Protein-glycosyl conjugate and preparation method therefor

Abstract

A method for performing glycosyl conjugation using enzyme catalysis, and a protein conjugate obtained via the method. In the method, various substrates having different molecular masses can be site-specifically conjugated to the core fucose position of a protein glycan by means of an enzyme-catalyzed reaction.

Description

Protein glycosyl conjugates and methods of making the same

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410850919.3, filed on June 27, 2024, and Chinese Patent Application No. 202411195192.6, filed on August 28, 2024, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular, to a conjugate constructed by protein glycosyl and a preparation method thereof. BACKGROUND

[0004] Conjugates obtained by connecting antibodies with other bioactive substances (such as toxin molecules) through linkers can simultaneously exert the high targeting of antibodies and the therapeutic effect of bioactive substances. As a representative, more than a dozen antibody-drug conjugates (ADC) have been approved for marketing for the treatment of cancer, and more conjugate molecules are in clinical or preclinical research stage.

[0005] In the approved conjugates, some are conjugated with toxin molecules through the natural lysine in the antibody, which leads to high heterogeneity of the product and may cause the decrease of drug efficacy and the narrowing of the therapeutic window. Another widely used method is to use the side chain thiol of cysteine for conjugation with maleimide groups through Michael addition. By completely reducing the disulfide bonds between the chains of the antibody, a product with a higher drug-antibody ratio (DAR value) can be obtained. However, this thiol conjugation is not stable and will undergo reverse reaction in the blood to cause the toxin molecules to fall off, thereby producing peripheral toxicity. Therefore, there is an urgent need for a conjugation method that can obtain uniform and stable products.

[0006] Recently, the sugