optionally interrupted by one or more Rs 2 (e.g., one or more Rs may be inserted in any of the preceding groups Rs 2 group, for example, a plurality of -O- can be inserted into the aforementioned alkylene to obtain a polyethylene glycol group).
  • Each of said Rs 1 can be independently selected from: halogen, -OH, -NH 2 and -COOH.
  • Each of said Rs 2 can be independently selected from: -O-, -S-, wherein Rs 3 is selected from the group consisting of 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 Substituted C 3 -C 24 cycloalkyl.
  • the L 1 ' can be selected from where each s2 is independently 0-50 (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 -CH2- is optionally replaced by -O-, but adjacent -CH2- are not simultaneously replaced by -O- (eg, there are no two or more linked -O-),
  • the right end of the structure can be connected to the AB2, and the left end of the structure can be connected to the Y1 or X1Y1 .
  • the right end of the L 1 ' structure may be connected (eg, by condensation reaction) via an amino group to -COOH at the C-terminal of the AB2 structure to form a peptide bond.
  • the L 1 ' can be selected from the following structures:
  • the left end of the structure is connected with Y1
  • the right end can be connected with the AB2 through an amino group.
  • the GalX is optionally substituted galactose. In certain instances, the GalX is galactose.
  • the GalX is a substituted galactose.
  • one or more hydroxyl groups at the C2, C3, C4 and C6 positions of the galactose may be substituted.
  • the hydroxyl group at the C2 position of the galactose may be substituted.
  • the GalX is a monosaccharide.
  • the monosaccharides can be, for example, molecules that cannot be further simply hydrolyzed into smaller sugars.
  • the monosaccharide may be a monosaccharide derivative, but contain only one core monosaccharide backbone.
  • GalNAz, GalNH2 or GalNAc are monosaccharides.
  • LacNAc which contains both Gal and GlcNAc
  • 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.
  • alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, (hetero)aryl, alkyl (hetero)aryl and/or (hetero)arylalkyl are independently may be optionally substituted by one or more substituent Rs 4 , and/or each independently may be optionally interrupted by one or more substituent Rs 5 (for example, one or more Rs may be inserted in any of the foregoing groups 5 groups, for example, a plurality of -O- can be inserted into the aforementioned alkyl group to obtain a polyethylene glycol group).
  • Each of said Rs 4 may be selected from the group consisting of halogen, -OH, -NH 2 , -SH, -N 3 , -COOH and -CN.
  • Each of said Rs 5 can be independently selected from the following group: -O-, -S-, wherein, the Rs 3 can 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.
  • the GalX can be substituted by Replacement, 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) ) aryl alkylene.
  • the alkylene, cycloalkylene, alkenylene, cycloalkenylene, alkynylene, cycloalkynylene, (hetero)arylene, alkyl (hetero)arylene and/or ( Hetero)arylalkylene groups can each independently be optionally substituted by one or more substituents Rs 4 and/or each independently be optionally interrupted by one or more substituents Rs 5 (for example, in any of the preceding groups).
  • One or more Rs 5 groups can be inserted in, for example, multiple -O- can be inserted into the aforementioned alkylene to obtain a polyethylene glycol group).
  • 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 (hetero)aryl, wherein the alkyl, cycloalkyl, alkynyl, cycloalkynyl and/or (hetero) ) aryl groups can each independently be optionally substituted by one or more Rs 4 .
  • Each of said Rs 4 can be independently selected from: halogen, -OH, -NH 2 , -SH, -N 3 , -COOH and -CN.
  • Each of said Rs 5 can be independently selected from: -O-, -S-, Said 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 GalX can be selected from the group consisting of:
  • the GalX* may have the following structure: GalX 2 Y 2 -(L 2 ') m -AB3.
  • GalX 2 Y 2 is the residual group after the connection reaction between GalX 2 and group Y 2 , wherein the GalX 2 contains X 2 , X 2 contains a functional group capable of participating in a bioorthogonal connection reaction, and the Y 2 Contains a functional group capable of bioorthogonal linkage reaction with the X2 .
  • the L 2 ' is a linker, and m can be 0 or 1.
  • the linker L 2 ' does not exist, and Y 2 or X 2 Y 2 can be directly connected to the AB3.
  • m is 1, the Y 2 or X 2 Y 2 and the AB3 may be connected through the L 2 ′.
  • the L 2 ' can be a C 3 -C 200 polypeptidene group, a C 1 -C 200 alkylene group, a C 3 -C 200 cycloalkylene group, a C 2 -C 200 alkenylene group, 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)arylene alkylene, C 3 -C 200 alkyl(hetero)arylene, their derivatives, or any combination thereof.
  • the subpolypeptidyl group, alkylene group, cycloalkylene group, alkenylene group, cycloalkenylene group, alkynylene group, cycloalkynylene group, (hetero)arylene group, (hetero)aryl alkylene group Or an alkyl(hetero)arylene group may be optionally substituted by one or more Rs 1 and/or optionally interrupted by one or more Rs 2 (e.g., one or more Rs may be inserted in any of the preceding groups Rs 2 group, for example, a plurality of -O- can be inserted into the aforementioned alkylene to obtain a polyethylene glycol group).
  • Each of said Rs 1 can be independently selected from: halogen, -OH, -NH 2 and -COOH.
  • Each of said Rs 2 can be independently selected from: -O-, -S-, wherein Rs 3 is selected from the group consisting of 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 Substituted C 3 -C 24 cycloalkyl.
  • the L2 ' can be selected from: where each s2 is independently 0-50 (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 by -O-, but adjacent -CH 2 - are not simultaneously Replaced by -O- (for example, there are no two or more connected -O-), the right end of the structure can be connected to the AB3, and the left end of the structure can be connected to the Y2 or X2Y2 connect. In some cases, the right end of the L 2 ' structure can be connected via an amino group to -COOH at the C-terminal of the AB3 structure to form a peptide bond (eg, through a condensation reaction).
  • each s2 is independently 0-50 (e.g. 0-45, 0-40, 0-35, 0-30, 0-25,
  • the L 2 ' can have the following structure: The left end of the structure is connected with Y2 , and the right end can be connected with the AB2 through an amino group.
  • the GalX 2 may be galactose in which one or more hydroxyl groups at positions C2, C3, C4 and C6 are substituted. In some cases, the GalX 2 can be galactose in which the hydroxyl group at the C2 position is substituted. In certain instances, the GalX 2 is a monosaccharide.
  • X 2 in the GalX 2 can be azide
  • the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound. In some cases, the GalX2 can be any organic compound.
  • the Y2 may contain
  • the X2 can be azide and the Y2 may contain
  • the X2Y2 may comprise a structure selected from the group consisting of:
  • the antibody-Fuc*' conjugate of the conjugator structure reacts with Y 1 -(L 1 ') n' -AB2 to produce the multispecific antibody described in this application with higher efficiency.
  • Fuc*' contains the structure Fuco-J-(L 1 ) n -X 1 , J is
  • structure conjugates have higher efficiency in reacting with Y 1 -(L 1 ') n' -AB2 to prepare the multispecific antibody described in this application.
  • the Q may be guanosine diphosphate (GDP), uridine diphosphate (UDP) and/or cytidine diphosphate (CDP).
  • the Q can be guanosine diphosphate (GDP).
  • the Q-Fuc*' may be GDP-Fuc*'.
  • GDP-fucose derivatives with a zygote structure such as GDP-Fuc*'
  • the GDP-fucose derivatives of the conjugate structure such as GDP-Fuc*'
  • the conversion rate on antibody-(GalNAz ⁇ 1,4)GlcNAc) was significantly improved.
  • the Q-Fuc*' can be selected from the following structures:
  • the catalyst in step i) may contain fucosyltransferase.
  • the fucosyltransferase may be an alpha-1,3-fucosyltransferase or a functional variant or fragment thereof.
  • the fucosyltransferase may be derived from bacteria, eg, it may be derived from Helicobacter pylori (eg Helicobacter pylori 26695).
  • the fucosyltransferase may be derived from ⁇ -1,3 fucosyltransferase with GenBank accession number AAB81031.1, GenBank accession number AAD07447.1 or GenBank accession number AAD07710.1, or their variants or fragments.
  • the fucosyltransferase may be derived from ⁇ -1,3 fucosyltransferase with GenBank accession number AAD07710.1.
  • the fucosyltransferase may be ⁇ -1,3 fucosyltransferase with GenBank accession number AAD07710.1, or a functional variant or fragment thereof.
  • the wild-type ⁇ -1,3 fucosyltransferase with GenBank accession number AAD07710.1 has a catalytically active region, 10 polypeptide repeats and a C-terminal tail structure, and its functional variants or fragments can contain catalytic activity Regions and 1-10 polypeptide repeats.
  • said fucosyltransferase in step i) may comprise a catalytically active region and at least one heptapeptide repeat (HPR)
  • said fucosyltransferase in step i) may comprise a catalytically active region and 1-10 heptad repeats (HPRs) (eg, 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.
  • the C-terminus of the catalytically active region can be linked to the HPR (eg, the N-terminus of the HPR).
  • the catalytically active region may comprise the amino acid sequence shown in SEQ ID NO: 1 or a functional variant or fragment thereof.
  • the catalytically active region may comprise the amino acid sequence shown in SEQ ID NO: 1 or have at least about 80% (e.g., at least about 82%, at least about 85%, at least about 88%, Amino acid sequences having at least about 90%, at least about 92%, at least about 95%, at least about 98%, at least about 99% or greater) sequence identity.
  • 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 higher) sequence identity.
  • sequence identity means, after aligning a candidate sequence (e.g., the sequence of a variant) with a reference sequence (e.g., the sequence of a parent) and introducing gaps (if necessary) to achieve a maximum sequence After percent identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference sequence. Sequence alignment can be carried out by means known to those skilled in the art to determine the percentage of amino acid sequence identity, for example, BLAST, BLAST-2, Clustal W, ALIGN-2, Megalign (DNASTAR) software or FASTA program package, etc. can be used . Appropriate parameters for aligning sequences can be determined by those skilled in the art.
  • the catalytically active region may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the HPR may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the fucosyltransferase may comprise the amino acid sequence shown in SEQ ID NO:3
  • the catalyst may comprise a fucosyltransferase and a tag sequence as described above.
  • the catalyst may comprise the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4.
  • 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.
  • the preparation method described in the present application may also include the following steps: treating the protein comprising sugar chains and the antibody part A with an endoglycosidase to obtain the treated protein; making the treated protein
  • the protein is contacted with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX(VI) shown in formula (VI), the structure of the protein is as follows: As shown in (VIII): Wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond , b is 0 or 1; and GalX is optionally substituted galactose, and the GalX is connected to the GlcNAc through a ⁇ -1,4 glycosidic bond
  • the suitable catalyst may be ⁇ 1,4 galactosyltransferase or a functional variant thereof.
  • the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the endoglycosidase can be Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, Endo H and/or their functional variants.
  • the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO:6.
  • b is 1 if the protein comprising sugar chains and the antibody part A has a core ⁇ -1,6 fucose modification, for example.
  • b is 0 if the protein comprising sugar chains and said antibody part A does not have core ⁇ -1,6 fucose modification, if any.
  • the preparation method described in the present application may further comprise the step of treating the protein comprising sugar chains and the antibody part A with endoglycosidase and ⁇ 1,6 fucosidase to obtain the treated protein; make the processed protein contact with UDP-GalX in the presence of a suitable catalyst to obtain a protein having a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX(VI) shown in formula (VI) Protein, the structure of this protein is shown in formula (VIII): (VIII), wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly linked to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through ⁇ -1, 6 glycosidic linkages, b is 0; and GalX is optionally substituted galactose, and the GalX and the GlcNAc are linked by ⁇ -1,4 glycos
  • the suitable catalyst may be ⁇ 1,4 galactosyltransferase or a functional variant thereof.
  • the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the endoglycosidase can be Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, Endo H and/or their functional variants.
  • the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the ⁇ 1,6 fucosidase may be BfFucH, Alfc, BKF and/or functional variants thereof.
  • the ⁇ 1,6 fucosidase may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the preparation method described in the present application may also include the following steps: treating the protein comprising sugar chains and the antibody part A with an endoglycosidase to obtain the treated protein; making the treated protein
  • the protein of the protein is contacted with UDP-GalX 2 under the condition that a suitable catalyst exists, so as to obtain a protein having a sugar chain comprising the structure-GlcNAc(Fuc) b -GalX 2 (IX) shown in the formula (IX), the structure of the protein As shown in formula (XI):
  • GlcNAc is N-acetylglucosamine, and the GlcNAc is directly connected to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through an ⁇ -1,6 glycosidic bond , b is 0 or 1; and GalX 2 is substituted galactose and contains X 2 , X 2 contains a functional group capable of participating in
  • the suitable catalyst may be ⁇ 1,4 galactosyltransferase or a functional variant thereof.
  • the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the endoglycosidase can be Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, Endo H and/or their functional variants.
  • the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO:6.
  • b is 1 if the protein comprising sugar chains and the antibody part A has a core ⁇ -1,6 fucose modification, for example.
  • b is 0 if the protein comprising sugar chains and said antibody part A does not have core ⁇ -1,6 fucose modification, if any.
  • the preparation method described in the present application may also include the following steps: treating the protein comprising sugar chains and the antibody part A with endoglycosidase and ⁇ 1,6 fucosidase to obtain the treated protein; make the processed protein contact with UDP-GalX in the presence of a suitable catalyst to obtain a sugar chain comprising the structure -GlcNAc(Fuc) b -GalX 2 (IX) shown in formula (IX)
  • the albumen, the structure of this albumen is shown in formula (XI): (XI), wherein, GlcNAc is N-acetylglucosamine, and the GlcNAc is directly linked to amino acid N297 of the Fc region; Fuc is fucose, and the Fuc is connected to the GlcNAc through ⁇ -1, 6 glycosidic linkages, b is 0; and GalX 2 is substituted galactose and contains X 2 , X 2 contains a functional group capable of participating in
  • the suitable catalyst may be ⁇ 1,4 galactosyltransferase or a functional variant thereof.
  • the suitable catalyst may comprise the amino acid sequence shown in SEQ ID NO:5.
  • the endoglycosidase can be Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D, Endo H and/or their functional variants.
  • the endoglycosidase may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the ⁇ 1,6 fucosidase may be BfFucH, Alfc, BKF and/or functional variants thereof.
  • the ⁇ 1,6 fucosidase may comprise the amino acid sequence shown in SEQ ID NO:7.
  • composition which may comprise one or more multispecific antibodies described herein.
  • compositions described herein may be pharmaceutical compositions.
  • it may comprise one or more pharmaceutically acceptable carriers or excipients.
  • the present application provides a method for preventing, alleviating and/or treating a disease or disorder, the method comprising administering the multispecific antibody described in the present application to a subject in need, and/or the present application said composition.
  • the present application provides the use of the multispecific antibody described in the present application and/or the composition described in the present application in the preparation of a medicament for preventing, alleviating and/or treating a disease or condition.
  • the disease or condition may include, for example, a tumor, cancer, or other proliferative disease.
  • the disease or condition can also include, for example, a disease or condition related to the immune system (eg, an autoimmune disease).
  • Protein molecular weight analysis was performed using a Xevo G2-XS QTOF mass spectrometer (Waters Corporation).
  • the mass spectrometer was equipped with an electrospray ionization source (ESI) and an Acuqity UPLC I-Class plus system (Waters Corporation).
  • the purified protein was purified by Waters ACQUITY UPLC Protein BEH C4 column ( 1.7 ⁇ m, 2.1mm x 100mm) for separation and desalination.
  • Mobile phase A was 0.1% formic acid/water solution
  • mobile phase B was acetonitrile containing 0.1% formic acid in water, and the flow rate was 0.2 mL/min.
  • Data analysis was performed with Waters Unify software (version 1.9.4, Waters Corporation).
  • BGalT1(Y289L) Bovine ⁇ -1,4-galactosyltransferase I, bovine ⁇ -1,4-galactosyltransferase with Y289L mutation
  • EndoS Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S
  • Cloning, expression and purification of Alfc Lactobacillus casei ⁇ -1,6-fucosidase Alfc, Lactobacillus casei ⁇ -1,6-fucosidase
  • Sortase 5M Sortase 5M (Staphylococcus aureus Sortase 5M, Staphylococcus aureus transpeptidase 5M)
  • HpFT-2HR which comprises a catalytically active region, two heptad repeat sequences and a C-terminal fusion His tag, whose amino acid sequence is shown in SEQ ID NO: 4
  • NdeI and BamHI enzymes Insert the nucleic acid sequence into the pET24b vector (Nanjing GenScript). Transform E.coli BL21(DE3) with the constructed recombinant plasmid.
  • the transformed recombinant bacteria were cultured in LB medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C until the OD 600 reached 0.6-0.8, and then IPTG (isopropyl- ⁇ -D-thiosemi Lactose) to a final concentration of 0.2 mM, and then continue to incubate at 25° C. and 200 rpm for 16 hours to induce protein expression.
  • the induced cells were centrifuged and suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 500 mM sodium chloride, 20 mM imidazole, 1 mM PMSF (phenylmethylsulfonyl fluoride)).
  • the suspended cells were lysed by sonication, and then purified with Ni-NTA packing (GE Health).
  • the main fractions were collected with a purity greater than 90% and then dialyzed into storage buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol).
  • the transformed recombinant bacteria were cultured in LB medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C until the OD 600 reached 0.6-0.8, and then IPTG (isopropyl- ⁇ -D-thiosemi Lactose) to a final concentration of 0.2 mM, and then continue to incubate at 25° C. and 200 rpm for 16 hours to induce protein expression.
  • the induced cells were centrifuged and suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 500 mM sodium chloride, 20 mM imidazole, 1 mM PMSF (phenylmethylsulfonyl fluoride)).
  • the suspended cells were lysed by sonication, and then purified with Ni-NTA (GE Health) filler.
  • Ni-NTA GE Health filler.
  • the main fractions were collected with a purity greater than 90% and then dialyzed into storage buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl).
  • GDP-FAz was synthesized with reference to the method reported by Wu P. et al. (Proc. Natl. Acad. Sci. USA 2009, 106, 16096). And through Bio-Gel P-2Gel column, using 50mM ammonium bicarbonate solution as the eluent to complete the purification.
  • the amino acid sequence of the light chain of Trastuzumab is shown in SEQ ID NO:15, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:16.
  • the amino acid sequence of the light chain of Durvalumab is shown in SEQ ID NO: 17, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18.
  • reaction solution containing the Ni-NTA filler was centrifuged at 4000rpm for 1 min to remove the filler, and the reaction solution was purified with an SP column (cation exchange chromatography column, Suzhou Nanowell) to obtain the modified target product.
  • SP column cation exchange chromatography column, Suzhou Nanowell
  • reaction solution containing the Ni-NTA filler was centrifuged at 4000rpm for 1 min to remove the filler, and the reaction solution was purified with an SP column to obtain the modified target product.
  • the mass spectrum identification showed that the main peak was DBCO-aPDL1 (15213Da).
  • reaction solution containing the Ni-NTA filler was centrifuged at 4000rpm for 1 min to remove the filler, and the reaction solution was purified with an SP column to obtain the modified target product.
  • the mass spectrometry showed that the main peak was DBCO-aCD3 (15099Da).
  • reaction solution containing the Ni-NTA filler was centrifuged at 4000rpm for 1 min to remove the filler, and the reaction solution was purified with an SP column to obtain the modified target product.
  • the mass spectrum identification showed that the main peak was BCN-aCD3 (14990Da).
  • the mass spectrum identification showed that the main peak was Trastuzumab-(GalNAz-DBCO-aPDL1)GlcNAc-FAmP 8 Tz-BCN-aCD3 (abbreviated as Tras-aPDL1-aCD3) (207930Da, MAR2+MAR2).
  • the results are shown in Fig. 3E, showing that the obtained trispecific antibodies have good homogeneity.
  • Example 26 Comparison of the efficiency of preparing the multispecific antibody described in this application by reacting antibody-Fuc*' conjugates containing different conjugators with Y 1 -(L 1 ') n' -AB2.
  • DBCO-aCD3 (final concentration 1 mg/mL) was added to 1 ⁇ PBS (pH 7.0) buffer, followed by Trastuzumab-(Gal ⁇ 1,4)GlcNAc-FAz, Trastuzumab-(Gal ⁇ 1,4)GlcNAc-FAmAz or Trastuzumab-( Gal ⁇ 1,4) GlcNAc-FAmP 4 Az (final concentration 0.5 mg/mL), and the above mixture was reacted at room temperature for 2 h. After reaching the reaction time point, 20 ⁇ L of samples were taken respectively, added with 5 ⁇ L of loading buffer, treated at 90°C for 5 min, and then samples of the same volume were taken for SDS-PAGE detection. The results are shown in Figure 9.
  • Affinity detection for Her2 antigen Add different concentrations of Tras-aCD3, Tras-aPDL1-aCD3 and Dur-ZHer-aCD3 into Her2-positive SKOV3 (Her2+/PDL1-) cells, and the cells were resuspended in FACs buffer (DPBS containing 2% bovine serum), 60000 cells/well in duplicate. After incubation at 4°C for 30 minutes, unbound multispecific antibodies on the cells were washed away with FACs buffer (DPBS containing 2% fetal bovine serum), and then the cells were resuspended in 30 ⁇ L of FACs buffer.
  • FACs buffer DPBS containing 2% bovine serum
  • Affinity test for CD3 antigen Add different concentrations of Tras-aCD3, Tras-aPDL1-aCD3 and Dur-ZHer-aCD3 to CD3+ Jurkat cells, resuspend the cells in FACs buffer, 60,000 cells/well, in duplicate share. After incubation at 4°C for 30 minutes, unbound multispecific antibodies on the cells were washed away with FACs buffer, and then the cells were resuspended in 30 ⁇ L of FACs buffer.
  • PDL-1 antigen Dilute the recombinant PDL-1 antigen (PDL-1, nearshore protein) with coating buffer (50mM Na 2 CO 3 /NaHCO 3 , pH9.6) to a final concentration of 250ng/mL , inoculate 96-well plates at a volume of 100 ⁇ L per well and incubate overnight in a 4°C refrigerator. The next day, after removing the coating solution, first block with blocking solution (PBS solution containing 3% (v/v) fetal bovine serum albumin) at 30° C. for 2 hours in an incubator, 200 ⁇ L per well.
  • PBS solution containing 3% (v/v) fetal bovine serum albumin
  • sample dilution buffer PBS solution containing 1% (v/v) fetal bovine serum albumin
  • Samples (Durvalumab, Dur-ZHer-aCD3, Tras-aPDL1-aCD3) 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 plate respectively, 100 ⁇ L per well. After adding the samples, place them in a 30°C incubator and continue to incubate for 1 hour.
  • Both Dur-ZHer-aCD3 and Tras-aPDL1-aCD3 can bind to PDL-1, and Dur-ZHer-aCD3 and Durvalumab have similar binding forces, which are slightly stronger than Tras-aPDL1-aCD3 .
  • RTCA-DP instrument Use the RTCA-DP instrument to test the killing of the effector cells on the target cells (the adherent cells will generate impedance on the metal electrode plate, after being killed, the adhesion effect will become worse and the impedance will decrease, and the RTCA will detect the number of living cells based on microelectronic impedance technology ).
  • the test is suspended, the E-Plate 16PET plate is removed from the instrument, and Tras-aCD3 and 50000 hPBMC (human peripheral Blood mononuclear cells), and control wells were set, and 50,000 hPBMCs, 30nM aCD3 and 50,000 hPBMCs, or 15nM Trastuzumab and 50,000 hPBMCs were added separately, in duplicate. Finally, the test was continued for 30 hours, the data was exported through the instrument, and graphed and analyzed with GraphPad Prism 8. The results are shown in Fig.
  • Killing of target cells by effector cells was tested using the RTCA-DP instrument.
  • Killing of target cells by effector cells was tested using the RTCA-DP instrument.
  • SKOV3 Her2+/PDL1-
  • Killing of target cells by effector cells was tested using the RTCA-DP instrument.
  • Killing of target cells by effector cells was tested using the RTCA-DP instrument.
  • hPBMC were obtained by separating human peripheral blood, and 100,000 hPBMC cells were planted in a flat-bottomed 96-well plate, and the following control groups were designed, which were the no-treatment group (without and with 20,000 SKOV3), and the aCD3 group with a final concentration of 30 nM (without Add and add 20000 SKOV3), final concentration of 15nM commercial anti-human CD3 antibody OKT3 (BioXCell, BE0001-2) group (without and plus 20000 SKOV3), and final concentration of 15nM Tras-aCD3, Tras-aPDL1 - aCD3 and Dur-ZHer-aCD3 groups (without and with 20,000 SKOV3), all experimental wells with a volume of 100 ⁇ L were made in five replicates.
  • the supernatant collected in the previous step was taken out from the -80°C refrigerator, thawed at room temperature, centrifuged at 1000*g for 10 min to remove particles and aggregates, and the supernatant was diluted 5 times.
  • Human IFN- ⁇ and human TNF- ⁇ in the samples were measured using Elisa kit (Beijing Sizhengbai).
  • 100 ⁇ L of supernatants from different control groups were incubated with the coated plate with human IFN- ⁇ and human TNF- ⁇ antibodies, and incubated in a 37° C. incubator for 90 minutes. After the incubation, wash the plate 3 times with washing solution, add 100 ⁇ L of biotin solution, and incubate for 60 min in a 37° C. incubator.
  • hPBMC Human peripheral blood mononuclear cells
  • the cells were resuspended in 30 ⁇ L FACs buffer, and 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 staining, in which CD4-PB, CD8a-PE is used to distinguish CD4+ and CD8+ T cells, CD69-APC was used to assess CD69 upregulation, CD25-Percp/Cy5.5 was used to assess CD25 upregulation, and PD-1-FITC was used to assess PD-1 upregulation.
  • CD4-PB CD8a-PE
  • CD69-APC was used to assess CD69 upregulation
  • CD25-Percp/Cy5.5 was used to assess CD25

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Abstract

本申请提供了一种靶向CD3的多特异性抗体及其应用。

Description

靶向CD3的多特异性抗体及其应用 技术领域
本申请涉及生物医药领域，具体的涉及多特异性抗体及其应用。
背景技术
过去几十年中，人们在新型抗体工程化方面的努力已经取得了不少进展，并由此初步展现了多特异性抗体的广阔应用前景。
目前常见的多特异性抗体(例如，多特异性)的结构包括，例如：四价IgG-scFv融合蛋白(Coloma M.J.等,Nat.Biotechnol,1997,15,159)、双特异性抗体体(diabody)(Holliger P.等,Proc.Natl.Acad.Sci.USA,1993,90,6444)、串联scFv分子(参见，例如，Bargou R.等,Science,2008,321,974)、四价IgG-样双可变结构域抗体(“DVD-Ig”，Wu C.等,Nat.Biotechnol,2007,25,1290)、四价Fab串联免疫球蛋白(“FIT-Ig”)(Wu C B.等,WO2015103072A1.)、二价大鼠/小鼠杂合多特异性IgG(Lindhofer H.等,J.Immunol,1995,155,219)和多特异性Crossmab结合蛋白(参见，例如，Auer J.等,WO2013026831A1.)。
为了治疗某些疾病或病症，可能通过设计和生产可结合两个不同表位和/或两种不同靶标抗原的工程化多特异性抗体，来避免组合疗法带来的复杂性和高昂成本。
其中，引起广泛关注的是用于治疗癌症的多特异性抗体，特别是能够使T细胞重新靶向而杀伤各种肿瘤细胞的多特异性抗体。
常见的T细胞受体(“TCR”)是由α链和β链共价连接的异源二聚体(“TCRαβ”)。CD3分子是T细胞共受体或者被称为T细胞辅助受体，其由五条不同的多肽链组成(即CD3γ链、CD3δ链、两条CD3ε链和两条ζ链)。这些多肽链通过彼此结合而形成三个二聚体的复合物(即εγ、εδ和ζζ复合物)。CD3复合物与TCR结合以在T淋巴细胞中产生激活信号。在缺少CD3的情况下，TCR不能恰当组装，并被降解。TCR复合物非常重要，其包含大约10个免疫受体酪氨酸基的激活基序(ITAMs)。在病理状态下，T细胞是各种器官特异性自身免疫性疾病，例如I型糖尿病、类风湿性关节炎和多发性硬化症的关键参与者。目前，人们认为激活T细胞需要两种信号，以避免单一信号造成不恰当地针对自体抗原的免疫应答。换言之，在T细胞与其他细胞的接触导致仅产生两种必要的信号中的一种时，T细胞不被激活并且不发生适应性免疫应答。
因此，可通过使多特异性抗体结合尚未成熟的T细胞上的T细胞受体(TCR)复合物中 的激活组分(如CD3)来招募或激活T细胞。多特异性抗体与两种细胞类型(例如，肿瘤细胞和T细胞)的同时结合，能够在靶细胞和T细胞之间建立暂时的关联，导致攻击靶向癌细胞的细胞毒性T淋巴细胞(CTL)被激活。
然而，许多多特异性形式的分子，如BiTE、双链抗体、DART和TandAb等，均使用单链形式，通过肽接头连接不同的可变结构域以实现多特异性。由于这些形式不含Fc区，因此它们通常具有非常短的体内半衰期并且在物理上不稳定。另一方面，目前供选择的多特异性抗体的分子构型种类仍较为单一，在一定程度上限制了多特异性分子的设计和应用。此外，肿瘤细胞可以通过抗原丢失来逃逸靶向CD3的多特异性抗体，开发能靶向多种肿瘤抗原的多特异性抗体，有助于提高其药效。
因此，仍需要设计改进的多特异性抗体，来满足对疗效、安全性和设计灵活性等方面的要求。
发明内容
本申请提供了一种多特异性抗体，其包含能够特异性结合第一靶标的抗体部分A；和2个包含式(I)
Figure PCTCN2022074201-appb-000001
所示结构的糖链部分；且所述多特异性抗体具有式(II)所示的结构：
Figure PCTCN2022074201-appb-000002
其中：所述A包含能够特异性结合所述第一靶标的第一抗原结合部分AB1和Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；所述Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000003
(III)，AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；所述第一靶标和所述第二靶标中至少有一个为CD3；并且所述氨基酸N297的位置根据Kabat中的EU索引编号确定。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标与所述第二靶标不相同。
在本申请的多特异性抗体的某些实施方式中，所述AB1和所述AB2各自独立地为抗体 的抗原结合片段。
在本申请的多特异性抗体的某些实施方式中，所述抗原结合片段为Fab，F(ab) 2，F(ab’)，F(ab’) 2，scFv，亲和体(affibody)和/或单域抗体。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标为肿瘤相关抗原，且所述第二靶标为CD3。在本申请的多特异性抗体的某些实施方式中，所述肿瘤相关抗原选自：Her2和PD-L1。
在本申请的多特异性抗体的某些实施方式中，所述A为IgG抗体。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含选自下组的抗体的抗原结合部分：曲妥珠单抗和度伐利尤单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR3(HCDR3)，且所述HCDR3包含SEQ ID NO：24和32中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR2(HCDR2)，且所述HCDR2包含SEQ ID NO：23和31中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR1(HCDR1)，且所述HCDR1包含SEQ ID NO：22和30中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR3(LCDR3)，且所述LCDR3包含SEQ ID NO：21和29中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR2(LCDR2)，且所述LCDR2包含SEQ ID NO：20和28中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR1(LCDR1)，且所述LCDR1包含SEQ ID NO：19和27中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链可变区VH，且所述VH包含SEQ ID NO：26和34中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链可变区VL，且所述VL包含SEQ ID NO：25和33中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述A为曲妥珠单抗或度伐利尤单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB2包含选自下组的抗体的CD3抗原结合部分：OKT3、M291、YTH12.5、博纳吐单抗和卡妥索单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB2包含SEQ ID NO：14所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标为CD3，且所述第二靶标为肿瘤相关抗原。在本申请的多特异性抗体的某些实施方式中，所述肿瘤相关抗原选自：Her2和PD-L1。
在本申请的多特异性抗体的某些实施方式中，所述Fuco-L-AB2中，L的结构为J-(L 1) n-X 1Y 1-(L 1’) n’，其中：所述L 1为第一连接子，n为0或1；所述L 1’为第二连接子，n’为0或1；所述J为直接与Fuco相连接的接合子；X 1Y 1为基团X 1与基团Y 1发生连接反应之后的残留基团，其中所述基团X 1包含能够参与生物正交连接反应的官能团，且所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团。
在本申请的多特异性抗体的某些实施方式中，所述X 1包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。
在本申请的多特异性抗体的某些实施方式中，所述X 1包含选自下组的官能团：
Figure PCTCN2022074201-appb-000004
Figure PCTCN2022074201-appb-000005
Figure PCTCN2022074201-appb-000006
其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
在本申请的多特异性抗体的某些实施方式中，所述X 1包含选自下组的官能团：
Figure PCTCN2022074201-appb-000007
Figure PCTCN2022074201-appb-000008
在本申请的多特异性抗体的某些实施方式中，所述X 1包含
Figure PCTCN2022074201-appb-000009
在本申请的多特异性抗体的某些实施方式中，所述Y 1包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。
在本申请的多特异性抗体的某些实施方式中，所述Y 1包含选自下组的官能团：
Figure PCTCN2022074201-appb-000010
Figure PCTCN2022074201-appb-000011
Figure PCTCN2022074201-appb-000012
其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
在本申请的多特异性抗体的某些实施方式中，所述X 1和所述Y 1包含选自以下各组的一组结构：
a)X 1包含
Figure PCTCN2022074201-appb-000013
且Y 1包含
Figure PCTCN2022074201-appb-000014
b)X 1包含
Figure PCTCN2022074201-appb-000015
且Y 1包含
Figure PCTCN2022074201-appb-000016
c)X 1包含
Figure PCTCN2022074201-appb-000017
且Y 1包含
Figure PCTCN2022074201-appb-000018
d)X 1包含
Figure PCTCN2022074201-appb-000019
且Y 1包含
Figure PCTCN2022074201-appb-000020
和
e)X 1包含
Figure PCTCN2022074201-appb-000021
且Y 1包含
Figure PCTCN2022074201-appb-000022
其中R 1和R 2如本申请前述所
定义的。
在本申请的多特异性抗体的某些实施方式中，所述X 1Y 1包含选自下组的结构：
Figure PCTCN2022074201-appb-000023
在本申请的多特异性抗体的某些实施方式中，所述J为
Figure PCTCN2022074201-appb-000024
Figure PCTCN2022074201-appb-000025
其中所述Rf为-CH 2-，-NH-或-O-，该J结构的左端与所述Fuco直接相连接。
在本申请的多特异性抗体的某些实施方式中，所述J为
Figure PCTCN2022074201-appb-000026
结构的左端和Fuco直接相连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1和所述L 1’各自独立地选自：C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000027
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在本申请的多特异性抗体的某些实施方式中，所述L 1选自：
Figure PCTCN2022074201-appb-000028
Figure PCTCN2022074201-appb-000029
其中每个s1独立地为1-50的整数，每个s2独立地为0-50的整数,每个所述-CH 2-任选地被-O-替代，但连续相邻的-CH 2-不同时被-O-替代，该结构的左端与所述J连接，且该结构的右端与所述X 1连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1选自：
Figure PCTCN2022074201-appb-000030
Figure PCTCN2022074201-appb-000031
Figure PCTCN2022074201-appb-000032
且该结构的右端与所述X 1连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1’选自：
Figure PCTCN2022074201-appb-000033
Figure PCTCN2022074201-appb-000034
Figure PCTCN2022074201-appb-000035
其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1’选自：
Figure PCTCN2022074201-appb-000036
Figure PCTCN2022074201-appb-000037
该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
在本申请的多特异性抗体的某些实施方式中，所述GalX为半乳糖。
在本申请的多特异性抗体的某些实施方式中，所述GalX为被取代的半乳糖，且所述半乳糖中位于C2、C3、C4和/或C6位置的一个或多个羟基被取代。
在本申请的多特异性抗体的某些实施方式中，所述GalX为被取代的半乳糖，且所述半乳糖中位于C2位置的羟基被取代。
在本申请的多特异性抗体的某些实施方式中，所述GalX为单糖。
在本申请的多特异性抗体的某些实施方式中，所述GalX被取代基Rg 1取代，Rg 1为选自下组的基团：氢、卤素、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24烯基、C 5-C 24环烯基、C 2-C 24炔基、C 6-C 24环炔基、C 3-C 24(杂)芳基、C 3-C 24烷基(杂)芳基和C 3-C 24(杂)芳基烷基；其中，所述烷基、环烷基、烯基、环烯基、炔基、环炔基、(杂)芳基、烷基(杂)芳基和/或(杂)芳基烷基各自独立地任选被一个或多个取代基Rs 4取代，和/或各自独立地任选被一个或多个取代基Rs 5间隔；其中，每个所述Rs 4各自独立地选自下组：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN；每个所述Rs 5各自独立地选自下组：-O-、-S-、
Figure PCTCN2022074201-appb-000038
其中，所述Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在本申请的多特异性抗体的某些实施方式中，所述GalX被取代基
Figure PCTCN2022074201-appb-000039
Figure PCTCN2022074201-appb-000040
取代，其中：t为0或1；Rg 2为选自下组的基团：C 1-C 24亚烷基、C 3-C 24亚环烷基、C 2-C 24亚烯基、C 5-C 24亚环烯基、C 2-C 24亚炔基、C 6-C 24亚环炔基、C 3-C 24(杂)亚芳基、C 3-C 24烷基(杂)亚芳基和C 3-C 24(杂)芳基亚烷基，其中，所述亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、烷基(杂)亚芳基和/或(杂)芳基亚烷基各自独立地任选被一个或多个取代基Rs 4取代，和/或各自独立地任选被一个或多个取代基Rs 5间隔，所述Rg 3选自：氢、卤素、-OH、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24炔基、C 5-C 24环炔基和C 2-C 24(杂)芳基，其中所述烷基、环烷基、炔基、环炔基和/或(杂)芳基各自独立地任选被一个或多个Rs 4取代，其中，每个所述Rs 5各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000041
所述Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基，且每个所述Rs 4各自独立地选自：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN。
在本申请的多特异性抗体的某些实施方式中，所述GalX选自下组：
Figure PCTCN2022074201-appb-000042
Figure PCTCN2022074201-appb-000043
在本申请的多特异性抗体的某些实施方式中，所述b是0。
另一方面，本申请提供了一种多特异性抗体，其包含：能够特异性结合第一靶标的抗体部分A；和2个包含式(IV)
Figure PCTCN2022074201-appb-000044
所示结构的糖链部分；且所述多特异性抗体具有式(V)所示的结构：
Figure PCTCN2022074201-appb-000045
其中：所述A包含能够特异性结合所述第一靶标的第一抗 原结合部分AB1和Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000046
AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；GalX*为被取代的半乳糖，所述GalX*与所述GlcNAc之间通过β-1,4糖苷键连接，且所述GalX*包含能够特异性结合第三靶标的第三抗原结合部分AB3；所述第一靶标、第二靶标和第三靶标中至少有一个为CD3；并且所述氨基酸N297的位置根据Kabat中的EU索引编号确定。
在本申请的多特异性抗体的某些实施方式中，所述GalX*具有以下结构GalX 2Y 2-(L 2’) m-AB3，其中GalX 2Y 2为基团GalX 2与基团Y 2发生连接反应之后的残留基团，其中所述GalX 2包含X 2，所述X 2包含能够参与生物正交连接反应的官能团，且所述Y 2包含能够与所述X 2发生生物正交连接反应的官能团；且所述L 2’为连接子，m为0或1。
在本申请的多特异性抗体的某些实施方式中，所述GalX 2是C2、C3、C4和/或C6位的一个或多个羟基被取代的半乳糖。
在本申请的多特异性抗体的某些实施方式中，所述GalX 2是C2位的羟基被取代的半乳糖。
在本申请的多特异性抗体的某些实施方式中，所述GalX 2为单糖。
在本申请的多特异性抗体的某些实施方式中，所述GalX 2中的X 2包含
Figure PCTCN2022074201-appb-000047
Figure PCTCN2022074201-appb-000048
在本申请的多特异性抗体的某些实施方式中，所述GalX 2中的X 2包含
Figure PCTCN2022074201-appb-000049
在本申请的多特异性抗体的某些实施方式中，所述GalX 2具有以下结构
Figure PCTCN2022074201-appb-000050
在本申请的多特异性抗体的某些实施方式中，所述Y 2包含
Figure PCTCN2022074201-appb-000051
Figure PCTCN2022074201-appb-000052
在本申请的多特异性抗体的某些实施方式中，所述X 2Y 2包含选自下组的结构：
Figure PCTCN2022074201-appb-000053
在本申请的多特异性抗体的某些实施方式中，所述L 2’选自：C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000054
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在本申请的多特异性抗体的某些实施方式中，所述L 2’选自：
Figure PCTCN2022074201-appb-000055
Figure PCTCN2022074201-appb-000056
其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB3连接，且该结构的左端与所述Y 2连接。
在本申请的多特异性抗体的某些实施方式中，所述L 2’为
Figure PCTCN2022074201-appb-000057
在本申请的多特异性抗体的某些实施方式中，所述Fuco-L-AB2中，L的结构为J-(L 1) n-X 1Y 1-(L 1’) n’，其中：所述L 1为第一连接子，n为0或1；所述L 1’为第二连接子，n’为0或1；所述J为直接与Fuco相连接的接合子；X 1Y 1为基团X 1与基团Y 1发生连接反应之后的残留基团，其中所述基团X 1包含能够参与生物正交连接反应的官能团，且所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团。
在本申请的多特异性抗体的某些实施方式中，所述X 1包含
Figure PCTCN2022074201-appb-000058
其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
在本申请的多特异性抗体的某些实施方式中，所述X 1包含
Figure PCTCN2022074201-appb-000059
在本申请的多特异性抗体的某些实施方式中，所述Y 1包含
Figure PCTCN2022074201-appb-000060
Figure PCTCN2022074201-appb-000061
在本申请的多特异性抗体的某些实施方式中，所述X 1Y 1包含选自下组的结构：
Figure PCTCN2022074201-appb-000062
在本申请的多特异性抗体的某些实施方式中，所述J为
Figure PCTCN2022074201-appb-000063
Figure PCTCN2022074201-appb-000064
其中所述Rf为-CH 2-，-NH-或-O-，其中所述J结构的左端与所述Fuco相连接。
在本申请的多特异性抗体的某些实施方式中，所述J为
Figure PCTCN2022074201-appb-000065
其中所述J结构的左端与所述Fuco相连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1和所述L 1’各自独立地选自：C 3-C 200 亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000066
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在本申请的多特异性抗体的某些实施方式中，所述L 1选自：
Figure PCTCN2022074201-appb-000067
Figure PCTCN2022074201-appb-000068
其中s1为1-50的整数，每个s2独立地为0-50的整数,每个所述-CH 2-任选地被-O-替代，但连续相邻的-CH 2-不同时被-O-替代，该结构的左端与所述J连接，且该结构的右端与所述X 1连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1选自：
Figure PCTCN2022074201-appb-000069
Figure PCTCN2022074201-appb-000070
Figure PCTCN2022074201-appb-000071
且该结构的右端与所述X 1连接，左端与所述J连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1’选自：
Figure PCTCN2022074201-appb-000072
Figure PCTCN2022074201-appb-000073
其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
在本申请的多特异性抗体的某些实施方式中，所述L 1’为
Figure PCTCN2022074201-appb-000074
该结构的左端与所述Y 1连接，且右端与所述AB2连接。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标、第二靶标和第三靶标彼此均不相同。
在本申请的多特异性抗体的某些实施方式中，所述AB1、所述AB2和所述AB3各自独立地为抗体的抗原结合片段。
在本申请的多特异性抗体的某些实施方式中，所述抗原结合片段为Fab，F(ab) 2，F(ab’)，F(ab’) 2，scFv，亲和体(affibody)和/或单域抗体。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标为肿瘤相关抗原，所述第二靶标为CD3，且所述第三靶标为肿瘤相关抗原。在本申请的多特异性抗体的某些实施方式中，所述肿瘤相关抗原选自：Her2和PD-L1。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标为Her2，所述第二靶标为CD3，且所述第三靶标为PD-L1。
在本申请的多特异性抗体的某些实施方式中，所述第一靶标为PD-L1，所述第二靶标为CD3，且所述第三靶标为Her2。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含选自下组的抗体的抗原结合部分：曲妥珠单抗和度伐利尤单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR3(HCDR3)，且所述HCDR3包含SEQ ID NO：24和32中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR2(HCDR2)，且所述HCDR2包含SEQ ID NO：23和31中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链CDR1(HCDR1)，且所述HCDR1包含SEQ ID NO：22和30中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR3(LCDR3)，且所述LCDR3包含SEQ ID NO：21和29中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR2(LCDR2)，且所述LCDR2包含SEQ ID NO：20和28中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链CDR1(LCDR1)， 且所述LCDR1包含SEQ ID NO：19和27中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体重链可变区VH，且所述VH包含SEQ ID NO：26和34中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB1包含抗体轻链可变区VL，且所述VL包含SEQ ID NO：25和33中任一项所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述A为度伐利尤单抗或曲妥珠单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB2包含选自下组的抗体的抗原结合部分：OKT3、M291、YTH12.5、博纳吐单抗和卡妥索单抗。
在本申请的多特异性抗体的某些实施方式中，所述AB2包含SEQ ID NO：14所示的氨基酸序列。
在本申请的多特异性抗体的某些实施方式中，所述AB3包含选自下组的抗体的抗原结合部分：度伐利尤单抗，阿替利珠单抗，恩沃利单抗,曲妥珠单抗,帕托珠单抗和ZHer2:342。
在本申请的多特异性抗体的某些实施方式中，所述AB3包含SEQ ID NO：10和12中任一项所示的氨基酸序列。
另一方面，本申请提供了制备本申请所述多特异性抗体的方法。
在某些实施方式中，所述方法包括：i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，其中所述糖链包含式(VI)所示的结构-GlcNAc(Fuc) b-GalX(VI)，以获得结构如式(VII)所示的蛋白
Figure PCTCN2022074201-appb-000075
其中：所述A包含所述AB1和所述Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；Q为二磷酸核糖核苷酸；且Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000076
所述J为直接与Fuco相连接的接合子，且所述J与式(III)的左端相连接；所述X 1包含能够参与生物正交连接反应的官能团；所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和ii)使所述结构如式(VII)所示的蛋白与Y 1-(L 1’) n’-AB2反应，以获得本申请所述的多特异性抗体；其中所述A,GalX，X 1,Y 1,J,L 1,L 1’,n,n’,AB1和AB2如本申请前述所限定的。
在某些实施方式中，所述方法还包括以下步骤：用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构-GlcNAc(Fuc) b-GalX(VI)的糖链的蛋白，该蛋白的结构如式(VIII)所示：
Figure PCTCN2022074201-appb-000077
其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。
在某些实施方式中，所述方法还包括以下步骤：用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构-GlcNAc(Fuc) b-GalX(VI)的糖链的蛋白，该蛋白的结构如式(VIII)所示：
Figure PCTCN2022074201-appb-000078
其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。
在某些实施方式中，所述方法包括：i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，其中所述糖链包含式(IX)所示的结构-GlcNAc(Fuc) b-GalX 2(IX)，以获得结构如式(X)所示的蛋白
Figure PCTCN2022074201-appb-000079
其中：所述A包含所述AB1和所述Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX 2为被取代的半乳糖且GalX 2包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接；Q为二磷酸核糖核苷酸；且Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000080
所述J为直接与Fuco相连接的接合子，且所述J连接在式(III)左端；所述X 1包含能够参与生物正交连接反应的官能团；所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和 ii)使所述结构如式(X)所示的蛋白与Y 1-(L 1’) n’-AB2及Y 2-(L 2’) m-AB3反应，以获得本申请所述的多特异性抗体；其中所述A,X 1,Y 1,J,L 1,L 1’,n,n’,AB1，AB2，X 2，GalX 2，AB3,L 2’,Y 2和m如本申请中所限定的。
在某些实施方式中，所述方法还包括以下步骤：用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX 2接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
Figure PCTCN2022074201-appb-000081
其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且GalX 2为被取代的半乳糖且其包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。
在某些实施方式中，所述方法还包括以下步骤：用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
Figure PCTCN2022074201-appb-000082
其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且GalX 2为被取代的半乳糖且其包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。
在本申请的方法的某些实施方式中，所述Q为二磷酸鸟苷(GDP)、二磷酸尿苷(UDP)和/或二磷酸胞苷(CDP)。
在本申请的方法的某些实施方式中，所述Q-Fuc*’为GDP-Fuc*’。
在本申请的方法的某些实施方式中，所述Q-Fuc*’选自以下结构：
Figure PCTCN2022074201-appb-000083
Figure PCTCN2022074201-appb-000084
在本申请的方法的某些实施方式中，所述催化剂包含岩藻糖基转移酶。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶为α1,3-岩藻糖基转移酶或其功能性变体或片段。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶源自细菌。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶源自幽门螺杆菌Helicobacter pylori。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶源自幽门螺杆菌Helicobacter pylori 26695。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶是源自GenBank登录号为AAD07710.1的幽门螺杆菌α-1,3岩藻糖基转移酶。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶包含催化活性区域和至少一个七肽重复片段，所述催化活性区域包含SEQ ID NO:1所示的氨基酸序列，且所述七肽重复片段包含SEQ ID NO:2所示的氨基酸序列。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶包含催化活性区域和1-10个七肽重复片段，所述催化活性区域包含SEQ ID NO:1中所示的氨基酸序列，且所述七肽重复片段包含SEQ ID NO:2所示的氨基酸序列。
在本申请的方法的某些实施方式中，所述岩藻糖基转移酶为α-1,3-岩藻糖基转移酶或其功能性变体或片段，且其包含SEQ ID NO:3中所示的氨基酸序列。
在本申请的方法的某些实施方式中，所述催化剂包含本申请所述的岩藻糖基转移酶和标签序列。
在本申请的方法的某些实施方式中，所述催化剂包含SEQ ID NO:3和4中任一项所示的氨基酸序列。
另一方面，本申请提供了一种组合物，其包含本申请所述的多特异性抗体。
在某些实施方式中，所述组合物还包含药学上可接受的载体。
另一方面，本申请提供了一种预防、缓解和/或治疗疾病或病症的方法，所述方法包括向有需要的受试者施用本申请所述的多特异性抗体，和/或本申请所述的组合物。
另一方面，本申请提供了本申请所述的多特异性抗体和/或本申请所述的组合物用于制备药物的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A-1B显示的是制备本申请的多特异性抗体的方法示意图，其中
Figure PCTCN2022074201-appb-000085
为N-乙酰葡萄糖胺，
Figure PCTCN2022074201-appb-000086
为α1,6岩藻糖，
Figure PCTCN2022074201-appb-000087
为抗体部分A，Fuc*’是Fuco-J-(L 1) n-X 1，Fuc*是J-(L 1) n-X 1Y 1-(L 1’) n’-AB2，GalX*是GalX 2Y 2-(L 2’) n”-AB3,AB2为靶向CD3的抗原结合部分，AB3是第三抗原结合部分。
图2显示的是本申请中Q-Fuc*’的一些示例性结构。
图3A-3E显示的是本申请的示例性靶向CD3的多特异性抗体的制备和表征。
图4A-4C显示的是本申请的示例性靶向CD3的多特异性抗体的结合亲和力。
图5A-5B显示的是本申请的示例性靶向CD3的多特异性抗体的体外杀伤活性。
图6A-6C显示的是本申请的示例性靶向CD3的多特异性抗体的体外杀伤活性。
图7A-7C显示的是本申请的示例性靶向CD3的多特异性抗体的抗原依赖性T细胞激活表征。
图8A-8B显示的是本申请的示例性靶向CD3的多特异性抗体的浓度依赖性T细胞激活表征。
图9显示的是含有不同接合子的抗体偶联物与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体的效率对比。
图10显示的是本申请中使用的一些化合物的分子结构。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“直接连接”通常是指连接位点不包含另外的化合物(例如，其他糖基)或接头。例如，一个分子或实体与另一个直接连接，可以指二者之间不存在另外的分子或实体。例如，直接连接可以指一个部分与另一部分连接而没有任何中间部分或接头。例如，GlcNAc与蛋白质(如抗体)的氨基酸残基直接连接一般是指GlcNAc通过共价键与该蛋白质的氨基酸残基连接，例如通过N-糖苷键与酰胺氮键连接该蛋白质的氨基酸(如天冬酰胺氨基酸)侧链上的原子。在本申请中，当GlcNAc与蛋白质的氨基酸“间接连接”时，
GlcNAc与蛋白质的氨基酸之间通常存在至少一个单糖部分。
在本申请中，术语“包含”通常是指包括该限定之后的内容，但并不排除没有具体提到的其他内容，即在本申请中通常应理解为一种开放式的限定。
除非本文另有说明或者上下文清楚地有相反含义，否则本文所用的术语“一个”、“一种”、“该/所述”以及本发明的上下文中(尤其是在权利要求书的上下文中)所使用的类似术语应理解为既包括单数，又包括复数。
本申请说明书和权利要求书中所公开的化合物可包含一个或多个不对称中心，并且可存在所述化合物的不同的非对映异构体和/或对映异构体。除非另有说明，对本申请说明书和权利要求书中任何化合物的描述意指包括所有非对映异构体及其混合物。此外，除非另有说明，对本说明书和权利要求书中任何化合物的描述意指包括单独的对映异构体以及对映异构体的任何混合物、外消旋体或其他形式。当化合物的结构被描述为具体的对映异构体时，应理解，本申请的发明不限于该具体的对映异构体。
所述化合物可以不同的互变异构的形式存在。除非另有说明，本发明的化合物意指包括所有的互变异构形式。当化合物的结构被描述为具体的互变异构体时，应理解，本申请的发明不限于该具体的互变异构体。
未取代的烷基具有通式C nH 2n+1并且可以是直链的或支链的。未取代的烷基也可以包含环状部分，并因此具有相应的通式C nH 2n-1。任选地，所述烷基被一个或多个在本申请中进一步指定的取代基取代。烷基的实例包括，例如甲基、乙基、丙基、2-丙基、叔丁基、1-己基、1-十二烷基等。
芳基可包含六至十二个碳原子并可包含单环和双环结构。任选地，所述芳基可以被一个或多个在本文中进一步指定的取代基取代。芳基的实例为苯基和萘基。
芳基烷基和烷基芳基可包含至少七个碳原子并且可包含单环和双环结构。任选地，所述芳基烷基和烷基芳基可被一个或多个在本申请中进一步指定的取代基取代。芳基烷基可以为，例如苄基。烷基芳基可以为，例如4-叔丁基苯基。
在本申请中，术语“杂芳基”通常是指5-12个原子的芳族单环或多环基团，其具有至少一个包含选自N，O，和S的一个、两个或三个环杂原子的芳环，并且余下的环原子是C。杂芳基的一个或两个环碳原子可以被羰基取代。
杂芳基烷基和烷基杂芳基包含至少三个碳原子(即至少C 3)并且可包含单环和双环结构。任选地，杂芳基可被一个或多个在本文中进一步指定的取代基取代。
当芳基表示为(杂)芳基时，该表示法意指包括芳基和杂芳基。类似地，烷基(杂)芳基意指包括烷基芳基和烷基杂芳基，(杂)芳基烷基意指包括芳基烷基和杂芳基烷基。因此C 2-C 24(杂)芳基应理解为包括C 2-C 24杂芳基和C 6-C 24芳基。类似地，C 3-C 24烷基(杂)芳基意指包括C 7-C 24烷基芳基和C 3-C 24烷基杂芳基，并且C 3-C 24(杂)芳基烷基意指包括C 7-C 24芳基烷基和C 3-C 24杂芳基烷基。
除非另有说明，烷基、烯基、烯烃、炔烃、(杂)芳基、(杂)芳基烷基和烷基(杂)芳基可被一个或多个选自下述的取代基取代：C 1-C 12烷基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 5-C 12环烯基、C 7-C 12环炔基、C 1-C 12烷氧基、C 2-C 12烯氧基、C 2-C 12炔氧基、C 3-C 12环烷氧基、卤素、氨基、氧代，其中烷基、烯基、炔基、环烷基、烷氧基、烯氧基、炔氧基和环烷氧基任选地被取代，烷基、烷氧基、环烷基和环烷氧基任选地被一个或多个选自O、N和S的杂原子间隔或中断。
在本申请中，术语“炔基”通常包含碳-碳三键。含有一个三键的未取代的炔基具有通式C nH 2n-3。末端炔基是其中三键位于碳链末端位置的炔基。任选地，炔基被一个或多个在本申 请中进一步指定的取代基取代，和/或被选自氧、氮和硫的杂原子间隔或中断。炔基的实例包括，例如乙炔基、丙炔基、丁炔基、辛炔基等。
在本申请中，术语“环炔基”通常是指具有规定数目碳原子和一或多个碳-碳三键的不饱和的单环、双环或三环烃环。例如，C 7-C 12环炔基是指具有7－12个碳原子的环炔基。在某些实施方案中，环炔基在环中具有一个碳-碳三键。在其它实施方案中，环炔基在环中具有一个以上的碳-碳三键。环炔基的代表性示例包括但不限于环庚炔基，环辛炔基等。
在本申请中，术语“杂环炔基”通常是被选自氧、氮和硫的杂原子间隔或中断的环炔基。任选地，杂环炔基被一个或多个在本文中进一步指定的取代基取代。杂环炔基的实例为氮杂环辛炔基(azacyclooctynyl))。
当烷基、(杂)芳基、烷基(杂)芳基、(杂)芳基烷基、(杂)环炔基被任选地取代时，所述基团独立地任选地被一个或多个独立地选自下述的取代基取代：C 1-C 12烷基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 1-C 12烷氧基、C 2-C 12烯氧基、C 2-C 12炔氧基、C 3-C 12环烷氧基、卤素、氨基、氧代基和甲硅烷基，其中所述烷基、烯基、炔基、环烷基、烷氧基、烯氧基、炔氧基和环烷氧基被任选地取代，所述烷基、烷氧基、环烷基和环烷氧基任选地被一个或多个选自O、N和S的杂原子间隔或中断。
在本申请中，术语“亚多肽基”，通常是指包含两个或两个以上氨基酸残基的多肽基，例如，其可以由两个及以上的氨基酸缩合而成，其可以通过两端的氨基或者羧基与其他结构相连接。例如
Figure PCTCN2022074201-appb-000088
是一个亚多肽基，其左端通过羧基与其他结构连接，其右端通过氨基与其他部分连接。
在本申请中，术语“糖”通常是指单糖，例如葡萄糖(Glc)、半乳糖(Gal)、甘露糖(Man)和岩藻糖(Fuc)。术语“糖衍生物”在申请中通常是指单糖的衍生物，即包含取代基和/或官能团的单糖。糖衍生物的实例包括氨基糖和糖酸，例如葡糖胺(GlcNH 2)、半乳糖胺(GalNH 2)、N-乙酰葡萄糖胺(GlcNAc)、N-乙酰半乳糖胺(GalNAc)、唾液酸(Sia)(也被称为N-乙酰神经氨酸(NeuNAc))、和N-乙酰胞壁酸(MurNAc)、葡糖醛酸(GlcA)和艾杜糖醛酸(IdoA)。糖衍生物的实例也包括本申请中表示为GalX的化合物，其可以为半乳糖或半乳糖衍生物。糖衍生物的实例也包括本申请中表示为Fuc*'的化合物，其可以为岩藻糖衍生物。
在本申请中，术语“核苷酸”通常是指由核碱基、五碳糖(核糖或2-脱氧核糖)和一个、两个或三个磷酸基组成的分子。不含磷酸基时，核碱基和糖构成了核苷。因此，核苷酸也被称为核苷单磷酸、核苷二磷酸或核苷三磷酸。核碱基可以为腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧 啶或胸腺嘧啶。核苷酸的实例包括二磷酸核糖核苷酸，例如尿苷二磷酸(UDP)、鸟苷二磷酸(GDP)、胸苷二磷酸(TDP)、胞苷二磷酸(CDP)和胞苷单磷酸(CMP)。
在本申请中，术语“蛋白质”通常是指包含约10个或更多个氨基酸的多肽。蛋白质可包括天然的氨基酸，但也可包括非天然的氨基酸。
在本申请中，术语“糖蛋白”以其常见的科学含义使用，并且指的是含有一个或多个共价键合至蛋白质上的单糖或低聚糖链(“糖链”)的蛋白质。糖链可以连接到蛋白质的羟基上(O-连接-糖链)，例如连接到丝氨酸、苏氨酸、酪氨酸、羟赖氨酸或羟脯氨酸的羟基上；或者连接到蛋白质的酰氨基上(N-糖蛋白)，例如天冬酰胺或精氨酸；或者连接到蛋白质的碳上(C-糖蛋白)，例如色氨酸。糖蛋白可以包含多于一个糖链，可以包含一个或多个单糖和一个或多个低聚糖糖链的组合，并且可以包含N-连接、O-连接和C-连接的糖链的组合。据估计，超过50％的所有蛋白质具有一些糖基化的形式，因此可被描述为糖蛋白。
在本申请中，术语“糖链”通常是指连接至蛋白质的单糖或低聚糖链。因此，术语糖链指的是糖蛋白的糖类部分。糖链可以通过一个糖的C1碳连接在蛋白质上，所述糖链可以不含其他取代基(单糖)或者可以在其一个或多个羟基上被进一步取代(低聚糖)。天然存在的聚糖通常包含1至约10个糖部分。然而，当更长的聚糖连接在蛋白质上时，所述聚糖在本申请中也被认为是糖链。
糖蛋白的糖链可以是单糖。通常，糖蛋白的单糖糖链由共价键合至蛋白质的单个N-乙酰葡萄糖胺(GlcNAc)、葡萄糖(Glc)、甘露糖(Man)或岩藻糖(Fuc)组成。糖链也可以为低聚糖。
糖蛋白的低聚糖链可以是直链的或支链的。在低聚糖中，直接连接在蛋白质上的糖称为核心糖。在低聚糖中，未直接连接在蛋白质上且连接在至少两个其他糖上的糖被称为内糖。在低聚糖中，未直接连接在蛋白质上但连接在单个其他糖上——即在其一个或多个其他羟基上没有与其他他糖连接——的糖被称为末端糖。为了避免疑义，在糖蛋白的低聚糖中可以存在多个末端糖，但是通常仅存在一个核心糖。
糖链可以为O-连接的糖链、N-连接的糖链或C-连接的糖链。在O-连接的糖链中，单糖或低聚糖糖链键合在所述蛋白质的氨基酸中的O原子上，通常经由丝氨酸(Ser)或苏氨酸(Thr)的羟基。在N-连接的糖链中，单糖或低聚糖糖链经由所述蛋白质的氨基酸中的N原子键合在蛋白质上，通常经由天冬酰胺(Asn)或精氨酸(Arg)侧链上的酰胺氮。在C-连接的糖链中，单糖或低聚糖糖链键合在所述蛋白质的氨基酸中的C原子上，通常键合在色氨酸(Trp)的C原子上。
直接连接在蛋白质上的低聚糖的末端被称作糖链的还原端。所述低聚糖的另一端被称作糖链的非还原端。
对于O-连接的糖链，存在很多不同的链。天然存在的O-连接的糖链通常具有丝氨酸或苏氨酸-连接的α-O-GalNAc部分，其还可以被半乳糖、唾液酸和/或岩藻糖取代。携带糖链取代基的羟基化氨基酸可以是蛋白质中任何氨基酸序列的一部分。
对于N-连接的糖链，存在很多不同的链。天然存在的N-连接的糖链通常具有天冬酰胺-连接的β-N-GlcNAc部分，转而在其C4上进一步与β-GlcNAc连接，然后在其C4上进一步与β-Man连接，继而在其C3和C6上进一步与α-Man连接，形成五糖Man 3GlcNAc 2。核心GlcNAc部分还可以在其C6上与α-Fuc连接。五糖Man 3GlcNAc 2是大多数N-连接的糖蛋白的常见的低聚糖框架并可以进一步和其他糖进行连接，包括但不限于Man、GlcNAc、Gal和唾液酸。在侧链上被糖链修饰的天冬酰胺通常是序列Asn-X-Ser/Thr的一部分，其中X为除了脯氨酸以外的任何氨基酸且Ser/Thr为丝氨酸或苏氨酸。
在本申请中，术语“抗体”通常是指通过免疫系统产生的能够识别并与特定抗原结合的蛋白质或其抗原结合片段。抗体是糖蛋白的一个实例。术语抗体在本申请中以其最广泛的意思使用并具体包括单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如多特异性抗体)、抗体片段以及双链和单链抗体。抗体也包括人抗体、人源化抗体、嵌合抗体和特异性结合癌抗原的抗体。在本申请中。术语“抗体”通常包括完整的抗体，但也包括抗体片段，例如来自经裂解的抗体的抗体Fab片段、(Fab’) 2、Fv片段或Fc片段，scFv-Fc片段，小抗体(minibody)，单域抗体(也称为纳米抗体，nanobody)、多特异性抗体(diabody)，亲和体(affibody)或scFv。此外，术语“抗体”也包括经工程化或遗传学改造的抗体和/或抗体的衍生物。在一些实施方式中，抗体被称为免疫球蛋白并且包括各种类别和同种型，例如IgA(IgA1和IgA2)、IgD、IgE、IgM和IgG(IgG1、IgG3和IgG4)等。在本申请中，术语“抗体”可包括多克隆抗体和单克隆抗体及其功能片段。抗体包括保留特异性结合表位的能力的经修饰或衍生的抗体变体。抗体能够选择性地结合靶抗原或表位。在本申请中，抗体可以来自任何来源，例如小鼠或人，包括其嵌合抗体，例如，抗体可以是人源化的。
在本申请中，术语“人源化抗体”通常是指含有来自非人动物抗体的部分或全部CDR的抗体，并且抗体的框架和恒定区含有来自人抗体序列的氨基酸残基。抗体、抗体片段和基因改造的抗体均可以通过本领域已知的方法获得。
在本申请中，术语“治疗”通常是指获得期望的药理和/或生理效果。该效果在完全地或部分地预防疾病或其症状方面可能是预防性的，和/或在疾病和/或由该疾病引起的副作用的部分 或完全治愈方面可能是治疗性的。本申请中所使用的“治疗”涵盖对哺乳动物(特别是人)的疾病的任何治疗，并包括预防疾病在受试者中发生，所述受试者可能易感染疾病但是还未被诊断出患有该疾病；抑制疾病，即阻止其发展；缓解疾病，即引起疾病的消退。
在本申请中，术语“Fc区”通常是指免疫球蛋白重链的C末端区域，它可以通过完整抗体的木瓜蛋白酶消化来产生。Fc区可以是天然序列Fc区或变体Fc区。免疫球蛋白的Fc区一般包含2个恒定结构域，即CH2结构域和CH3结构域，且任选包含CH4结构域和/或铰链区。在本申请中，术语“Fc区”包括任何多肽(或编码此类多肽的核酸)，无论其生产方式如何。
在本申请中，术语“GlcNAc”或“N-乙酰葡萄糖胺”可以互换地使用，通常是指单糖葡萄糖的酰胺衍生物。
糖基化通常是指碳水化合物，即糖基供体，与另一个分子(糖基受体)的羟基或其他官能团相连的反应。在一些实施方案中，糖基化主要特别是指将聚糖连接到蛋白质或其他有机分子的酶促过程。蛋白质中的糖基化可以在糖基键、糖基结构、糖基组成和/或糖基长度等方面进行修饰。糖基化可以包括N-连接糖基化、O-连接糖基化、磷酸丝氨酸糖基化、C-甘露糖基化、形成GPI锚(glypiation)和/或化学糖基化相应地，蛋白质的糖基化寡糖可以是N-连接寡糖、O-连接寡糖、磷酸丝氨酸寡糖、C-甘露糖基化寡糖、糖基化寡糖和/或化学寡糖。
在本申请中，术语“单克隆抗体”通常是指从基本上同质的抗体群体获得的抗体，即，除了可能以少量存在的、可能天然发生的突变和/或翻译后修饰(例如，异构化、酰胺化)之外，构成该群体的个体抗体是相同的。
在本申请中，术语“IgG”通常是指免疫球蛋白或其功能衍生物。本领域技术人员将理解免疫球蛋白重链被分为γ、μ、α、δ和ε(γ、μ、α、δ、ε)以及其中的一些亚型(例如γ1-γ4或α1-α2)。正是这条链的性质决定了抗体的“同种型”，分别为IgG、IgM、IgD、IgA或IgE。免疫球蛋白亚类(亚型)，例如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2等已被很好地表征并且已知赋予功能特异性。人IgG的典型特征是Fc区重链CH2区Asn297位(根据Kabat的EU编号)的糖基化。
在本申请中，术语“Asn297”或“N297”可互换地使用，通常是指抗体Fc区第297位(根据Kabat的EU编号规则编号)的天冬酰胺。Asn297可以与一种或多种寡糖连接。
在本申请中，术语“Fuc*α1,3GlcNAc连接”通常是指Fuc*的Fuco和GlcNAc之间的连接，如将Fuco的C1连接到GlcNAc的C3。
在本申请中，术语“GalXβ1,4GlcNAc连接”通常是指任选被取代的半乳糖GalX和GlcNAc 之间的连接，如将GalX的C1连接到GlcNAc的C4。
在本申请中，术语“官能团”通常是指能够与另一个基团反应的基团。官能团可用于将试剂(例如，没有反应活性或具有低反应活性的试剂)连入本申请的多特异性抗体中。例如，官能团可以是化学基团或具有化学和/或酶促反应性的残基。在一些实施例中，官能团可以是能够在连接反应中反应的基团。
在本申请中，术语“标签序列”通常是指被整合(例如，连接)到目标蛋白质(例如，抗体)中的另一个分子实体(例如，另一段氨基酸序列)。例如，所述标签序列可以是可检测的标记，例如经放射标记的氨基酸或者经生物素化的多肽，其能被标记的抗生物素蛋白检测到(例如带有荧光标记或酶活性的抗生物素蛋白链菌素，可通过光学方法或比色法检测到)。例如，标签序列可以包括但不限于：放射性同位素或放射性核素(例如3H、14C、15N、35S、90Y、99Tc、111In、125I、131I)，荧光标签(例如FITC、罗丹明、镧磷光剂)，酶标签(例如辣根过氧化物酶、β-半乳糖苷酶、萤光素酶、碱性磷酸酶)，化学发光标记，生物素基团，预先选定的能被第二个受体识别的多肽抗原表位(例如亮氨酸拉链互补序列、二抗的结合位点、金属结合结构域、附加表位)，磁性试剂例如钆螯合物。在某些实施方式中，所述标签序列可以是亲和纯化标签，例如多聚组氨酸标签(His标签)，Arg标签、FLAG标签、3xFlag标签、链霉亲和素标签、纳米标签、SBP标签、c-myc标签、S标签、钙调蛋白结合肽、纤维素结合结构域、壳多糖结合结构域、GST标签和/或MBP标签。在某些实施方式中，标签序列可以融合在目的蛋白的N端和/或C端。
在本申请中，术语“岩藻糖基转移酶”通常是指可以将L-岩藻糖从岩藻糖供体底物(如鸟苷二磷酸-岩藻糖)转移到受体底物的酶或其功能片段或变体。受体底物可以是另一种糖，例如包含GlcNAc-Gal(LacNAc)的糖，如在N-糖基化或在O-糖基化的情况下。术语“岩藻糖基转移酶”可以包括其任何功能片段，或其催化结构域，以及具有催化活性结构域的功能变体(例如、突变体、同种型)。岩藻糖基转移酶的例子可以是α-1,3岩藻糖基转移酶。术语“岩藻糖基转移酶”可以源自各种物种，例如哺乳动物(例如人类)、细菌、线虫或吸虫。在一些实施方案中,岩藻糖基转移酶是源自细菌的α-1,3岩藻糖基转移酶。在一些实施例方案中，岩藻糖基转移酶是源自幽门螺杆菌的α-1,3岩藻糖基转移酶。在一些实施方案中，岩藻糖基转移酶是源自Helicobacter pylori 26695的α-1,3岩藻糖基转移酶。在一些实施方案中，其中所述岩藻糖基转移酶是源自GenBank登录号为AAD07710.1、GenBank登录号为AAD07447.1或GenBank登录号为AAB81031.1的α-1,3岩藻糖基转移酶。在一些实施方案中，其中所述岩藻糖基转移酶是GenBank登录号为AAD07710.1、GenBank登录号为AAD07447.1或GenBank 登录号为AAB81031.1的岩藻糖转移酶。在一些实施方案中，其中所述岩藻糖基转移酶为GenBank登录号为AAD07710.1、GenBank登录号为AAD07447.1或GenBank登录号为AAB81031.1的α-1,3岩藻糖基转移酶的功能性变体或片段。在一些实施方案中，岩藻糖基转移酶包含如GenBank登录号AAD07710.1中所列的氨基酸序列，或其功能变体或片段。例如，岩藻糖基转移酶可以包含基因库登录号AAD07710.1中所列的氨基酸序列，或者岩藻糖基转移酶可以包含具有超过80％(例如，超过83％，超过88％、超过90％、超过95％、超过96％、超过97％、超过98％、超过99％或更多)如GenBank登录号AAD07710.1中所述的氨基酸序列,或其功能变体或片段。例如，岩藻糖基转移酶可以包含催化活性区域和至少一个七肽重复片段(例如1-10个)(SEQ ID NO:2)，催化活性区域包含SEQ ID NO:1中所示的氨基酸序列或者包含与SEQ ID NO:1中所示的氨基酸序列具有至少80％(例如，超过83％，超过88％、超过90％、超过95％、超过96％、超过97％、超过98％、超过99％或更多)序列同源性的氨基酸序列。又例如，岩藻糖基转移酶可包含如SEQ ID NO:3以及4中任一项所示的氨基酸序列，或者岩藻糖基转移酶可包含与SEQ ID NO:3中项所示的氨基酸序列具有至少80％(例如，超过88％、90％、95％、96％、97％、98％、99％或更多)同源性或同一性的氨基酸序列。
在本申请中，术语“生物正交连接反应”通常是指用于制备本申请所述蛋白偶联物的化学反应。该反应特异性发生在位于蛋白质特定位置处(例如位于蛋白质的寡糖上)的第一功能官能团和与所述功能官能团部分对应的第二功能官能团之间。所述第一功能官能团与所述第二功能官能团通常被称为是一对生物正交连接反应对。通常，位于蛋白质特定位置的第一个功能官能团很容易与蛋白质其他部分的其他基团区分开来。通常，除了特定位置的第一功能官能团外，第二功能官能团不会与该蛋白质的其他部分反应。例如，叠氮基是能够参与生物正交连接反应的功能官能团。与之互补的DBCO或BCN基团可以与叠氮基特异性反应，而不会与蛋白质上的其他基团发生交叉反应。许多具有合适反应性、化学选择性和/或生物相容性的化学反应性功能官能团可用于生物正交连接反应。能够参与生物正交连接反应的基团可以选自但不限于叠氮基、末端炔基、环状炔基、四嗪基、1,2,4-三嗪基、末端烯基、环状烯基、酮基、醛基、羟基氨基、巯基、N-马来酰亚胺基及它们的功能衍生物(参见Bertozzi C.R.等Angew.Chem.Int.Ed.,2009,48,6974；Chin J.W.等ACS Chem.Biol.2014,9,16；van Del F.L.等Nat.Commun.,2014,5,5378；Prescher J.A.等Acc.Chem.Res.2018,51,1073；Devaraj N.K.等ACS Cent.Sci.2018,4,952；Liskamp R.M.J.等Chem.Sci.,2020,11,9011)。此处的功能衍生物可以指是经过修饰的功能官能团，其具有与未修饰的功能官能团相近或者更高的反应活性。
在本申请中，亲本多肽或蛋白的“功能性变体”通常与亲本多肽或蛋白质具有实质或显著的序列同一性或相似性，该功能性变体保留其亲本多肽或蛋白质的至少一部分功能。例如，酶的功能性变体以与亲本酶相似的程度、相同的程度或更高的程度保留酶活性。就亲本多肽或蛋白质而言，功能性变体可以是例如与其在氨基酸序列上具有约80％或更多、约90％或更多、约95％或更多、约96％或更多、约97％或更多、约98％或更多，或约99％或更多的序列同一性。在一些情况下，功能性变体可以是与亲本多肽或蛋白有至少一个氨基酸不同的多肽。例如，功能性变体可以是通过在亲本多肽或蛋白质上添加、缺失或取代一个或多个氨基酸获得的，例如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或1-2个氨基酸。
在本申请中，亲本多肽或蛋白的“功能性片段”通常是指下述肽或多肽(包括但不限于酶)，其包含亲本多肽或蛋白质的至少5个连续氨基酸残基、至少10个连续氨基酸残基、至少15个连续氨基酸残基、至少20个连续氨基酸残基、至少25个连续氨基酸残基、至少40个连续氨基酸残基、至少50个连续氨基酸残基、至少60个连续氨基酸残基、至少70个连续氨基酸残基、至少80个连续氨基酸残基、至少90个连续氨基酸残基、至少100个连续氨基酸残基、至少125个连续氨基酸残基、至少150个连续氨基酸残基、至少175个连续氨基酸残基、至少200个连续氨基酸残基、至少250个连续氨基酸残基或至少350个连续氨基酸残基，其中所述功能性片段具有其亲本多肽或蛋白的至少一部分功能。例如，亲本酶的“功能性片段”以与亲本酶相似的程度、相同的程度或更高的程度保留酶活性。
在本申请中，术语“CD3”通常是指CD3蛋白多亚基复合体(例如，人CD3蛋白多亚基复合体)。CD3蛋白质多亚基复合体可以由6条不同的多肽链构成。这些多肽链包括CD3γ链(SwissProt P09693)、CD3δ链(SwissProt P04234)、两条CD3ε链(SwissProt P07766)和一条CD3ζ链同源二聚体(SwissProt 20963)，并且该复合体与T细胞受体α和β链相关。除非另有说明，否则术语“CD3”包括由细胞(包括T细胞)天然表达或者可以在用编码那些多肽的基因或cDNA转染的细胞上表达的任何CD3变体、同种型和物种同源物。
在本申请中，术语“包含”也涵盖“是”、“由….组成”等限定。例如，一种蛋白包含SEQ ID NO:1所示的氨基酸序列应理解为不仅记载了一种蛋白，其氨基酸序列中包括但不限于SEQ ID NO:1所示的氨基酸序列。还应理解为也记载了一种蛋白体，其氨基酸序列由SEQ ID NO:1组成。
发明详述
多特异性抗体及其制备方法
一方面，本申请提供了一种多特异性抗体，其包含：能够特异性结合第一靶标的抗体部分A；和2个包含式(I)
Figure PCTCN2022074201-appb-000089
所示结构的糖链部分；且所述多特异性抗体具有式(II)所示的结构：
Figure PCTCN2022074201-appb-000090
其中：所述抗体部分A包含能够特异性结合所述第一靶标的第一抗原结合部分AB1和Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX为任选被取代的半乳糖(GalX为半乳糖或者为被取代的半乳糖)，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；所述Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000091
AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；所述第一靶标和所述第二靶标中至少有一个为CD3；并且所述氨基酸N297的位置根据Kabat中的EU索引编号确定。例如，在某些情形中，所述第一靶标是CD3。在某些情形中，所述第二靶标可以为CD3。在某些情形中，所述第一靶标和所述第二靶标可以均为CD3。在某些情形中，b为0。例如，b为0，式(II)
Figure PCTCN2022074201-appb-000092
等同于
Figure PCTCN2022074201-appb-000093
例如，所述GlcNAc直接与所述Fc区的氨基酸N297通过N-糖苷键相连接。例如，所述GlcNAc通过C1位与所述Fc区的氨基酸N297通过N-糖苷键相连接。
另一方面，本申请提供了一种多特异性抗体，其包含能够特异性结合第一靶标的抗体部分A；和2个包含式(IV)
Figure PCTCN2022074201-appb-000094
所示结构的糖链部分；且所述多特异性抗体具有式(V)所示的结构：
Figure PCTCN2022074201-appb-000095
其中：所述A包含能够特异性结合所述第一靶标的第一抗原结合部分AB1和Fc区；GlcNAc为N-乙酰葡萄糖胺， 且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000096
AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；GalX*为被取代的半乳糖，所述GalX*与所述GlcNAc之间通过β-1,4糖苷键连接，且所述GalX*包含能够特异性结合第三靶标的第三抗原结合部分AB3；所述第一靶标、第二靶标和第三靶标中至少有一个为CD3；并且所述氨基酸N297的位置根据Kabat中的EU索引编号确定。例如，在某些情形中，所述第一靶标是CD3。在某些情形中，所述第二靶标可以为CD3。在某些情形中，所述第三靶标可以为CD3。在某些情形中，所述第一靶标、所述第二靶标和所述第三靶标可以均为CD3。在某些情形中，b为0。例如，b为0，式(V)
Figure PCTCN2022074201-appb-000097
等同于
Figure PCTCN2022074201-appb-000098
在某些情形下，所述GalX*具有以下结构GalX 2Y 2-(L 2’) m-AB3，其中GalX 2Y 2为基团GalX 2与基团Y 2发生连接反应之后的残留基团，其中所述GalX 2包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述Y 2包含能够与所述X 2发生生物正交连接反应的官能团，且所述L 2’为连接子，m为0或1。
另一方面，本申请提供了制备本申请所述多特异性抗体的方法。该方法可以包括：i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，其中所述糖链包含式(VI)所示的结构-GlcNAc(Fuc) b-GalX(VI)，以获得结构如式(VII)所示的蛋白
Figure PCTCN2022074201-appb-000099
其中：所述A包含所述AB1和所述Fc区；GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；Q为二磷酸核糖核苷酸；且Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000100
所述J为直接与Fuco相连接的接合子，且所述J与式(III)左端连接；所述X 1包含能够参与生物正交 连接反应的官能团；所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和ii)使所述结构如式(VII)所示的蛋白与Y 1-(L 1’) n’-AB2反应，以获得本申请所述的多特异性抗体，其中，所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团，AB2为能够特异性结合第二靶标的第二抗原结合部分，所述L 1为第一连接子，n为0或1；且所述L 1’为第二连接子，n’为0或1。在某些情形中，b为0。例如，b为0，式(VII)
Figure PCTCN2022074201-appb-000101
等同于
Figure PCTCN2022074201-appb-000102
另一方面，本申请提供了制备本申请所述多特异性抗体的方法。该方法可以包括：i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，其中所述糖链包含式(IX)所示的结构-GlcNAc(Fuc) b-GalX 2(IX)，以获得结构如式(X)所示的蛋白
Figure PCTCN2022074201-appb-000103
其中：所述A包含所述AB1和所述Fc区；GlcNAc为N-乙酰葡糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；GalX 2为被取代的半乳糖且其包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接；Q为二磷酸核糖核苷酸；且Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
Figure PCTCN2022074201-appb-000104
所述J为直接与Fuco相连接的接合子，且所述J与式(III)左端相连；所述X 1包含能够参与生物正交连接反应的官能团；所述L 1为第一连接子，n为0或1；所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和ii)使所述结构如式(X)所示的蛋白与Y 1-(L 1’) n’-AB2及Y 2-(L 2’) m-AB3反应，以获得本申请所述的多特异性抗体；其中，所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团，AB2为能够特异性结合第二靶标的第二抗原结合部分，所述L 1’为第二连接子，n’为0或1；所述AB3为能够特异性结合所述第三靶标的第三抗原结合部分，所述Y 2包含能够与所述X 2发生生物正交连接反应的官能团，所述L 2’为连接子，且m为0或1。在某些情形中，b为0。例如， b为0，式(X)
Figure PCTCN2022074201-appb-000105
等同于
Figure PCTCN2022074201-appb-000106
在本申请中，所述第一靶标与所述第二靶标可以相同或不相同。例如，所述第一靶标可以与所述第二靶标相同。在有些情形中，所述第一靶标可以与所述第二靶标不同，例如，所述第一靶标与所述第二靶标可以是不同的抗原蛋白，或者可以是同一个抗原蛋白的不同表位(例如，不完全相同的表位多肽)。
在本申请中，所述第三靶标与所述第一靶标可以相同或不相同。例如，所述第三靶标可以与所述第一靶标相同。在有些情形中，所述第三靶标可以与所述第一靶标不同，例如，所述第三靶标与所述第一靶标可以是不同的抗原蛋白，或者可以是同一个抗原蛋白的不同表位(例如，不完全相同的表位多肽)。
在本申请中，所述第三靶标与所述第二靶标可以相同或不相同。例如，所述第三靶标可以与所述第二靶标相同。在有些情形中，所述第三靶标可以与所述第二靶标不同，例如，所述第三靶标与所述第二靶标可以是不同的抗原蛋白，或者可以是同一个抗原蛋白的不同表位(例如，不完全相同的表位多肽)。
在某些情形中，所述第一靶标、所述第二靶标及所述第三靶标彼此均不相同。例如，三者可以是不同的抗原蛋白，或者可以是同一个抗原蛋白的不同表位(例如，不完全相同的表位多肽)。
在某些情形中，所述抗体部分A可以是IgG抗体。例如，其可以包含第一轻链，第一重链，第二重链和第二轻链。所述第一轻链可以包含第一轻链可变区VL1和第一轻链恒定区CL1。所述第二轻链可以包含第二轻链可变区VL2和第二轻链恒定区CL2。所述第一重链可以包含第一重链可变区VH1和第一重链恒定区(其可包含第一重链Fc结构域)。所述第二重链可以包含第二重链可变区VH2和第二重链恒定区(其可包含第二重链Fc结构域)。在某些情形中，所述抗体部分A可以包含scFv(例如，作为本申请的AB1)和抗体Fc区(例如，源自IgG的Fc区)。在某些情形中，所述抗体部分A可以包含单域抗体VHH和抗体Fc区(例如，源自IgG的Fc区)。在某些情形中，所述抗体部分A可以是多特异性(例如，双特异性)抗体，其除了包含能够特异性结合所述第一靶标的AB1部分外，还可以包含能够特异性结合其他目标分子的抗原结合片段。例如，所述抗体部分A可以包含第一Fab和第二Fab，其第一Fab可以为所述AB1，其第二Fab可以特异性结合其他目标分子。
例如，所述抗体部分A可以包含Fc区。例如，所述Fc区可以是源自IgG的Fc区，例 如，所述Fc区可以是源自IgG1，IgG2，IgG3或IgG4的Fc区，或其功能性变体。例如，所述Fc区可以是源自人IgG的Fc区，例如，其可以是源自人IgG1，IgG2，IgG3或IgG4的Fc区，或其功能性变体。例如，所述Fc区可至少包含CH2结构域和CH3结构域。例如，所述Fc区可包含人IgG1重链保守区(UniProtKB：P01857-1)序列中的99-330的氨基酸序列，或其功能性变体或片段。例如，所述Fc区可包含人IgG2重链保守区(UniProtKB：P01859-1)序列中的99-326的氨基酸序列，或其功能性变体或片段。例如，所述Fc区可包含人IgG3重链保守区(UniProtKB：P01860-1)序列中的99-377的氨基酸序列，或其功能性变体或片段。例如，所述Fc区可包含人IgG4重链保守区(UniProtKB：P01861-1)序列中的99-327的氨基酸序列，或其功能性变体或片段。
在本申请中，AB1可以是抗体的抗原结合片段，其能够特异性结合所述第一靶标。
在本申请中，AB2可以是抗体的抗原结合片段，其能够特异性结合所述第二靶标。
在本申请中，AB3可以是抗体的抗原结合片段，其能够特异性结合所述第三靶标。
在本申请中，抗体的抗原结合片段可以为Fab，F(ab) 2，F(ab’)，F(ab’) 2，scFv，亲和体(affibody)和/或单域抗体(在本申请中，单域抗体也称为纳米抗体(nanobody)或VHH)。
在某些情形中，所述第一靶标为肿瘤相关抗原(此时，所述AB1为靶向这些肿瘤相关抗原的抗体的抗原结合片段)，且所述第二靶标为CD3(此时，所述AB2为靶向CD3的抗体的抗原结合片段)。例如，所述第一靶标可以源自下列靶蛋白：Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM。在某些实施方式中，所述第一靶标源自Her2，且所述第二靶标为CD3。在某些实施方式中，所述第一靶标源自PD-L1，且所述第二靶标为CD3。因此，所述AB1可以为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段；且所述AB2可以为靶向CD3的抗体的抗原结合片段。在某些情形中，所述AB1为靶向Her2的抗体的抗原结合片段，且所述AB2为靶向CD3的抗体的抗原结合片段。在某些情形中，所述AB1为靶向PD-L1的抗体的抗原结合片段，且所述AB2为靶向CD3的抗体的抗原结合片段。
在某些情形中，所述第一靶标为CD3(此时，所述AB1为靶向CD3的抗体的抗原结合片段)，且所述第二靶标为肿瘤相关抗原(此时，所述AB2为靶向这些肿瘤相关抗原的抗体的抗原结合片段)。例如，所述第二靶标可以源自下列靶蛋白：Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、 ErbB3、ErbB4、PD-L1和/或EpCAM。在某些实施方式中，所述第一靶标为CD3，且所述第二靶标源自Her2。在某些实施方式中，所述第一靶标为CD3。且所述第二靶标源自PD-L1。因此，所述AB1可以为靶向CD3的抗体的抗原结合片段，且所述AB2可以为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段。在某些情形中，所述AB1为靶向CD3的抗体的抗原结合片段，且所述AB2为靶向Her2的抗体的抗原结合片段。在某些情形中，所述AB1为靶向CD3的抗体的抗原结合片段，且所述AB2为靶向PD-L1的抗体的抗原结合片段。
在某些情形中，所述第一靶标为肿瘤相关抗原(此时，所述AB1为靶向这些肿瘤相关抗原的抗体的抗原结合片段)，所述第二靶标为CD3(此时，所述AB2为靶向CD3的抗体的抗原结合片段)，且所述第三靶标为肿瘤相关抗原(此时，所述AB3为靶向这些肿瘤相关抗原的抗体的抗原结合片段)。在某些情形中，所述第一靶标为CD3(此时，所述AB1为靶向CD3的抗体的抗原结合片段)，所述第二靶标为肿瘤相关抗原(此时，所述AB2为靶向这些肿瘤相关抗原的抗体的抗原结合片段)，且所述第三靶标为肿瘤相关抗原(此时，所述AB3为靶向这些肿瘤相关抗原的抗体的抗原结合片段)。在某些情形中，所述第一靶标为肿瘤相关抗原(此时，所述AB1为靶向这些肿瘤相关抗原的抗体的抗原结合片段)，所述第二靶标为肿瘤相关抗原(此时，所述AB2为靶向这些肿瘤相关抗原的抗体的抗原结合片段)，且所述第三靶标为CD3(此时，所述AB3为靶向CD3的抗体的抗原结合片段)。例如，所述肿瘤相关抗原可以是Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM。
在某些实施方式中，所述第一靶标为Her2，所述第二靶标为CD3，且所述第三靶标为PD-L1。在某些实施方式中，所述第一靶标为PD-L1，所述第二靶标为CD3，且所述第三靶标为Her2。
例如，所述AB1可以为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段；所述AB2可以为靶向CD3的抗体的抗原结合片段，且所述AB3可以为为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段。
例如，所述AB1可以为靶向CD3的抗体的抗原结合片段，所述AB2可以为靶向Her2、 Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段，且所述AB3可以为为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段。
例如，所述AB1可以为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段，所述AB2可以为为靶向Her2、Her3、Trop2、EGFR、VEGFR、VEGFR2、BCMA、Nectin-4、MUC1、c-Met、PSMA、GD2、GPC3、CEA、CD20、ErbB3、ErbB4、PD-L1和/或EpCAM的抗体的抗原结合片段，且所述AB3可以为靶向CD3的抗体的抗原结合片段。
在某些情形中，所述AB1为靶向Her2的抗体的抗原结合片段，所述AB2为靶向CD3的抗体的抗原结合片段，且所述AB3为靶向PD-L1的抗体的抗原结合片段。
在某些情形中，所述AB1为靶向PD-L1的抗体的抗原结合片段，所述AB2为靶向CD3的抗体的抗原结合片段，且所述AB3为靶向Her2的抗体的抗原结合片段。
在本申请中，所述靶向Her2的抗体可以是任何能够特异性结合Her2(例如，人Her2)的抗体或其抗原结合片段。例如，所述够特异性结合Her2的抗体可以选自：曲妥珠单抗(trastuzumab)，帕托珠单抗(pertuzumab)和ZHer2:342。在某些实施方式中，所述能够特异性结合Her2的抗体为曲妥珠单抗。在某些实施方式中，所述能够特异性结合Her2的抗体为ZHer2:342。
例如，所述靶向Her2的抗体可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：32所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含重链CDR2(HCDR2)，且所述HCDR2可包含SEQ ID NO：31所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：30所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2，和SEQ ID NO:30所示的HCDR1,或者它们的功能性变体或片段。
例如，所述靶向Her2的抗体可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：29所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：28所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：27所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2，SEQ ID NO:30所示的HCDR1，SEQ ID NO:29所示的LCDR3，SEQ ID NO:28所示的LCDR2和SEQ ID NO:27所示的LCDR1,或者它们的功能性变体或片段。
例如，所述靶向Her2的抗体可以包含重链可变区VH，且所述VH可包含SEQ ID NO：34所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：33所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含SEQ ID NO:34所示的VH和SEQ ID NO:33所示的VL,或者它们的功能性变体或片段。
例如，所述靶向Her2的抗体可以包含重链H，且所述重链H可包含SEQ ID NO:16所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含轻链L，且所述轻链L可包含SEQ ID NO:15所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向Her2的抗体可以包含SEQ ID NO:16所示的重链和SEQ ID NO:15所示的轻链,或者它们的功能性变体或片段。
例如，所述靶向Her2的抗体可以为亲和体(affibody)，其可包含SEQ ID NO:10所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述靶向PD-L1的抗体可以是任何能够特异性结合PD-L1(例如，人PD-L1)的抗体或其抗原结合片段。例如，所述能够特异性结合PD-L1的抗体可以选自：阿替利珠单抗(atezolimumab)，度伐利尤单抗(durvalumab)和恩沃利单抗(KN035)。在某些实施方式中，所述能够特异性结合PD-L1的抗体为度伐利尤单抗。在某些实施方式中，所述能够特异性结合PD-L1的抗体为恩沃利单抗。例如，所述能够特异性结合PD-L1的抗体可以是单域抗体VHH。
例如，所述靶向PD-L1的抗体可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：24所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含重链CDR2(HCDR2)，且所述HCDR2可包含 SEQ ID NO：23中所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：22中所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2和SEQ ID NO:22所示的HCDR1，或者它们的功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：21所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：20所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：19所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2，SEQ ID NO:22所示的HCDR1，SEQ ID NO:21所示的LCDR3，SEQ ID NO:20所示的LCDR2和SEQ ID NO:19所示的LCDR1,或者它们的功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含重链可变区VH，且所述VH可包含SEQ ID NO：26所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：25所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含SEQ ID NO:26所示的VH和SEQ ID NO:25所示的VL,或者它们的功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含重链H，且所述重链H可包含SEQ ID NO:18所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含轻链L，且所述轻链L可包含SEQ ID NO:17所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向PD-L1的抗体可以包含SEQ ID NO:18所示的重链和SEQ ID NO:17所示的轻链,或者它们的功能性变体或片段。
例如，所述靶向PD-L1的抗体可以为单域抗体VHH，其可包含SEQ ID NO:12所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述靶向CD3的抗体可以是任何能够特异性结合CD3(例如，人CD3)的抗体或其抗原结合片段。例如，所述能够特异性结合CD3的抗体可以选自：OKT3、M291、 YTH12.5、博纳吐单抗(blinatumomab)和卡妥索单抗(catumaxomab)。例如，所述能够特异性结合CD3的抗体可以是单域抗体VHH。
例如，所述靶向CD3的抗体可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：42所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含重链CDR2(HCDR2)，且所述HCDR2可包含SEQ ID NO：41所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：40所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含SEQ ID NO:42所示的HCDR3，SEQ ID NO:41所示的HCDR2和SEQ ID NO:40所示的HCDR1，或者它们的功能性变体或片段。
例如，所述靶向CD3的抗体可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：39所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：38所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：37所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含SEQ ID NO:42所示的HCDR3，SEQ ID NO:41所示的HCDR2，SEQ ID NO:40所示的HCDR1，SEQ ID NO:39所示的LCDR3，SEQ ID NO:38所示的LCDR2和SEQ ID NO:37所示的LCDR1，或者它们的功能性变体或片段。
例如，所述靶向CD3的抗体可以包含重链可变区VH，且所述VH可包含SEQ ID NO：36所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：35所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含SEQ ID NO:36所示的VH和SEQ ID NO:35所示的VL,或者它们的功能性变体或片段。
例如，所述靶向CD3的抗体可以包含重链H，且所述重链H可包含SEQ ID NO:44中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含轻链L，且所述轻链L可包含SEQ ID NO:43中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述靶向CD3的抗体可以包含SEQ ID NO:44所示的重链和SEQ ID NO:43所示 的轻链,或者它们的功能性变体或片段。
例如，所述靶向CD3的抗体可以为单域抗体VHH，其可包含SEQ ID NO:14所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：24和32中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含重链CDR2(HCDR2)，且所述HCDR2可包含SEQ ID NO：23和31中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：22和30中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2和SEQ ID NO:22所示的HCDR1，或者它们的功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2和SEQ ID NO:30所示的HCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB1可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：21和29中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：20和28中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：19和27中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2，SEQ ID NO:30所示的HCDR1，SEQ ID NO:29所示的LCDR3，SEQ ID NO:28所示的LCDR2和SEQ ID NO:27所示的LCDR1，或者它们的功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2，SEQ ID NO:22所示的HCDR1，SEQ ID NO:21所示的LCDR3，SEQ ID NO:20所示的LCDR2和SEQ ID NO:19所示的LCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB1可以包含重链可变区VH，且所述VH可包含SEQ ID NO：26和34中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：25和33中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:26所示的VH和SEQ ID NO:25所示的VL,或者 它们的功能性变体或片段。
例如，所述AB1可以包含SEQ ID NO:34所示的VH和SEQ ID NO:33所示的VL,或者它们的功能性变体或片段。
在本申请中，所述AB1可以为单域抗体VHH，其可包含SEQ ID NO:12所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB1可以为亲合体，其可包含SEQ ID NO:10所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：42所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含重链CDR2(HCDR2)，且所述HCDR2可包含SEQ ID NO：41所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：40所示的氨基酸序列，其功能性变体或片段。
例如，所述AB2可以包含SEQ ID NO:42所示的HCDR3，SEQ ID NO:41所示的HCDR2和SEQ ID NO:40所示的HCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB2可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：39所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：38所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：37所示的氨基酸序列，其功能性变体或片段。
例如，所述AB2可以包含SEQ ID NO:42所示的HCDR3，SEQ ID NO:41所示的HCDR2，SEQ ID NO:40所示的HCDR1，SEQ ID NO:39所示的LCDR3，SEQ ID NO:38所示的LCDR2和SEQ ID NO:37所示的LCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB2可以包含重链可变区VH，且所述VH可包含SEQ ID NO：36所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB2可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：35所示的氨基酸序列，其功能性变体或片段。
例如，所述AB2可以包含SEQ ID NO:36所示的VH和SEQ ID NO:35所示的VL,或者它们的功能性变体或片段。
在本申请中，所述AB2可以为单域抗体VHH，其可包含SEQ ID NO:14所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含重链CDR3(HCDR3)，且所述HCDR3可包含SEQ ID NO：24和32中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含重链CDR2(HCDR2)，且所述HCDR2可包含SEQ ID NO：23和31中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含重链CDR1(HCDR1)，且所述HCDR1可包含SEQ ID NO：22和30中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2和SEQ ID NO:22所示的HCDR1，或者它们的功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2和SEQ ID NO:30所示的HCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB3可以包含轻链CDR3(LCDR3)，且所述LCDR3可包含SEQ ID NO：21和29中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含轻链CDR2(LCDR2)，且所述LCDR2可包含SEQ ID NO：20和28中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含轻链CDR1(LCDR1)，且所述LCDR1可包含SEQ ID NO：19和27中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:32所示的HCDR3，SEQ ID NO:31所示的HCDR2，SEQ ID NO:30所示的HCDR1，SEQ ID NO:29所示的LCDR3，SEQ ID NO:28所示的LCDR2和SEQ ID NO:27所示的LCDR1，或者它们的功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:24所示的HCDR3，SEQ ID NO:23所示的HCDR2，SEQ ID NO:22所示的HCDR1，SEQ ID NO:21所示的LCDR3，SEQ ID NO:20所示的LCDR2和SEQ ID NO:19所示的LCDR1，或者它们的功能性变体或片段。
在本申请中，所述AB3可以包含重链可变区VH，且所述VH可包含SEQ ID NO：26和34中任一项所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以包含轻链可变区VL，且所述VL可包含SEQ ID NO：25和33中任一项所示的氨基酸序列，其功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:26所示的VH和SEQ ID NO:25所示的VL,或者它们的功能性变体或片段。
例如，所述AB3可以包含SEQ ID NO:34所示的VH和SEQ ID NO:33所示的VL,或者它们的功能性变体或片段。
在本申请中，所述AB3可以为单域抗体VHH，其可包含SEQ ID NO:12所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述AB3可以为亲合体，其可包含SEQ ID NO:10所示的氨基酸序列，其功能性变体或片段。
在本申请中，所述Fuc*可包含结构Fuco-L-AB2。在所述Fuco-L-AB2中，L的结构可以为J-(L 1) n-X 1Y 1-(L 1’) n’。
在本申请中，所述Fuc*’可包含结构Fuco-J-(L 1) n-X 1。
在本申请中，所述Fuco的结构可以如式(III)所示：
Figure PCTCN2022074201-appb-000107
在本申请中，所述基团X 1包含能够参与生物正交连接反应的官能团。例如，所述X 1可包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。例如，所述X 1可包含选自下组的官能团：
Figure PCTCN2022074201-appb-000108
Figure PCTCN2022074201-appb-000109
其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。例如，所述X 1可包含选自下组的官能团：
Figure PCTCN2022074201-appb-000110
例如，所述X 1可包含
Figure PCTCN2022074201-appb-000111
例如，所述X 1可包含
Figure PCTCN2022074201-appb-000112
例如，所述X 1可以是
Figure PCTCN2022074201-appb-000113
例如，所述X 1可以是
Figure PCTCN2022074201-appb-000114
在本申请中，所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团。例如，所述Y 1可包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、 环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。例如，所述Y 1可包含选自下组的官能团：
Figure PCTCN2022074201-appb-000115
Figure PCTCN2022074201-appb-000116
其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。例如，所述Y 1可包含选自下组的官能团：
Figure PCTCN2022074201-appb-000117
例如，所述Y 1可包含
Figure PCTCN2022074201-appb-000118
例如，Y 1可以是
Figure PCTCN2022074201-appb-000119
例如，所述X 1和所述Y 1可包含选自以下各组的一组结构：
a)X 1包含
Figure PCTCN2022074201-appb-000120
且Y 1包含
Figure PCTCN2022074201-appb-000121
b)X 1包含
Figure PCTCN2022074201-appb-000122
且Y 1包含
Figure PCTCN2022074201-appb-000123
c)X 1包含
Figure PCTCN2022074201-appb-000124
且Y 1包含
Figure PCTCN2022074201-appb-000125
d)X 1包含
Figure PCTCN2022074201-appb-000126
且Y 1包含
Figure PCTCN2022074201-appb-000127
和
e)X 1包含
Figure PCTCN2022074201-appb-000128
且Y 1包含
Figure PCTCN2022074201-appb-000129
例如，所述X 1可包含
Figure PCTCN2022074201-appb-000130
且所述Y 1可包含
Figure PCTCN2022074201-appb-000131
例如，所述X 1可包含
Figure PCTCN2022074201-appb-000132
且所述Y 1可包含
Figure PCTCN2022074201-appb-000133
例如，所述X 1可包含
Figure PCTCN2022074201-appb-000134
且所述Y 1可包含
Figure PCTCN2022074201-appb-000135
在本申请中，所述X 1Y 1为基团X 1与基团Y 1发生连接反应之后的残留基团。例如，所述X 1Y 1可包含选自下组的结构：
Figure PCTCN2022074201-appb-000136
Figure PCTCN2022074201-appb-000137
在本申请中，所述J为直接与Fuco相连接的接合子。所述J与式(III)左端相连。例如，所述J可以为
Figure PCTCN2022074201-appb-000138
其中所述Rf可以为-CH 2-，-NH-或-O-。在某些情形中，所述J可以是
Figure PCTCN2022074201-appb-000139
其中J结构的左端与所述Fuco相连。
在本申请中，所述L 1为第一连接子，n可以为0或1。当n为0时，所述第一连接子L 1不存在，所述J可以直接与所述X 1或X 1Y 1连接。当n为1时，所述J与所述X 1或X 1Y 1之间通过所述L 1连接。在某些情形中，所述L 1可以为C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物，或者它们的任意组合。其中，所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基可任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔(例如，在任意前述基团中可以插入一个或多个Rs 2基团，例如，多个-O-可以被插入到前述亚烷基中而获得亚聚乙二醇基)。
每个所述Rs 1可各自独立地选自：卤素、-OH、-NH 2和-COOH。
每个所述Rs 2可各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000140
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取 代的C 3-C 24环烷基。
例如，所述L 1可选自：
Figure PCTCN2022074201-appb-000141
其中s1为1-50(例如1-45，1-40，1-35，1-30，1-25，1-20，1-15，14，13，12，11，10，9，8，7，6，5，4，3，2或1)的整数；其中每个s2独立地为0-50(例如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或0)的整数。每个所述-CH 2-(括号中的-CH 2-)任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代(例如，不存在两个或更多个相连的-O-)；该结构的左端可与所述J连接，且该结构的右端可与所述X 1连接。在某些情形中，所述L 1为C 2-C 20亚聚乙二醇(PEG)基。例如，L 1可以为-(CH 2OCH 2) s1’-，其中s1’可以为0-20的整数。在某些情形中，所述L 1为C 1-C 50亚烷基。
在本申请中，所述L 1可以选自下组：
Figure PCTCN2022074201-appb-000142
Figure PCTCN2022074201-appb-000143
Figure PCTCN2022074201-appb-000144
且该结构的右端与所述X 1连接。
在本申请中，所述L 1’为第二连接子，n’可以为0或1。当n’为0时，所述第二连接子L 1’不存在，Y 1或X 1Y 1可直接与所述AB2连接。当n’为1时，所述Y 1或X 1Y 1与所述AB2之间通过所述L 1’连接。在某些情形中，所述L 1’可以为C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物，或者它们的任意组合。其中，所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基可任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔(例如，在任意前述基团中可以插入一个或多个Rs 2基团，例如，多个-O-可以被插入到前述亚烷基中而获得亚聚乙二醇基)。
每个所述Rs 1可各自独立地选自：卤素、-OH、-NH 2和-COOH。
每个所述Rs 2可各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000145
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
例如，所述L 1’可选自
Figure PCTCN2022074201-appb-000146
Figure PCTCN2022074201-appb-000147
Figure PCTCN2022074201-appb-000148
其中每个s2独立地为0-50(例如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或0)的整数。每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代(例如，不存在两个或更多个相连的-O-)，该结构的右端可与所述AB2连接，且该结构的左端可与所述Y 1或X 1Y 1连接。在某些情形中，所述L 1’结构的右端可通过氨基与所述AB2结构C端的-COOH形成肽键相连接(例如，通过缩合反应)连接。
例如，所述L 1'可从下述结构中进行选择：
Figure PCTCN2022074201-appb-000149
Figure PCTCN2022074201-appb-000150
其中结构的左端与Y 1相连接，右端可通过氨基与所述AB2相连接。
在本申请中，所述GalX为任选被取代的半乳糖。在某些情形中，所述GalX为半乳糖。
在某些情形中，所述GalX为被取代的半乳糖。例如，所述半乳糖中位于C2、C3、C4和C6位置的一个或多个羟基可以被取代。在某些情形中，所述半乳糖中位于C2位置的羟基可以被取代。在某些情形中，所述GalX为单糖。所述单糖可以是，例如，不能再被简单水解成更小的糖类的分子。例如，所述单糖可以为单糖衍生物，但仅含有一个核心单糖骨架。例如GalNAz，GalNH 2或GalNAc是单糖。又例如，同时含有Gal和GlcNAc的LacNAc不是单糖。在某些情形中，所述GalX中，所述半乳糖可以被取代基Rg 1取代。所述Rg 1可选自下组：氢、卤素、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24烯基、C 5-C 24环烯基、C 2-C 24炔基、C 6-C 24环炔基、C 3-C 24(杂)芳基、C 3-C 24烷基(杂)芳基和C 3-C 24(杂)芳基烷基。其中，所述烷基、环烷基、烯基、环烯基、炔基、环炔基、(杂)芳基、烷基(杂) 芳基和/或(杂)芳基烷基各自独立地可任选被一个或多个取代基Rs 4取代，和/或各自独立地可任选被一个或多个取代基Rs 5间隔(例如，在任意前述基团中可以插入一个或多个Rs 5基团，例如，多个-O-可以被插入到前述烷基中而获得聚乙二醇基)。
每个所述Rs 4可选自下组：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN。
每个所述Rs 5可各自独立地选自下组：-O-、-S-、
Figure PCTCN2022074201-appb-000151
其中，所述Rs 3可选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在某些情形中，所述GalX可被取代基
Figure PCTCN2022074201-appb-000152
取代，其中，t为0或1。
所述Rg 2可选自下组：C 1-C 24亚烷基、C 3-C 24亚环烷基、C 2-C 24亚烯基、C 5-C 24亚环烯基、C 2-C 24亚炔基、C 6-C 24亚环炔基、C 3-C 24(杂)亚芳基、C 3-C 24烷基(杂)亚芳基和C 3-C 24(杂)芳基亚烷基。其中，所述亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、烷基(杂)亚芳基和/或(杂)芳基亚烷基可各自独立地任选被一个或多个取代基Rs 4取代，和/或各自独立地任选被一个或多个取代基Rs 5间隔(例如，在任意前述基团中可以插入一个或多个Rs 5基团，例如，多个-O-可以被插入到前述亚烷基中而获得亚聚乙二醇基)。
所述Rg 3可选自下组：氢、卤素、-OH、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24炔基、C 5-C 24环炔基和C 2-C 24(杂)芳基，其中所述烷基、环烷基、炔基、环炔基和/或(杂)芳基可各自独立地任选被一个或多个Rs 4取代。
每个所述Rs 4可各自独立地选自：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN。
每个所述Rs 5可各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000153
所述Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
在某些情形中，所述GalX可选自下组：
Figure PCTCN2022074201-appb-000154
Figure PCTCN2022074201-appb-000155
在本申请中，所述GalX*可具有以下结构：GalX 2Y 2-(L 2’) m-AB3。其中，GalX 2Y 2为GalX 2与基团Y 2发生连接反应之后的残留基团，其中所述GalX 2包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述Y 2包含能够与所述X 2发生生物正交连接反应的官能团。
根据本申请的任一方面，所述L 2’为连接子，m可以为0或1。当m为0时，所述连接子L 2’不存在，Y 2或X 2Y 2可直接与所述AB3连接。当m为1时，所述Y 2或X 2Y 2与所述AB3之间可通过所述L 2’连接。在某些情形中，所述L 2’可以为C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物，或者它们的任意组合。其中，所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基可任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔(例如，在任意前述基团中可以插入一个或多个Rs 2基团，例如，多个-O-可以被插入到前述亚烷基中而获得亚聚乙二醇基)。
每个所述Rs 1可各自独立地选自：卤素、-OH、-NH 2和-COOH。
每个所述Rs 2可各自独立地选自：-O-、-S-、
Figure PCTCN2022074201-appb-000156
其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
例如，所述L 2’可选自：
Figure PCTCN2022074201-appb-000157
Figure PCTCN2022074201-appb-000158
Figure PCTCN2022074201-appb-000159
其中每个s2独立地为0-50(例如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或0)的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代(例如，不存在两个或更多个相连的-O-)，该结构的右端可与所述AB3连接，且该结构的左端可与所述Y 2或X 2Y 2连接。在某些情形中，所述L 2’结构的右端可通过氨基与所述AB3结构C端的-COOH形成肽键(例如，通过缩合反应)连接。
例如，所述L 2'可具有以下结构：
Figure PCTCN2022074201-appb-000160
其中结构的左端与Y 2相连接，右端可通过氨基与所述AB2相连接。
在本申请中，所述GalX 2可以是C2、C3、C4和C6位的一个或多个羟基被取代的半乳糖。在某些情形中，所述GalX 2可以是C2位的羟基被取代的半乳糖。在某些情形中，所述GalX 2是单糖。
例如，所述GalX 2中的X 2可以为叠氮
Figure PCTCN2022074201-appb-000161
在某些情形中，所述GalX 2可以是
Figure PCTCN2022074201-appb-000162
在某些情形中，所述Y 2可包含
Figure PCTCN2022074201-appb-000163
例如，所述X 2可以为叠氮
Figure PCTCN2022074201-appb-000164
且所述Y 2可包含
Figure PCTCN2022074201-appb-000165
Figure PCTCN2022074201-appb-000166
在某些情形中，所述X 2Y 2可包含选自下组的结构：
Figure PCTCN2022074201-appb-000167
Figure PCTCN2022074201-appb-000168
在本申请中，在某些情形下，具有
Figure PCTCN2022074201-appb-000169
接合子结构的抗体-Fuc*'偶联物与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体上有着更高的效率。例如，具有
Figure PCTCN2022074201-appb-000170
或者
Figure PCTCN2022074201-appb-000171
(其中Fuc*’包含结构Fuco-J-(L 1) n-X 1，J为
Figure PCTCN2022074201-appb-000172
)结构的偶联物在与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体上有着更高的效率。例如，具有
Figure PCTCN2022074201-appb-000173
或者
Figure PCTCN2022074201-appb-000174
(其中Fuc*’包含结构Fuco-J-(L 1) n-X 1，J为
Figure PCTCN2022074201-appb-000175
X 1为
Figure PCTCN2022074201-appb-000176
)结构的偶联物在与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体上有着更高的效率。例如，实施例26中展示了具有不同接合子的抗体-Fuc*'偶联物与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体效率对比。
在本申请中，所述Q可以为二磷酸鸟苷(GDP)、二磷酸尿苷(UDP)和/或二磷酸胞苷(CDP)。在某些情形中，所述Q可以为二磷酸鸟苷(GDP)。
在本申请中，所述Q-Fuc*’可以为GDP-Fuc*’。
在本申请中，在某些情形中，具有
Figure PCTCN2022074201-appb-000177
接合子结构的Q-岩藻糖衍生物(如Q-Fuc*')相比于具有
Figure PCTCN2022074201-appb-000178
接合子结构的Q-岩藻糖衍生物(如Q-Fuc*')在岩藻糖基转移酶催化的条件下转移至抗体上的转化率有着显著提升。例如，具有
Figure PCTCN2022074201-appb-000179
接合子结构的GDP-岩藻糖衍生物(如GDP-Fuc*')相比于具有
Figure PCTCN2022074201-appb-000180
接合子结构的GDP-岩藻糖衍生物(如GDP-Fuc*')在幽门螺杆菌α1,3-岩藻糖基转移酶催化下转移至抗体(如，抗体-(Galβ1,4)GlcNAc、抗体-(GalNAzβ1,4)GlcNAc)上的转化率有着显著提升。例如，实施例27 中展示了，具有
Figure PCTCN2022074201-appb-000181
接合子结构的GDP-FAmP 4Biotin相比于具有
Figure PCTCN2022074201-appb-000182
接合子结构的GDP-FAzP 4Biotin在幽门螺杆菌α1,3-岩藻糖基转移酶催化下转移至trastuzumab-(Galβ1,4)GlcNAc和trastuzumab-(GalNAzβ1,4)GlcNAc上的转化率有着显著的提升。
在本申请中，所述Q-Fuc*’可以选自以下结构：
Figure PCTCN2022074201-appb-000183
在本申请的所述制备方法中，步骤i)中的所述催化剂可以包含岩藻糖基转移酶。例如，所述岩藻糖基转移酶可以为α-1,3-岩藻糖基转移酶或其功能性变体或片段。例如，所述岩藻糖基转移酶可以源自细菌，例如，其可以源自幽门螺杆菌Helicobacter pylori(如Helicobacter pylori 26695)。例如，所述岩藻糖基转移酶可源自GenBank登录号为AAB81031.1，GenBank登录号为AAD07447.1或GenBank登录号为AAD07710.1的α-1,3岩藻糖基转移酶，或者它们的变体或片段。例如，所述岩藻糖基转移酶可源自GenBank登录号为AAD07710.1的α-1,3岩藻糖基转移酶。例如，所述岩藻糖基转移酶可以是GenBank登录号为AAD07710.1的α-1,3岩藻糖基转移酶，或者其功能性变体或片段。例如，GenBank登录号为AAD07710.1的野生型α-1,3岩藻糖基转移酶具有催化活性区域，10个多肽重复片段和C端尾巴结构，其功能性变体或片段可以包含催化活性区域和1-10个多肽重复片段。
例如，步骤i)中的所述岩藻糖基转移酶可包含催化活性区域和至少一个七肽重复序列(HPR)例如，步骤i)中的所述岩藻糖基转移酶可包含催化活性区域和1-10个七肽重复序列(HPR)(例如，1,2,3,4,5,6,7,8,9或10个HPRs)。所述催化活性区域可位于所述HRP的N端。例如，所述催化活性区域的C末端可与HPR(例如，HPR的N末端)相连。
例如，所述催化活性区域可包含SEQ ID NO:1中所示的氨基酸序列或其功能性变体或片段。
例如，所述催化活性区域可包含SEQ ID NO:1中所示的氨基酸序列或与SEQ ID NO:1具有至少约80％(例如，至少约82％，至少约85％，至少约88％，至少约90％,至少约92％,至少约95％,至少约98％,至少约99％或更高)序列同一性的氨基酸序列。
与亲本岩藻糖基转移酶(例如，本申请上述的那些岩藻糖基转移酶或催化活性区域)的氨基酸序列相比，所述变体的氨基酸序列可具有至少约80％(例如，至少约82％，至少约85％，至少约88％，至少约90％,至少约92％,至少约95％,至少约98％,至少约99％或更高)的序列同一性。
在本申请中，“序列同一性”是指，在比对候选序列(例如，变体的序列)与参考序列(例如，亲本的序列)并引入空位gap(若有必要)以实现最大的序列同一性百分比之后，候选序列中的氨基酸残基与参考序列中的氨基酸残基相同的百分比。可通过本领域技术人员已知的方式来进行序列比对以确定氨基酸序列同一性百分比，例如，可使用BLAST，BLAST-2，Clustal W，ALIGN-2，Megalign(DNASTAR)软件或FASTA程序包等。本领域技术人员可确定用于比对序列的适当参数。
例如，所述催化活性区域可包含SEQ ID NO:1中所示的氨基酸序列。
例如，所述HPR可包含SEQ ID NO:2所示的氨基酸序列。
例如，所述岩藻糖基转移酶可包含SEQ ID NO:3所示的氨基酸序列
例如，所述催化剂可以包含上述的岩藻糖基转移酶和标签序列。
例如，所述催化剂可以包含SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列。
在某些情形中，步骤i)中的所述催化剂可包含SEQ ID NO:3所示的氨基酸序列。在某些情形中，步骤i)中的所述催化剂可包含SEQ ID NO:4所示的氨基酸序列。
在某些情形中，本申请所述的制备方法还可包括下述步骤：用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构-GlcNAc(Fuc) b-GalX(VI)的糖 链的蛋白，该蛋白的结构如式(VIII)所示：
Figure PCTCN2022074201-appb-000184
其中，GlcNAc为N-乙酰葡糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。例如，所述合适的催化剂可以为β1,4半乳糖转移酶或其功能性变体。例如，所述合适的催化剂可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述内切糖苷酶可以为Endo S,Endo S2,Endo A,Endo F,Endo M,Endo D，Endo H和/或他们的功能性变体。例如，所述内切糖苷酶可以包含SEQ ID NO:6所示的氨基酸序列。在某些情形中，如果包含糖链和所述抗体部分A的蛋白如有带有核心α-1,6岩藻糖修饰，则b为1。在某些情形中，如果包含糖链和所述抗体部分A的蛋白如有不带有核心α-1,6岩藻糖修饰，则b为0。
在某些情形中，本申请所述的制备方法还可包含下述步骤：用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构-GlcNAc(Fuc) b-GalX(VI)的糖链的蛋白，该蛋白的结构如式(VIII)所示：
Figure PCTCN2022074201-appb-000185
(VIII)，其中，GlcNAc为N-乙酰葡糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。例如，b为0时，式(VIII)
Figure PCTCN2022074201-appb-000186
等同于
Figure PCTCN2022074201-appb-000187
例如，所述合适的催化剂可以为β1,4半乳糖转移酶或其功能性变体。例如，所述合适的催化剂可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述内切糖苷酶可以为Endo S,Endo S2,Endo A,Endo F,Endo M,Endo D，Endo H和/或他们的功能性变体。例如，所述内切糖苷酶可以包含SEQ ID NO:6所示的氨基酸序列。例如，所述α1,6岩藻糖苷酶可以为BfFucH、Alfc，BKF和/或它们的功能性变体。例如，所述α1,6岩藻糖苷酶可以包含SEQ ID NO:7所示的氨基酸序列。
在某些情形中，本申请所述的制备方法还可包括下述步骤：用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在 的条件下与UDP-GalX 2接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
Figure PCTCN2022074201-appb-000188
其中，GlcNAc为N-乙酰葡糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且GalX 2为被取代的半乳糖且包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。例如，所述合适的催化剂可以为β1,4半乳糖转移酶或其功能性变体。例如，所述合适的催化剂可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述内切糖苷酶可以为Endo S,Endo S2,Endo A,Endo F,Endo M,Endo D，Endo H和/或他们的功能性变体。例如，所述内切糖苷酶可以包含SEQ ID NO:6所示的氨基酸序列。在某些情形中，如果包含糖链和所述抗体部分A的蛋白如有带有核心α-1,6岩藻糖修饰，则b为1。在某些情形中，如果包含糖链和所述抗体部分A的蛋白如有不带有核心α-1,6岩藻糖修饰，则b为0。
在某些情形中，本申请所述的制备方法还可包括下述步骤：用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
Figure PCTCN2022074201-appb-000189
(XI)，其中，GlcNAc为N-乙酰葡糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且GalX 2为被取代的半乳糖且包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。例如，b为0，
Figure PCTCN2022074201-appb-000190
等同为
Figure PCTCN2022074201-appb-000191
例如，所述合适的催化剂可以为β1,4半乳糖转移酶或其功能性变体。例如，所述合适的催化剂可以包含SEQ ID NO:5所示的氨基酸序列。例如，所述内切糖苷酶可以为Endo S,Endo S2,Endo A,Endo F,Endo M,Endo D，Endo H和/或他们的功能性变体。例如，所述内切糖苷酶可以包含SEQ ID NO:6所示的氨基酸序列。例如，所述α1,6岩藻糖苷酶可以为BfFucH、Alfc，BKF和/或它们的功能性变体。例如，所述α1,6岩藻糖苷酶 可以包含SEQ ID NO:7所示的氨基酸序列。
组合物及医药用途
另一方面，本申请提供了一种组合物，其可包含一种或多种本申请所述的多特异性抗体。
本申请所述的组合物可以为药物组合物。例如，其可包含一种或多种药学上可接受的载体或赋形剂。
另一方面，本申请提供了一种预防、缓解和/或治疗疾病或病症的方法，所述方法包括向有需要的受试者施用本申请所述的多特异性抗体，和/或本申请所述的组合物。
另一方面，本申请提供了本申请所述的多特异性抗体和/或本申请所述的组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
在本申请中，所述疾病或病症可包括，例如，肿瘤、癌症或其他增生性疾病。所述疾病或病症还可包括，例如，免疫系统相关疾病或病症(例如，自体免疫疾病)。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的多特异性抗体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
材料和方法
蛋白质分子量质谱分析
利用Xevo G2-XS QTOF质谱仪(Waters Corporation)，进行蛋白的分子量分析。该质谱仪配备有电喷雾电离源(ESI)及Acuqity UPLC I-Class plus系统(Waters Corporation)。纯化后的蛋白用Waters ACQUITY UPLC Protein BEH C4柱(
Figure PCTCN2022074201-appb-000192
1.7μm,2.1mm x 100mm)进行分离和脱盐。流动相A为0.1％甲酸/水溶液，流动相B为含0.1％甲酸水的乙腈，流速为0.2mL/min。用Waters Unify软件(version 1.9.4,Waters Corporation)进行数据分析。
BGalT1(Y289L)(Bovineβ-1,4-galactosyltransferase I，牛β-1,4-半乳糖基转移酶带有Y289L突变)，EndoS(Streptococcus pyogenes endoglycosidase S，酿脓链球菌内切糖苷酶S)，Alfc(Lactobacillus caseiα-1,6-fucosidase Alfc，干酪乳杆菌α-1,6-岩藻糖苷酶)及Sortase 5M(Staphylococcus aureus Sortase 5M，金黄色葡萄球菌转肽酶5M)的克隆、表达及纯化
参考Qasba P.K等报道的方法(J.Biol.Chem.2002,277,20833；Prot.Expr.Fur.2003,30,219)克隆、表达及纯化BGalT1(Y289L)(SEQ ID NO:5)，参考Collin,M.等报道的方法(EMBO J.2001,20,3046；Infect.Immun.2001,69,7187)克隆、表达及纯化EndoS(SEQ ID NO:6)，参考Wang L.,等报道的方法(Methods Mol.Biol.2018,19,367)克隆、表达及纯化 Alfc(SEQ ID NO:7)，以及参考Liu D.等报道的方法(Proc.Natl.Acad.Sci.USA 2011,108,11399)克隆、表达及纯化Sortase 5M(SEQ ID NO:8)。
HpFT-2HR的克隆、表达以及纯化
通过基因合成编码HpFT-2HR(其包含催化活性区域、2个七肽重复序列以及C端融合的His标签，其氨基酸序列如SEQ ID NO:4所示)的核酸序列，并通过NdeI和BamHI酶切位点，将该核酸序列插入至pET24b载体中(南京金斯瑞)。用构建好的重组质粒转化E.coli BL21(DE3)。在含有50μg/mL卡那霉素的LB培养基中培养经转化后的重组细菌，在37℃下培养至OD 600达到0.6-0.8时，添加IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为0.2mM，然后继续在25℃，200rpm的条件下孵育16小时进行蛋白诱导表达。诱导后的菌体经离心后，悬浮于裂解缓冲液中(25mM Tris-HCl，pH 7.5，500mM氯化钠，20mM咪唑，1mM PMSF(苯甲基磺酰氟))。将悬浮后的细胞经超声裂解，然后用Ni-NTA填料(GE Health)进行纯化。收集纯度大于90％主组分，然后透析至保存缓冲液中(25mM Tris-HCl，pH 7.5，150mM氯化钠，5％甘油)。
ZHer 2:342亲合体，aPDL1以及aCD3的克隆、表达及纯化
通过基因合成编码ZHer 2:342亲合体(序列参考文献Eigenbrot,C.等，Proc.Natl.Acad.Sci.USA,2010,107,15039)(其包含C端融合LPETGG及His标签，其氨基酸序列如SEQ ID NO:9所示)，并使用NdeI和BamHI酶切位点，将该核酸序列插入至pET24b载体中(南京金斯瑞)。通过基因合成编码PDL1纳米抗体(aPDL1)(序列参考文献Zhang F.等，Cell Discov,2017,3,17004.)(其包含C端融合LPETGG及His标签，其氨基酸序列如SEQ ID NO:11所示)及CD3纳米抗体(aCD3)(序列参考文献Annelies R.等,，US 2019/0382485A1)(其包含C端融合LPETGG及His标签，其氨基酸序列如SEQ ID NO:13所示)的核酸序列，并使用NcoI和BamHI酶切位点，将该核酸序列插入至pET26b载体中(南京金斯瑞)。用构建好的重组质粒转化E.coli BL21(DE3)。在含有50μg/mL卡那霉素的LB培养基中培养经转化后的重组细菌，在37℃下培养至OD 600达到0.6-0.8时，添加IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为0.2mM，然后继续在25℃，200rpm的条件下孵育16小时进行蛋白诱导表达。诱导后的菌体经离心后，悬浮于裂解缓冲液中(25mM Tris-HCl，pH 7.5，500mM氯化钠，20mM咪唑，1mM PMSF(苯甲基磺酰氟))。将悬浮后的细胞经超声裂解，然后用Ni-NTA(GE Health)填料进行纯化。收集纯度大于90％主组分，然后透析至保存缓冲液中(25mM Tris-HCl，pH 7.5，150mM氯化钠)。
实施例1 GDP-FAz的合成
参考Wu P.等报道的方法(Proc.Natl.Acad.Sci.USA 2009,106,16096)合成GDP-FAz。并通过Bio-Gel P-2Gel柱，使用50mM碳酸氢铵溶液为洗脱液完成纯化。
HRMS(ESI-):C 16H 24N 8O 15P 2(M-H +)计算值为629.0764,实测值629.0785。
实施例2 GDP-FAm的合成
Figure PCTCN2022074201-appb-000193
将100mg GDP-FAz溶于8.75mL甲醇/水(体积比1:1.5)溶液中，然后向体系中加入5mg钯碳，抽真空置换氢气，并将体系压力保持在0.28MPa。在室温下搅拌反应4h，过滤、浓缩和冻干得到白色固体(84.8mg，88％)。HRMS(ESI-):C 16H 26N 6O 15P 2(M-H +)计算值603.0859,实测值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).
实施例3合成GDP-FAmAz
向600μL GDP-FAm(100mM水溶液)中依次加入200μL NaHCO 3(200mM水溶液)，780μL THF(四氢呋喃)和220μL NHS-azide(西安点化科技)(100mM THF溶液)。在室温下搅拌反应过夜，并通过薄层色谱法监测。通过制备型高效液相色谱(Pre-HPLC)纯化，得到白色固体产物(8.7mg，63％)。HRMS(ESI-):C 18H 27N 9O 16P 2(M-H +)计算值686.0978，实测值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).
实施例4合成GDP-FAmP 4Az
向200μL GDP-FAm(100mM水溶液)中依次加入200μL NaHCO 3(200mM水溶液)、780μL THF、220μL NHS-PEG 4-azide(西安点化科技)(100mM THF溶液)，以及600μL H 2O。在室温下搅拌反应过夜，通过薄层色谱法监测。通过制备型高效液相色谱(Pre-HPLC)纯化，得到白色固体产物(9mg，51％)。HRMS(ESI-):C 27H 45N 9O 20P 2(M-H +)计算值876.2183,实测值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).
实施例5合成GDP-FAmP 8Tz
向200μL GDP-FAm(100mM水溶液)中依次加入200μL NaHCO 3(200mM水溶液),780μL THF以及220μL NHS-PEG 8-Tz(西安点化科技)(100mM THF溶液)。在室温搅拌反应4h，通过薄层色谱法进行监测。通过Pre-HPLC纯化，得到粉色固体产物(9.2mg，38％)。HRMS(ESI-):C 44H 68N 10O 25P 2(M-2H +)/2计算值598.1844,实测值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).
实施例6合成GDP-FAmP 4Tz
向200μL GDP-FAm(100mM水溶液)中依次加入200μL NaHCO 3(200mM水溶液),780μL THF以及220μL NHS-PEG 4-Tz(Click Chemistry Tools)(100mM THF溶液)。在室温搅拌反应4h，通过薄层色谱法进行监测。通过Pre-HPLC纯化，得到粉色固体产物(9.9mg，48％)。HRMS(ESI-):C 36H 52N 10O 21P 2(M-H +)计算值1021.2711,实测值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).
实施例7合成GDP-FAmP 4BCN
向200μL GDP-FAm(100mM水溶液)中依次加入200μL NaHCO 3(200mM水溶液),560μL THF以及440μL NHS-PEG 4-BCN(西安点化科技)(50mM THF溶液)。在室温搅拌反应4h，通过薄层色谱法进行监测。通过Pre-HPLC纯化，得到白色固体产物(7.4mg，36％)。HRMS(ESI-):C 38H 59N 7O 22P 2(M-2H +)/2计算值512.6522,实测值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).
实施例8合成GDP-FAmP 4TCO
向400μL GDP-FAm(100mM水溶液)中依次加入400μL NaHCO 3(200mM水溶液),1.36mL THF以及440μL NHS-PEG 4-TCO(Click Chemistry Tools)(50mM THF溶液)。在室温搅拌反应4h，通过薄层色谱法进行监测。通过Pre-HPLC纯化，得到白色固体产物(8.2mg，20％)。HRMS(ESI-):C 36H 59N 7O 22P 2(M-H +)/2计算值1002.3116,实测值1002.3134。
实施例9合成GDP-FAmP 4Biotin
Figure PCTCN2022074201-appb-000194
向500μL GDP-FAm(100mM水溶液)中依次加入500μL NaHCO 3(200mM水溶液)、1.95mL THF和550μL NHS-PEG 4-Biotin(Click Chemistry Tools)(100mM THF溶液)。在室温下搅拌反应4h，通过薄层色谱法监测。通过Pre-HPLC纯化，得到白色固体产物(20.5mg，38％)。HRMS(ESI-):C 37H 61N 9O 22P 2S(M-H +)计算值1076.3054,实测值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).
实施例10合成GDP-FAzP 4Biotin
Figure PCTCN2022074201-appb-000195
向400μL GDP-FAz(50mM水溶液)中依次加入400μL CuSO 4/BTTP(5mM/10mM水溶液)、210μL propargyl-PEG 4-Biotin(Click Chemistry Tools)(100mM甲醇溶液)和40μL抗坏血酸钠(250mM水溶液)，在室温下搅拌反应5h，通过薄层色谱法监测。然后，加入2mM BCS(2,9-二甲基-4,7-二苯基-1,10-邻二氮杂菲二磺酸二钠盐)淬灭反应。通过Pre-HPLC纯化，得到白色固体产物(14.4mg，60％)。HRMS(ESI-):C 37H 59N 11O 21P 2S(M-H +)计算值1086.3010，实测值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).
实施例11制备Trastuzumab-(Galβ1,4)GlcNAc
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入曲妥珠单抗(Trastuzumab)(终浓度5mg/mL)，EndoS(终浓度0.05mg/mL)，以及Alfc(终浓度1.5mg/mL)，并将上述混合液于37℃反应24h。然后，向以上反应溶液中依次加入UDP-galactose(二磷酸尿苷半乳糖)(终浓度5mM)，氯化锰(终浓度5mM)，以及BGalT1(Y289L)(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应24h。随后通过protein A树脂(金斯瑞)对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(Galβ1,4)GlcNAc(145909Da,>90％)。
Trastuzumab的轻链的氨基酸序列如SEQ ID NO:15所示，重链的氨基酸序列如SEQ ID NO:16所示。
实施例12制备Trastuzumab-(GalNAzβ1,4)GlcNAc
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Trastuzumab(终浓度5mg/mL)，EndoS(终浓度0.05mg/mL)，以及Alfc(终浓度1.5mg/mL)，并将上述混合液于37℃反应24h。然后，向以上反应溶液中依次加入UDP-GalNAz(二磷酸尿苷-N-乙酰叠氮氨基半乳糖)(终浓度5mM)，氯化锰(终浓度5mM)，以及BGalT1(Y289L)(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应16h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(GalNAzβ1,4)GlcNAc(146077Da,>90％)。
实施例13制备Durvalumab-(GalNAzβ1,4)GlcNAc
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入度伐利尤单抗(Durvalumab)(终浓度5mg/mL)，EndoS(终浓度0.05mg/mL)，以及Alfc(终浓度1.5mg/mL)，并将上述混合液于37℃反应24h。然后，向以上反应溶液中依次加入UDP-GalNAz(二磷酸尿苷-N-乙酰叠氮氨基半乳糖)(终浓度5mM)，氯化锰(终浓度5mM)，以及BGalT1(Y289L)(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应16h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为durvalumab-(GalNAzβ1,4)GlcNAc(146963Da,>90％)。
Durvalumab的轻链的氨基酸序列如SEQ ID NO:17所示，重链的氨基酸序列如SEQ ID NO:18所示。
实施例14制备Trastuzumab-(Galβ1,4)GlcNAc-FAz
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Trastuzumab-(Galβ1,4)GlcNAc(终浓度5mg/mL)，GDP-FAz(终浓度5mM)，氯化镁(终浓度10mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应16h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(Galβ1,4)GlcNAc-FAz(146268Da，>90％)。
实施例15制备Trastuzumab-(Galβ1,4)GlcNAc-FAmAz
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Trastuzumab-(Galβ1,4)GlcNAc(终浓度5mg/mL)，GDP-FAmAz(终浓度5mM)，氯化镁(终浓度10mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应16h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(Galβ1,4)GlcNAc-FAmAz(146383Da，>90％)。
实施例16制备Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Trastuzumab-(Galβ1,4)GlcNAc(终浓度5mg/mL)，GDP-FAmP 4Az(终浓度5mM)，氯化镁(终浓度10mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应16h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az(146773Da，>90％)。
实施例17制备Trastuzumab-(GalNAzβ1,4)GlcNAc-FAmP 8Tz
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Trastuzumab-(GalNAzβ1,4)GlcNAc(终浓度5mg/mL)，GDP-FAmP 8Tz(终浓度5mM)，氯化镁(终浓度10mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应24h。随后通过protein A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为trastuzumab-(GalNAzβ1,4)GlcNAc-FAmP 8Tz(147578Da，>90％)。
实施例18制备Durvalumab-(GalNAzβ1,4)GlcNAc-FAmP 8Tz
首先，在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入Durvalumab-(GalNAzβ1,4)GlcNAc(终浓度5mg/mL)，GDP-FAmP 8Tz(终浓度5mM)，氯化镁(终浓度10mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃继续反应24h。随后通过protein  A树脂对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为durvalumab-(GalNAzβ1,4)GlcNAc-FAmP 8Tz(148475Da，>90％)。
实施例19制备DBCO-ZHer 2:342
在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入ZHer 2:342亲合体(终浓度1.5mg/mL)，DBCO-PEG 5-GGG(终浓度2mM)(西安点化科技)，氯化钠(终浓度150mM)，氯化钙(终浓度5mM)以及Sortase 5M(终浓度0.2mg/mL)，并将上述混合液于25℃反应3h。到达反应时间点后，向反应溶液中加入适量Ni-NTA填料，并于室温颠倒混匀孵育1h，去除Sortase 5M和未反应的ZHer 2:342亲合体。孵育结束后，将含Ni-NTA填料的反应液于4000rpm离心1min，去除填料，并将反应液用SP柱(阳离子交换层析柱，苏州纳微)进行纯化，进而获得修饰后的目标产物。经质谱鉴定，显示主峰为DBCO-ZHer 2:342(7923Da)。
实施例20制备DBCO-aPDL1
在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入aPDL1(终浓度1.5mg/mL)，DBCO-PEG 5-GGG(终浓度2mM)(西安点化科技)，氯化钠(终浓度150mM)，氯化钙(终浓度5mM)以及Sortase 5M(终浓度0.2mg/mL)，并将上述混合液于25℃反应3h。到达反应时间点后，向反应溶液中加入适量Ni-NTA填料，并于室温颠倒混匀孵育1h，去除Sortase5M和未反应的aPDL1。孵育结束后，将含Ni-NTA填料的反应液于4000rpm离心1min，去除填料，并将反应液用SP柱进行纯化，进而获得修饰后的目标产物。经质谱鉴定，显示主峰为DBCO-aPDL1(15213Da)。
实施例21制备DBCO-aCD3
在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入aCD3(终浓度1.5mg/mL)，DBCO-PEG 5-GGG(终浓度2mM)(西安点化科技)，氯化钠(终浓度150mM)，氯化钙(终浓度5mM)以及Sortase 5M(终浓度0.2mg/mL)，并将上述混合液于25℃反应3h。到达反应时间点后，向反应溶液中加入适量Ni-NTA填料，并于室温颠倒混匀孵育1h，去除Sortase 5M和未反应的aCD3纳米抗体。孵育结束后，将含Ni-NTA填料的反应液于4000rpm离心1min，去除填料，并将反应液用SP柱进行纯化，进而获得修饰后的目标产物。经质谱鉴定，显示主峰为DBCO-aCD3(15099Da)。
实施例22制备BCN-aCD3
在50mM Tris-HCl(pH 7.0)缓冲液中，依次加入aCD3(终浓度1.5mg/mL)，BCN-PEG 5-GGG(终浓度2mM)(西安点化科技)，氯化钠(终浓度150mM)，氯化钙(终浓度5mM)以及Sortase 5M(终浓度0.2mg/mL)，并将上述混合液于25℃反应3h。到达反应时 间点后，向反应溶液中加入适量Ni-NTA填料，并于室温颠倒混匀孵育1h，去除Sortase 5M和未反应的aCD3。孵育结束后，将含Ni-NTA填料的反应液于4000rpm离心1min，去除填料，并将反应液用SP柱进行纯化，进而获得修饰后的目标产物。经质谱鉴定，显示主峰为BCN-aCD3(14990Da)。
实施例23制备Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az-DBCO-aCD3
在1×PBS(pH 7.0)缓冲液中，依次加入Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az(终浓度1mg/mL)，DBCO-aCD3(终浓度3mg/mL)，并将上述混合液于室温反应8h。随后通过SP柱对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az-DBCO-aCD3(简写为Tras-aCD3)(176973Da,MAR2)。MAR(molecule of interest to antibody ratio,兴趣分子对抗体的比例)。结果展示在图3E中，显示获得的双特异性抗体具有很好的均一性。
实施例24制备Trastuzumab-(GalNAz-DBCO-aPDL1)GlcNAc-FAmP 8Tz-BCN-aCD3
在1×PBS(pH 7.0)缓冲液中，依次加入Trastuzumab-(GalNAz)GlcNAc-FAmP 8Tz(终浓度1mg/mL)，DBCO-aPDL1(终浓度3mg/mL)，并将上述混合液于室温反应8h。然后再向反应溶液中加入BCN-aCD3(终浓度2mg/mL)，并将反应液于室温继续反应16h。随后通过SP柱对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为Trastuzumab-(GalNAz-DBCO-aPDL1)GlcNAc-FAmP 8Tz-BCN-aCD3(简写为Tras-aPDL1-aCD3)(207930Da,MAR2+MAR2)。结果展示在图3E中，显示获得的三特异性抗体具有很好的均一性。
实施例25制备Durvalumab-(GalNAz-DBCO-ZHer 2:342)GlcNAc-FAmP 8Tz-BCN-aCD3
在1×PBS(pH 7.0)缓冲液中，依次加入Durvalumab-(GalNAz)GlcNAc-FAmP 8Tz(终浓度1mg/mL)，DBCO-ZHer 2:342(终浓度1.5mg/mL)，并将上述混合液于室温反应8h。然后再向反应溶液中加入BCN-aCD3(终浓度2mg/mL)，并将反应液于室温继续反应16h。随后通过SP柱对反应液进行纯化，进而获得修饰后的目标抗体。经质谱鉴定，显示主峰为Durvalumab-(GalNAz-DBCO-ZHer 2:342)GlcNAc-FAmP 8Tz-BCN-aCD3(简写为Dur-ZHer-aCD3)(194249Da,MAR2+MAR2)。结果显示在图3E中，显示获得的三特异性抗体具有很好的均一性。
实施例26含有不同接合子的抗体-Fuc*'偶联物与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体效率对比。
在1×PBS(pH 7.0)缓冲液中加入DBCO-aCD3(终浓度1mg/mL)，随后加入 Trastuzumab-(Galβ1,4)GlcNAc-FAz、Trastuzumab-(Galβ1,4)GlcNAc-FAmAz或Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az(终浓度0.5mg/mL)，并将上述混合液于室温反应2h。达到反应时间点后，分别取20μL样品，加入5μL上样缓冲液，90℃处理5min，然后取相同体积样品，进行SDS-PAGE检测。结果展示在图9中。结果显示，具有
Figure PCTCN2022074201-appb-000196
接合子结构的抗体-Fuc*’偶联物在与Y 1-(L 1’) n’-AB2反应制备本申请所述的多特异性抗体有着更高的效率。通过对比反应后产物条带(HC(重链)+aCD3)的量可知，Trastuzumab-(Galβ1,4)GlcNAc-FAmAz和Trastuzumab-(Galβ1,4)GlcNAc-FAmP 4Az(具有
Figure PCTCN2022074201-appb-000197
接合子结构)与DBCO-aCD3的反应效率要显著高于Trastuzumab-(Galβ1,4)GlcNAc-FAz与DBCO-aCD3的反应效率。
实施例27含有不同接合子的GDP-岩藻糖衍生物在岩藻糖转移酶催化下转移至抗体上的转化效率对比
在50mM Tris-HCl(pH 7.5)缓冲液中，依次加入trastuzumab-(Galβ1,4)GlcNAc(终浓度2mg/mL)，GDP-FAmP 4Biotin或GDP-FAzP 4Biotin(终浓度1mM)，氯化镁(终浓度5mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃反应10min，每组设置三个平行。到达反应时间点后，向反应溶液中加入10mM LacNAc并通过脱盐柱(Zeba TMSpin Desalting Columns/2ml/40K，赛默飞世尔科技)终止反应，最后通过质谱检测，并计算转化率。
在50mM Tris-HCl(pH 7.5)缓冲液中，依次加trastuzumab-(GalNAzβ1,4)GlcNAc(终浓度2mg/mL)，GDP-FAmP 4Biotin或GDP-FAzP 4Biotin(终浓度1mM)，氯化镁(终浓度5mM)，以及HpFT-2HR(终浓度0.5mg/mL)，并将上述混合液于30℃反应4h，每组设置三个平行。到达反应时间点后，向反应溶液中加入10mM LacNAc并通过脱盐柱(Zeba TMSpin Desalting Columns/2ml/40K，赛默飞世尔科技)终止反应，最后通过质谱检测，并计算转化率。转化率＝(MAR0峰强度*0+MAR1峰强度*1+MAR2峰强度*2)/2*(MAR0峰强度+MAR1峰强度+MAR2峰强度)*100％。
结果展示在下表中。结果显示具有
Figure PCTCN2022074201-appb-000198
接合子结构的GDP-岩藻糖衍生物(如GDP-FAmP 4Biotin)相比于具有
Figure PCTCN2022074201-appb-000199
接合子结构的GDP-岩藻糖衍生物(如GDP-FAzP 4Biotin)在抗体上(如Trastuzumab-(Galβ1,4)GlcNAc或Trastuzumab- (GalNAzβ1,4)GlcNAc)有着显著提升的转化率。
Figure PCTCN2022074201-appb-000200
实施例28 Tras-aCD3,Tras-aPDL1-aCD3和Dur-ZHer-aCD3抗原结合力检测
对Her2抗原的亲和力检测：将不同浓度的Tras-aCD3,Tras-aPDL1-aCD3和Dur-ZHer-aCD3加入Her2阳性的SKOV3(Her2+/PDL1-)胞中，细胞重悬在FACs缓冲液(DPBS含2％牛血清)中，60000个细胞/孔，一式两份。在4℃下孵育30分钟后，用FACs缓冲液(DPBS含2％胎牛血清)洗去细胞上未结合的多特异性抗体，之后将细胞重悬在30μL FACs缓冲液中。然后，在细胞中加入靶向人IgG Fc-PE(Biolegend,Clone HP6017,409304)的流式抗体，在4℃下孵育30分钟后，用FACs缓冲液洗去未结合在多特异性抗体上的流式抗体，然后将细胞重悬到100μL FACs缓冲液中。然后，使用流式细胞仪(Angilent Novocyte quanteon)采集样品，并使用PE通道分析，最后通过FlowJo 10.5.3和GraphPad Prism 8进行分析。结果如图4A所示，三种多特异性抗体都可以结合Her2抗原，其中Tras-aCD3与Tras-aPDL1-aCD3对Her2抗原有相似的结合亲和力，Dur-ZHer-aCD3比前两者略弱。
对CD3抗原的亲和力检测：将不同浓度的Tras-aCD3,Tras-aPDL1-aCD3和Dur-ZHer-aCD3加入CD3+的Jurkat细胞中，细胞重悬在FACs缓冲液中，60000个细胞/孔，一式两份。在4℃下孵育30分钟后，用FACs缓冲液洗去细胞上未结合的多特异性抗体，之后将细胞重悬在30μL FACs缓冲液中。在细胞中加入靶向人IgG Fc-PE的流式抗体，在4℃下孵育30分钟后，用FACs缓冲液洗去未结合在多特异性抗体上的流式抗体，然后将细胞重悬到100μL FACs缓冲液中。然后，使用流式细胞仪(Angilent Novocyte quanteon)采集样品，并使用PE通道分析，最后通过FlowJo 10.5.3和GraphPad Prism 8进行数据处理。如图4C所示，三种多特异性抗体都可以结合CD3抗原，其中Tras-aPDL1-aCD3与Dur-ZHer-aCD3对CD3抗原有相似的结合亲和力，Tras-aCD3较前两者略好。
对PDL1抗原的亲和力检测：用包被缓冲液(50mM Na 2CO 3/NaHCO 3,pH9.6)将重组的PDL-1抗原(PDL-1，近岸蛋白)稀释至终浓度为250ng/mL，按每孔100μL体积接种到96孔板并于4℃冰箱孵育过夜。第二天在去除包被液后，首先用封闭液(含3％(v/v)胎牛 血清白蛋白的PBS溶液)在30℃恒温箱下封闭2小时，每孔200μL。然后用洗板缓冲液PBST(含0.03％tween-20的PBS溶液)洗涤3次后，再利用样品稀释缓冲液(含有1％(v/v)胎牛血清白蛋白的PBS溶液)将待测样品(Durvalumab,Dur-ZHer-aCD3,Tras-aPDL1-aCD3)稀释至一系列最终浓度(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)并分别添加到板中，每孔100μL。样品加完后放于30℃恒温箱继续孵育1小时，待结束后继续用PBST缓冲液洗板3次，每孔加入辣根过氧化物酶(HRP)偶联的山羊抗人IgG抗体(碧云天)，并于30℃孵育半小时。孵育结束后每孔继续用PBST缓冲液洗涤5次，然后加入100μL 3,3',5,5'-四甲基联苯胺底物进行共处理显色。孵育5-10分钟后，通过添加100μL 3M HCl终止反应。最后在Synergy TM LX读板器上于450nm处读取吸光度。结果如图4B所示，Dur-ZHer-aCD3和Tras-aPDL1-aCD3均可以与PDL-1结合，其中Dur-ZHer-aCD3和Durvalumab有着相似的结合力，较Tras-aPDL1-aCD3相比略强。
实施例29 Tras-aCD3细胞杀伤活性检测
1.SKOV3的细胞毒性测试
使用RTCA-DP仪器测试效应细胞对靶细胞的杀伤(利用贴壁细胞在金属电极板上会产生阻抗，被杀伤后贴壁效果变差导致阻抗减小，RTCA基于微电子阻抗技术检测活细胞数)。首先，在E-Plate 16PET板(RTCA-DP仪器配套)中每孔加入50μL DMEM(Gibco)后，放入RTCA-DP仪器中测量基线。然后，取出E-Plate 16PET板，在每个孔接种10000个SKOV3细胞，在室温放置30min后，将E-Plate 16PET板放入RTCA-DP仪器中，开始测试细胞的生长曲线。25h后，当细胞曲线生长到细胞对数生长期后期时，暂停测试，从仪器上取下E-Plate 16PET板，在实验孔中加入终浓度为15nM的Tras-aCD3和50000个hPBMC(人外周血单核细胞)，并设置对照孔，分别单独加50000个hPBMC，30nM aCD3和50000个hPBMC，或15nM的Trastuzumab和50000个hPBMC，一式两份。最后继续测试30小时，通过仪器导出数据，用GraphPad Prism 8作图分析。结果如图5A所示，在SKOV3中加入hPBMC，hPBMC和aCD3，都不会显著抑制SKOV3细胞的生长。同时加入hPBMC和trastuzumab的实验组轻微的抑制SKOV3细胞的生长。对比之下，同时加入hPBMC和Tras-aCD3的实验组能显著并高效地抑制SKOV3细胞的生长，说明只有将Trastuzumab和aCD3偶联在一起，才会促使T细胞杀伤SKOV3。
2.MDA-MB-468的细胞毒性测试
使用RTCA-DP仪器测试效应细胞对靶细胞的杀伤。首先，在E-Plate 16PET板中每孔 加入50μL DMEM后，放入RTCA-DP仪器中测量基线。然后，取出E-Plate 16PET板，在每个孔接种10000个MDA-MB-468(Her2-)细胞，在室温放置30min后，将E-Plate 16PET板放入RTCA-DP仪器中，开始测试细胞的生长曲线。42h后，当细胞曲线生长到细胞对数生长期后期时，暂停测试，从仪器上取下E-Plate 16PET板，在实验孔中加入终浓度为15nM的Tras-aCD3和50000个hPBMC，并设置对照孔，分别单独加50000个hPBMC，30nM aCD3和50000个hPBMC，或15nM的Trastuzumab和50000个hPBMC，一式两份。最后继续测试38小时，通过仪器导出数据，用GraphPad Prism 8作图分析。结果如图5B所示，所有实验组都不会显著影响MDA-MB-468细胞的生长。同时，相较于Her2阳性的SKOV3细胞的实验结果，双抗在存在hPBMC的环境中并不会对Her2阴性细胞造成杀伤，说明双抗的杀伤具有抗原依懒性。
实施例30 Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3和的T细胞毒性测试
1.SKOV3的细胞毒性测试
使用RTCA-DP仪器测试效应细胞对靶细胞的杀伤。首先，在E-Plate 16PET板中每孔加入50μL DMEM后，放入RTCA-DP仪器中测量基线。然后，取出E-Plate 16PET板，在每个孔接种10000个SKOV3(Her2+/PDL1-)细胞，在室温放置30min后，将E-Plate 16PET板放入RTCA-DP仪器中，开始测试细胞的生长曲线。25h后，当细胞曲线生长到细胞对数生长期后期时，暂停测试，从仪器上取下E-Plate 16PET板，在实验孔中分别加入50000个hPBMC和终浓度为1.5pM的Tras-aCD3，Tras-aPDL1-aCD3或Dur-ZHer-aCD3，一式四份。最后继续测试157h，通过仪器导出数据，用GraphPad Prism 8作图分析。结果如图6A所示，在hPBMC存在的条件下，三种多特异性抗体均对SKOV3细胞有着显著的杀伤。其中Tras-aCD3和Tras-aPDL1-aCD3对SKOV3杀伤强于Dur-ZHer-aCD3。
2.JIMT1的细胞毒性测试
使用RTCA-DP仪器测试效应细胞对靶细胞的杀伤。首先，在E-Plate 16PET板中每孔加入50μL DMEM后，放入RTCA-DP仪器中测量基线。然后，取出E-Plate 16PET板，在每个孔接种20000个JIMT1(Her2+/PDL1+)细胞，在室温放置30min后，将E-Plate 16PET板放入RTCA-DP仪器中，开始测试细胞的生长曲线。25h后，当细胞曲线生长到细胞对数生长期后期时，暂停测试，从仪器上取下E-Plate 16PET板，在实验孔中分别加入100000个hPBMC和终浓度为1.5pM的Tras-aCD3，Tras-aPDL1-aCD3或Dur-ZHer-aCD3，一式四份。最后继续测试160h，通过仪器导出数据，用GraphPad Prism 8作图分析。结果如图6B所示，在hPBMC存在的情况下，三种多特异性抗体对JIMT1均具有显著的杀伤。值得一 提的是，相比于Tras-aCD3和Tras-aPDL1-aCD3，Dur-ZHer-aCD3在JIMT1(Her2+/PDL1+)上的杀伤显著强于在SKOV3(Her2+/PDL1-)上的杀伤。这可能是由于Dur-ZHer-aCD3中针对PDL1靶向起到了增强作用。
3.MDA-MB-468的细胞毒性测试
使用RTCA-DP仪器测试效应细胞对靶细胞的杀伤。首先，在E-Plate 16PET板中每孔加入50μL DMEM后，放入RTCA-DP仪器中测量基线。然后，取出E-Plate 16PET板，在每个孔接种20000个MDA-MB-468(Her2-/PDL1-)细胞，在室温放置30min后，将E-Plate 16PET板放入RTCA-DP仪器中，开始测试细胞的生长曲线。42h后，当细胞曲线生长到细胞对数生长期后期时，暂停测试，从仪器上取下E-Plate 16PET板，在实验孔中分别加入100000个hPBMC和终浓度为1.5pM的Tras-aCD3，Tras-aPDL1-aCD3或Dur-ZHer-aCD3，一式四份。最后继续测试38h，通过仪器导出数据，用GraphPad Prism 8作图分析。结果如图6C所示，显示所有实验组都不会显著抑制MDA-MB-468细胞的生长。
实施例31 Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3的免疫激活检测
1.T细胞活化检测
通过分离人外周血得到hPBMC，将100000个hPBMC细胞种植在平底96孔板，并设计了以下对照组，分别是不处理组(不加和加20000个SKOV3)，终浓度30nM的aCD3组(不加和加20000个SKOV3)，终浓度15nM商业化的抗人CD3的抗体OKT3(BioXCell，BE0001-2)组(不加和加20000个SKOV3)，以及终浓度15nM的Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3组(不加和加20000个SKOV3)，所有实验孔体积为100μL，一式五份。36小时后，1000g离心10min，收集细胞上清液并冷冻在-80℃冰箱，用于后续分析。同时，将离心后的细胞重悬于30μL FACs缓冲液(DPBS含2％胎牛血清)中，并用抗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)的流式抗体染色，其中CD4-PB，CD8a-PE是用于区分CD4+和CD8+的T细胞，CD69-APC用于评估CD69的上调，CD25-Percp/Cy5.5用于评估CD25的上调，和PD-1-FITC用于评估PD-1的上调。使用流式细胞仪(Angilent Novocyte quanteon)采集样品，并通过FlowJo 10.5.3和GraphPad Prism 8进行分析。结果如图7A-B所示，Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3，在加入Her2阳性的SKOV3细胞后，均会引起CD4+和CD8+T细胞的CD69，CD25和PD-1显著上调。这说明这三种多特异性抗体导致的T细胞活化具有抗原依懒性。
2.细胞因子测量
将上一步中收集上清液从-80℃冰箱取出，放在室温解冻后，1000*g离心10min去除颗粒和聚合物并将上清液稀释5倍。使用Elisa试剂盒(北京四正柏)对样品中的人IFN-γ和人TNF-α进行测量。首先，将100μL不同对照组的上清液与带有人IFN-γ和人TNF-α抗体的包被板进行孵育，在37℃恒温箱中孵育90min。孵育结束后，用洗涤液洗板3次后，加入100μL生物素溶液，在37℃恒温箱中孵育60min。孵育结束后，用洗涤液洗板3次后，加入100μL酶结合物，在37℃恒温箱中孵育30min。孵育结束后，用洗涤液洗板3次，最后加入显色剂避光孵育15min，最终加入终止液立即测试OD450的值。通过origin拟合出标准曲线，并用GraphPad Prism 8进行分析。如图7C所示，Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3在加入Her2阳性的SKOV3细胞后，均会引起显著的细胞因子释放上调，这说明这三种多特异性抗体导致的细胞因子释放是具有抗原依懒性。
实施例32 Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3的浓度依赖性T细胞激活检测
通过分离人外周血得到人外周血单核细胞(hPBMC)，将100000个hPBMC细胞和20000个SKOV3(E/T＝5)种植在平底96孔板，并将不同浓度(1.5fM,15fM,150fM,1500fM,15000fM,150000fM,1500000fM)的Tras-aCD3，Tras-aPDL1-aCD3或Dur-ZHer-aCD3加入细胞溶液中，所有实验孔体积为100μL，一式三份。16小时后，收集细胞，并用FACs缓冲液清洗细胞。最后将细胞重悬在30μL FACs缓冲液中，并用抗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)的流式抗体染色，其中CD4-PB，CD8a-PE是用于区分CD4+和CD8+的T细胞，CD69-APC用于评估CD69的上调，CD25-Percp/Cy5.5用于评估CD25的上调，和PD-1-FITC用于评估PD-1的上调。使用流式细胞仪(Angilent Novocyte quanteon)采集样品，并通过FlowJo 10.5.3和GraphPad Prism 8进行分析。结果如图8所示，随着药物浓度的增加，Tras-aCD3，Tras-aPDL1-aCD3和Dur-ZHer-aCD3导致CD4+和CD8+T细胞的CD69，CD25和PD-1上调的程度也在增加，具有浓度依赖性激活T细胞的能力。

Claims (116)

  1. 多特异性抗体，其包含：
    能够特异性结合第一靶标的抗体部分A；和
    2个包含式(I)
    Figure PCTCN2022074201-appb-100001
    所示结构的糖链部分；
    且所述多特异性抗体具有式(II)所示的结构：
    Figure PCTCN2022074201-appb-100002
    其中：
    所述A包含能够特异性结合所述第一靶标的第一抗原结合部分AB1和Fc区；
    GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；
    GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；
    所述Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
    Figure PCTCN2022074201-appb-100003
    Figure PCTCN2022074201-appb-100004
    AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；
    所述第一靶标和所述第二靶标中至少有一个为CD3；并且
    所述氨基酸N297的位置根据Kabat中的EU索引编号确定。
  2. 根据权利要求1所述的多特异性抗体，其中所述第一靶标与所述第二靶标不相同。
  3. 根据权利要求1-2中任一项所述的多特异性抗体，其中所述AB1和所述AB2各自独立地为抗体的抗原结合片段。
  4. 根据权利要求3所述的多特异性抗体，其中所述抗原结合片段为Fab，F(ab) 2，F(ab’)，F(ab’) 2，scFv，亲和体(affibody)和/或单域抗体。
  5. 根据权利要求1-4中任一项所述的多特异性抗体，其中所述第一靶标为肿瘤相关抗原，且所述第二靶标为CD3。
  6. 根据权利要求5所述的多特异性抗体，其中所述肿瘤相关抗原选自：Her2和PD-L1。
  7. 根据权利要求1-6中任一项所述的多特异性抗体，其中所述A为IgG抗体。
  8. 根据权利要求1-7中任一项所述的多特异性抗体，其中所述AB1包含选自下组的抗体的抗原结合部分：曲妥珠单抗和度伐利尤单抗。
  9. 根据权利要求1-8中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR3 (HCDR3)，且所述HCDR3包含SEQ ID NO：24和32中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR2(HCDR2)，且所述HCDR2包含SEQ ID NO：23和31中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR1(HCDR1)，且所述HCDR1包含SEQ ID NO：22和30中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR3(LCDR3)，且所述LCDR3包含SEQ ID NO：21和29中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR2(LCDR2)，且所述LCDR2包含SEQ ID NO：20和28中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR1(LCDR1)，且所述LCDR1包含SEQ ID NO：19和27中任一项所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的多特异性抗体，其中所述AB1包含抗体重链可变区VH，且所述VH包含SEQ ID NO：26和34中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链可变区VL，且所述VL包含SEQ ID NO：25和33中任一项所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的多特异性抗体，其中所述A为曲妥珠单抗或度伐利尤单抗。
  18. 根据权利要求1-17中任一项所述的多特异性抗体，其中所述AB2包含选自下组的抗体的CD3抗原结合部分：OKT3、M291、YTH12.5、博纳吐单抗和卡妥索单抗。
  19. 根据权利要求1-18中任一项所述的多特异性抗体，其中所述AB2包含SEQ ID NO：14所示的氨基酸序列。
  20. 根据权利要求1-4和7中任一项所述的多特异性抗体，其中所述第一靶标为CD3，且所述第二靶标为肿瘤相关抗原。
  21. 根据权利要求20所述的多特异性抗体，其中所述肿瘤相关抗原选自：Her2和PD-L1。
  22. 根据权利要求1-21中任一项所述的多特异性抗体，其中所述Fuco-L-AB2中，L的结构为J-(L 1) n-X 1Y 1-(L 1’) n’，其中：
    所述L 1为第一连接子，n为0或1；
    所述L 1’为第二连接子，n’为0或1；
    所述J为直接与Fuco相连接的接合子；
    X 1Y 1为基团X 1与基团Y 1发生连接反应之后的残留基团，其中所述基团X 1包含能够参 与生物正交连接反应的官能团，且所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团。
  23. 根据权利要求22所述的多特异性抗体，其中所述X 1包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。
  24. 根据权利要求22-23中任一项所述的多特异性抗体，其中所述X 1包含选自下组的官能团：
    Figure PCTCN2022074201-appb-100005
    Figure PCTCN2022074201-appb-100006
    其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
  25. 根据权利要求22-24中任一项所述的多特异性抗体，其中所述X 1包含选自下组的官能团：
    Figure PCTCN2022074201-appb-100007
  26. 根据权利要求22-25中任一项所述的多特异性抗体，其中所述X 1包含
    Figure PCTCN2022074201-appb-100008
  27. 根据权利要求22-26中任一项所述的多特异性抗体，其中所述Y 1包含选自下组的官能团：叠氮基、末端炔基、环炔基、四嗪基、1,2,4-三嗪基、末端烯基、环烯基、酮基、醛基、羟胺基、巯基、马来酰亚胺基以及它们的功能性衍生物。
  28. 根据权利要求22-27中任一项所述的多特异性抗体，其中所述Y 1包含选自下组的官能团：
    Figure PCTCN2022074201-appb-100009
    Figure PCTCN2022074201-appb-100010
    其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
  29. 根据权利要求22-28中任一项所述的多特异性抗体，其中所述X 1和所述Y 1包含选自以下各组的一组结构：
    a)X 1包含
    Figure PCTCN2022074201-appb-100011
    且Y 1包含
    Figure PCTCN2022074201-appb-100012
    b)X 1包含
    Figure PCTCN2022074201-appb-100013
    且Y 1包含
    Figure PCTCN2022074201-appb-100014
    c)X 1包含
    Figure PCTCN2022074201-appb-100015
    且Y 1包含
    Figure PCTCN2022074201-appb-100016
    d)X 1包含
    Figure PCTCN2022074201-appb-100017
    且Y 1包含
    Figure PCTCN2022074201-appb-100018
    和
    e)X 1包含
    Figure PCTCN2022074201-appb-100019
    且Y 1包含
    Figure PCTCN2022074201-appb-100020
  30. 根据权利要求22-29中任一项所述的多特异性抗体，其中所述X 1Y 1包含选自下组的结构：
    Figure PCTCN2022074201-appb-100021
    Figure PCTCN2022074201-appb-100022
  31. 根据权利要求22-30中任一项所述的多特异性抗体，其中所述J为
    Figure PCTCN2022074201-appb-100023
    Figure PCTCN2022074201-appb-100024
    其中所述Rf为-CH 2-，-NH-或-O-，该J结构的左端与所述Fuco直接相连接。
  32. 根据权利要求22-31中任一项所述的多特异性抗体，其中所述J为
    Figure PCTCN2022074201-appb-100025
    结构的左端和所述Fuco直接相连接。
  33. 根据权利要求22-32中任一项所述的多特异性抗体，其中所述L 1和所述L 1’各自独立地选自：C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷 基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
    Figure PCTCN2022074201-appb-100026
    其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
  34. 根据权利要求22-33中任一项所述的多特异性抗体，其中所述L 1选自：
    Figure PCTCN2022074201-appb-100027
    Figure PCTCN2022074201-appb-100028
    其中每个s1独立地为1-50的整数，每个s2独立地为0-50的整数,每个所述-CH 2-任选地被-O-替代，但连续相邻的-CH 2-不同时被-O-替代，该结构的左端与所述J连接，且该结构的右端与所述X 1连接。
  35. 根据权利要求22-34中任一项所述的多特异性抗体，其中所述L 1选自：
    Figure PCTCN2022074201-appb-100029
    Figure PCTCN2022074201-appb-100030
    且该结构的右端与所述X 1连接。
  36. 根据权利要求22-35中任一项所述的多特异性抗体，其中所述L 1’选自：
    Figure PCTCN2022074201-appb-100031
    Figure PCTCN2022074201-appb-100032
    其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
  37. 根据权利要求22-36中任一项所述的多特异性抗体，其中所述L 1’选自：
    Figure PCTCN2022074201-appb-100033
    Figure PCTCN2022074201-appb-100034
    该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
  38. 根据权利要求1-37中任一项所述的多特异性抗体，其中所述GalX为半乳糖。
  39. 根据权利要求1-37中任一项所述的多特异性抗体，其中所述GalX为被取代的半乳糖，且所述半乳糖中位于C2、C3、C4和/或C6位置的一个或多个羟基被取代。
  40. 根据权利要求1-37和39中任一项所述的多特异性抗体，其中所述GalX为被取代的半乳糖，且所述半乳糖中位于C2位置的羟基被取代。
  41. 根据权利要求1-40中任一项所述的多特异性抗体，其中所述GalX为单糖。
  42. 根据权利要求1-37和39-41中任一项所述的多特异性抗体，其中所述GalX被取代基Rg 1取代，Rg 1选自下组：氢、卤素、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24烯基、C 5-C 24环烯基、C 2-C 24炔基、C 6-C 24环炔基、C 3-C 24(杂)芳基、C 3-C 24烷基(杂)芳基和C 3-C 24(杂)芳基烷基；
    其中，所述烷基、环烷基、烯基、环烯基、炔基、环炔基、(杂)芳基、烷基(杂)芳基和/或(杂)芳基烷基各自独立地任选被一个或多个取代基Rs 4取代，和/或各自独立地任选被一个或多个取代基Rs 5间隔；
    其中，每个所述Rs 4各自独立地选自：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN；
    每个所述Rs 5各自独立地选自：-O-、-S-、
    Figure PCTCN2022074201-appb-100035
    其中，所述Rs 3包含氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
  43. 根据权利要求1-37和39-42中任一项所述的多特异性抗体，所述GalX被取代基
    Figure PCTCN2022074201-appb-100036
    取代，
    其中：
    t为0或1；
    所述Rg 2选自下组：C 1-C 24亚烷基、C 3-C 24亚环烷基、C 2-C 24亚烯基、C 5-C 24亚环烯基、 C 2-C 24亚炔基、C 6-C 24亚环炔基、C 3-C 24(杂)亚芳基、C 3-C 24烷基(杂)亚芳基和C 3-C 24(杂)芳基亚烷基，其中，所述亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、烷基(杂)亚芳基和/或(杂)芳基亚烷基各自独立地任选被一个或多个取代基Rs 4取代，和/或各自独立地任选被一个或多个取代基Rs 5间隔，
    所述Rg 3选自下组：氢、卤素、-OH、-NH 2、-SH、-N 3、-COOH、-CN、C 1-C 24烷基、C 3-C 24环烷基、C 2-C 24炔基、C 5-C 24环炔基和C 2-C 24(杂)芳基，其中所述烷基、环烷基、炔基、环炔基和/或(杂)芳基各自独立地任选被一个或多个Rs 4取代，
    其中，
    每个所述Rs 5各自独立地选自：-O-、-S-、
    Figure PCTCN2022074201-appb-100037
    所述Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基，且
    每个所述Rs 4各自独立地选自：卤素、-OH、-NH 2、-SH、-N 3、-COOH和-CN。
  44. 根据权利要求1-43任一项所述的多特异性抗体，其中所述GalX选自下组：
    Figure PCTCN2022074201-appb-100038
  45. 根据权利要求1-44中任一项所述的多特异性抗体，其中所述b是0。
  46. 多特异性抗体，其包含：
    能够特异性结合第一靶标的抗体部分A；和
    2个包含式(IV)
    Figure PCTCN2022074201-appb-100039
    所示结构的糖链部分；
    且所述多特异性抗体具有式(V)所示的结构：
    Figure PCTCN2022074201-appb-100040
    其中：
    所述A包含能够特异性结合所述第一靶标的第一抗原结合部分AB1和Fc区；
    GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；
    Fuc*包含结构Fuco-L-AB2，其中，Fuco的结构如式(III)所示：
    Figure PCTCN2022074201-appb-100041
    Figure PCTCN2022074201-appb-100042
    AB2为能够特异性结合第二靶标的第二抗原结合部分，L为连接子，且所述式(III)左端与L相连接，所述Fuc*与所述GlcNAc之间通过α-1,3糖苷键连接；
    GalX*为被取代的半乳糖，所述GalX*与所述GlcNAc之间通过β-1,4糖苷键连接，且所述GalX*包含能够特异性结合第三靶标的第三抗原结合部分AB3；
    所述第一靶标、第二靶标和第三靶标中至少有一个为CD3；并且
    所述氨基酸N297的位置根据Kabat中的EU索引编号确定。
  47. 根据权利要求46所述的多特异性抗体，其中所述GalX*具有以下结构GalX 2Y 2-(L 2’) m-AB3，其中GalX 2Y 2为基团GalX 2与基团Y 2发生连接反应之后的残留基团，其中所述GalX 2包含X 2，所述X 2包含能够参与生物正交连接反应的官能团，且所述Y 2包含能够与所述X 2发生生物正交连接反应的官能团；且
    所述L 2’为连接子，m为0或1。
  48. 根据权利要求47所述的多特异性抗体，其中所述GalX 2是C2、C3、C4和/或C6位的一个或多个羟基被取代的半乳糖。
  49. 根据权利要求47-48中任一项所述的多特异性抗体,其中所述GalX 2是C2位的羟基被取代的半乳糖。
  50. 根据权利要求47-49中任一项所述的多特异性抗体，其中所述GalX 2为单糖。
  51. 根据权利要求47-50中任一项所述的多特异性抗体,其中所述GalX 2中的X 2包含
    Figure PCTCN2022074201-appb-100043
    Figure PCTCN2022074201-appb-100044
  52. 根据权利要求47-51中任一项所述的多特异性抗体,其中所述GalX 2中的X 2包含
    Figure PCTCN2022074201-appb-100045
  53. 根据权利要求47-52中任一项所述的多特异性抗体,其中所述GalX 2具有以下结构
    Figure PCTCN2022074201-appb-100046
  54. 根据权利要求47-53中任一项所述的多特异性抗体，其中所述Y 2包含
    Figure PCTCN2022074201-appb-100047
    Figure PCTCN2022074201-appb-100048
  55. 根据权利要求47-54中任一项所述的多特异性抗体，其中所述X 2Y 2包含选自下组的结构：
    Figure PCTCN2022074201-appb-100049
    Figure PCTCN2022074201-appb-100050
  56. 根据权利要求47-55中任一项所述的多特异性抗体，其中所述L 2’选自：C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
    Figure PCTCN2022074201-appb-100051
    其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
  57. 根据权利要求47-56中任一项所述的多特异性抗体，其中所述L 2’选自：
    Figure PCTCN2022074201-appb-100052
    Figure PCTCN2022074201-appb-100053
    其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB3连接，且该结构的左端与所述Y 2连接。
  58. 根据权利要求47-57中任一项所述的多特异性抗体，其中所述L 2’为
    Figure PCTCN2022074201-appb-100054
  59. 根据权利要求46-58中任一项所述的多特异性抗体，其中所述Fuco-L-AB2中，L的结构为J-(L 1) n-X 1Y 1-(L 1’) n’，其中：
    所述L 1为第一连接子，n为0或1；
    所述L 1’为第二连接子，n’为0或1；
    所述J为直接与Fuco相连接的接合子；
    X 1Y 1为基团X 1与基团Y 1发生连接反应之后的残留基团，其中所述基团X 1包含能够参与生物正交连接反应的官能团，且所述Y 1包含能够与所述X 1发生生物正交连接反应的官能团。
  60. 根据权利要求59所述的多特异性抗体，其中所述X 1包含
    Figure PCTCN2022074201-appb-100055
    其中R 1选自：C 1-C 10亚烷基，C 5-C 10(杂)亚芳基,C 6-C 10烷基(杂)亚芳基和C 6-C 10(杂)芳基亚烷基，且R 2选自：氢、C 1-C 10烷基，C 5-C 10(杂)芳基，C 6-C 10烷基(杂)芳基和C 6-C 10(杂)芳基烷基。
  61. 根据权利要求59-60中任一项所述的多特异性抗体，其中所述X 1包含
    Figure PCTCN2022074201-appb-100056
  62. 根据权利要求59-61中任一项所述的多特异性抗体，其中所述Y 1包含
    Figure PCTCN2022074201-appb-100057
    Figure PCTCN2022074201-appb-100058
  63. 根据权利要求59-62中任一项所述的多特异性抗体，其中所述X 1Y 1包含选自下组的结 构：
    Figure PCTCN2022074201-appb-100059
  64. 根据权利要求59-63中任一项所述的多特异性抗体，其中所述J为
    Figure PCTCN2022074201-appb-100060
    Figure PCTCN2022074201-appb-100061
    其中所述Rf为-CH 2-，-NH-或-O-，其中所述J结构的左端与所述Fuco相连接。
  65. 根据权利要求59-64中任一项所述的多特异性抗体，其中所述J为
    Figure PCTCN2022074201-appb-100062
    其中所述J结构的左端与所述Fuco相连接。
  66. 根据权利要求59-65中任一项所述的多特异性抗体，其中所述L 1和所述L 1’各自独立地选自：C 3-C 200亚多肽基，C 1-C 200亚烷基，C 3-C 200亚环烷基，C 2-C 200亚烯基，C 5-C 200亚环烯基，C 2-C 200亚炔基，C 6-C 200亚环炔基，C 2-C 200(杂)亚芳基，C 3-C 200(杂)芳基亚烷基，C 3-C 200烷基(杂)亚芳基，它们的衍生物及它们的任意组合,其中所述亚多肽基、亚烷基、亚环烷基、亚烯基、亚环烯基、亚炔基、亚环炔基、(杂)亚芳基、(杂)芳基亚烷基或烷基(杂)亚芳基任选地被一个或多个Rs 1取代和/或任选地被一个或多个Rs 2间隔，其中每个所述Rs 1各自独立地选自：卤素、-OH、-NH 2和-COOH，每个所述Rs 2各自独立地选自：-O-、-S-、
    Figure PCTCN2022074201-appb-100063
    其中Rs 3选自：氢、任选被取代的C 1-C 24烷基、任选被取代的C 2-C 24烯基、任选被取代的C 2-C 24炔基和任选被取代的C 3-C 24环烷基。
  67. 根据权利要求59-66中任一项所述的多特异性抗体，其中所述L 1选自：
    Figure PCTCN2022074201-appb-100064
    Figure PCTCN2022074201-appb-100065
    其中s1为1-50的整数，每个s2独立地为0-50的整数,每个所述-CH 2-任选地被-O-替代，但连续相邻的-CH 2-不同时被-O-替代，该结构的左端与所述J连接，且该结构的右端与所述X 1连接。
  68. 根据权利要求59-67中任一项所述的多特异性抗体，其中所述L 1选自：
    Figure PCTCN2022074201-appb-100066
    Figure PCTCN2022074201-appb-100067
    且该结构的右端与所述X 1连接，左端与所述J连接。
  69. 根据权利要求59-68中任一项所述的多特异性抗体，其中所述L 1’选自：
    Figure PCTCN2022074201-appb-100068
    Figure PCTCN2022074201-appb-100069
    其中每个s2独立地为0-50的整数，每个所述-CH 2-任选地被-O-替代，但相邻的-CH 2-不同时被-O-替代，该结构的右端与所述AB2连接，且该结构的左端与所述Y 1连接。
  70. 根据权利要求59-69中任一项所述的多特异性抗体，其中所述L 1’为
    Figure PCTCN2022074201-appb-100070
    该结构的左端与所述Y 1连接，且右端与所述AB2连接。
  71. 根据权利要求46-70中任一项所述的多特异性抗体，其中所述第一靶标、第二靶标和第三靶标彼此均不相同。
  72. 根据权利要求46-71中任一项所述的多特异性抗体，其中所述AB1、所述AB2和所述AB3各自独立地为抗体的抗原结合片段。
  73. 根据权利要求72所述的多特异性抗体，其中所述抗原结合片段为Fab，F(ab) 2，F(ab’)，F(ab’) 2，scFv，亲和体(affibody)和/或单域抗体。
  74. 根据权利要求46-73中任一项所述的多特异性抗体，其中所述第一靶标为肿瘤相关抗原，所述第二靶标为CD3，且所述第三靶标为肿瘤相关抗原。
  75. 根据权利要求74所述的多特异性抗体，其中所述肿瘤相关抗原选自：Her2和PD-L1。
  76. 根据权利要求46-75中任一项所述的多特异性抗体，其中所述第一靶标为Her2，所述第二靶标为CD3，且所述第三靶标为PD-L1。
  77. 根据权利要求46-75中任一项所述的多特异性抗体，其中所述第一靶标为PD-L1，所述第二靶标为CD3，且所述第三靶标为Her2。
  78. 根据权利要求46-77中任一项所述的多特异性抗体，其中所述AB1包含选自下组的抗体的抗原结合部分：曲妥珠单抗和度伐利尤单抗。
  79. 根据权利要求46-78中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR3(HCDR3)，且所述HCDR3包含SEQ ID NO：24和32中任一项所示的氨基酸序列。
  80. 根据权利要求46-79中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR2(HCDR2)，且所述HCDR2包含SEQ ID NO：23和31中任一项所示的氨基酸序列。
  81. 根据权利要求46-80中任一项所述的多特异性抗体，其中所述AB1包含抗体重链CDR1(HCDR1)，且所述HCDR1包含SEQ ID NO：22和30中任一项所示的氨基酸序列。
  82. 根据权利要求46-81中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR3(LCDR3)，且所述LCDR3包含SEQ ID NO：21和29中任一项所示的氨基酸序列。
  83. 根据权利要求46-82中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR2(LCDR2)，且所述LCDR2包含SEQ ID NO：20和28中任一项所示的氨基酸序列。
  84. 根据权利要求46-83中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链CDR1(LCDR1)，且所述LCDR1包含SEQ ID NO：19和27中任一项所示的氨基酸序列。
  85. 根据权利要求46-84中任一项所述的多特异性抗体，其中所述AB1包含抗体重链可变区VH，且所述VH包含SEQ ID NO：26和34中任一项所示的氨基酸序列。
  86. 根据权利要求46-85中任一项所述的多特异性抗体，其中所述AB1包含抗体轻链可变区VL，且所述VL包含SEQ ID NO：25和33中任一项所示的氨基酸序列。
  87. 根据权利要求46-86中任一项所述的多特异性抗体，其中所述A为度伐利尤单抗或曲妥珠单抗。
  88. 根据权利要求46-87中任一项所述的多特异性抗体，其中所述AB2包含选自下组的抗体的抗原结合部分：OKT3、M291、YTH12.5、博纳吐单抗和卡妥索单抗。
  89. 根据权利要求46-88中任一项所述的多特异性抗体，其中所述AB2包含SEQ ID NO：14所示的氨基酸序列。
  90. 根据权利要求46-89中任一项所述的多特异性抗体，其中所述AB3包含选自下组的抗体的抗原结合部分：度伐利尤单抗，阿替利珠单抗，恩沃利单抗,曲妥珠单抗,帕托珠单抗和ZHer2:342。
  91. 根据权利要求46-90中任一项所述的多特异性抗体，其中所述AB3包含SEQ ID NO：10和12中任一项所示的氨基酸序列。
  92. 制备权利要求1-91中任一项所述的多特异性抗体的方法。
  93. 根据权利要求92所述的方法，所述方法包括：
    i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，
    其中所述糖链包含式(VI)所示的结构-GlcNAc(Fuc) b-GalX(VI)，以获得结构如式(VII)所示的蛋白
    Figure PCTCN2022074201-appb-100071
    其中：
    所述A包含所述AB1和所述Fc区；
    GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；
    GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接；
    Q为二磷酸核糖核苷酸；且
    Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
    Figure PCTCN2022074201-appb-100072
    Figure PCTCN2022074201-appb-100073
    所述J为直接与Fuco相连接的接合子，且所述J与式(III)的左端相连接；
    所述X 1包含能够参与生物正交连接反应的官能团；
    所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和
    ii)使所述结构如式(VII)所示的蛋白与Y 1-(L 1’) n’-AB2反应，以获得权利要求1-45中任一项所述的多特异性抗体；
    其中所述A，GalX，X 1,Y 1,J,L 1,L 1’,n,n’,AB1和AB2如权利要求1-45中任一项所限定的。
  94. 根据权利要求92-93中任一项所述的方法，其还包括以下步骤：
    用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；
    使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构：-GlcNAc(Fuc) b-GalX(VI)的糖链的蛋白，该蛋白的结构如式(VIII)所示：
    Figure PCTCN2022074201-appb-100074
    其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且
    GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。
  95. 根据权利要求92-93中任一项所述的方法，其还包括以下步骤：
    用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；
    使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(VI)所示结构-GlcNAc(Fuc) b-GalX(VI)的糖链的蛋白，该蛋白的结构如式(VIII)所示：
    Figure PCTCN2022074201-appb-100075
    其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且
    GalX为任选被取代的半乳糖，所述GalX与所述GlcNAc之间通过β-1,4糖苷键连接。
  96. 根据权利要求92所述的方法，所述方法包括：
    i)在催化剂存在的条件下，使供体Q-Fuc*’与包含糖链和所述抗体部分A的蛋白接触，其中所述糖链包含式(IX)所示的结构-GlcNAc(Fuc) b-GalX 2(IX)，以获得结构如式(X)所示的蛋白
    Figure PCTCN2022074201-appb-100076
    其中：
    所述A包含所述AB1和所述Fc区；
    GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；
    GalX 2为被取代的半乳糖且GalX 2包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接；
    Q为二磷酸核糖核苷酸；且
    Fuc*’包含结构Fuco-J-(L 1) n-X 1，其中，Fuco的结构如式(III)所示：
    Figure PCTCN2022074201-appb-100077
    Figure PCTCN2022074201-appb-100078
    所述J为直接与Fuco相连接的接合子，且所述J与式(III)左端相连接；
    所述X 1包含能够参与生物正交连接反应的官能团；
    所述氨基酸N297的位置根据Kabat中的EU索引编号确定；和
    ii)使所述结构如式(X)所示的蛋白与Y 1-(L 1’) n’-AB2及Y 2-(L 2’) m-AB3反应，以获得权 利要求46-91中任一项所述的多特异性抗体；
    其中所述A,X 1,Y 1,J,L 1,L 1’,n,n’,AB1，AB2，X 2，GalX 2，AB3,L 2’,Y 2和m如权利要求46-91中任一项所限定的。
  97. 根据权利要求96所述的方法，其还包括以下步骤：
    用内切糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；
    使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX 2接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
    Figure PCTCN2022074201-appb-100079
    其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0或1；且
    GalX 2为被取代的半乳糖且其包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。
  98. 根据权利要求96所述的方法，其还包括以下步骤：
    用内切糖苷酶和α1,6岩藻糖苷酶处理包含糖链和所述抗体部分A的蛋白，以得到经处理的蛋白；
    使所述经处理的蛋白在合适的催化剂存在的条件下与UDP-GalX接触，以获得具有包含式(IX)所示结构-GlcNAc(Fuc) b-GalX 2(IX)的糖链的蛋白，该蛋白的结构如式(XI)所示：
    Figure PCTCN2022074201-appb-100080
    其中，GlcNAc为N-乙酰葡萄糖胺，且所述GlcNAc直接与所述Fc区的氨基酸N297相连；
    Fuc为岩藻糖，所述Fuc与所述GlcNAc之间通过α-1,6糖苷键连接，b为0；且
    GalX 2为被取代的半乳糖且其包含X 2，X 2包含能够参与生物正交连接反应的官能团，且所述GalX 2与所述GlcNAc之间通过β-1,4糖苷键连接。
  99. 根据权利要求93-98中任一项所述的方法，其中所述Q为二磷酸鸟苷(GDP)、二磷酸 尿苷(UDP)和/或二磷酸胞苷(CDP)。
  100. 根据权利要求93-99中任一项所述的方法，其中所述Q-Fuc*’为GDP-Fuc*’。
  101. 根据权利要求93-100中任一项所述的方法，其中所述Q-Fuc*’选自以下结构：
    Figure PCTCN2022074201-appb-100081
  102. 根据权利要求93-101中任一项所述的方法，其中所述催化剂包含岩藻糖基转移酶。
  103. 根据权利要求102所述的方法，其中所述岩藻糖基转移酶为α1,3-岩藻糖基转移酶或其功能性变体或片段。
  104. 根据权利要求102-103中任一项所述的方法，其中所述岩藻糖基转移酶源自细菌。
  105. 根据权利要求102-104中任一项所述的方法，其中所述岩藻糖基转移酶源自幽门螺杆菌Helicobacter pylori。
  106. 根据权利要求102-105中任一项所述的方法，其中所述岩藻糖基转移酶源自幽门螺杆菌Helicobacter pylori 26695。
  107. 根据权利要求102-106中任一项所述的方法，其中所述岩藻糖基转移酶是源自GenBank 登录号为AAD07710.1的幽门螺杆菌α-1,3岩藻糖基转移酶。
  108. 根据权利要求102-107中任一项所述的方法，其中所述岩藻糖基转移酶包含催化活性区域和至少一个七肽重复片段，所述催化活性区域包含SEQ ID NO:1所示的氨基酸序列，且所述七肽重复片段包含SEQ ID NO:2所示的氨基酸序列。
  109. 根据权利要求102-108中任一项所述的方法，其中所述岩藻糖基转移酶包含催化活性区域和1-10个七肽重复片段，所述催化活性区域包含SEQ ID NO:1中所示的氨基酸序列，且所述七肽重复片段包含SEQ ID NO:2所示的氨基酸序列。
  110. 根据权利要求102-109中任一项所述的方法，其中所述岩藻糖基转移酶为α-1,3-岩藻糖基转移酶或其功能性变体或片段，且其包含SEQ ID NO:3中所示的氨基酸序列。
  111. 根据权利要求93-110中任一项所述的方法，其中所述催化剂包含权利要求102-110中任一项所述的岩藻糖基转移酶和标签序列。
  112. 根据权利要求93-111中任一项所述的方法，其中所述催化剂包含SEQ ID NO:3和4中任一项所示的氨基酸序列。
  113. 组合物，其包含权利要求1-91中任一项所述的多特异性抗体。
  114. 根据权利要求113所述的组合物，其还包含药学上可接受的载体。
  115. 预防、缓解和/或治疗疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-91中任一项所述的多特异性抗体，和/或权利要求113-114中任一项所述的组合物。
  116. 权利要求1-91中任一项所述的多特异性抗体和/或权利要求113-114中任一项所述的组合物用于制备药物的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
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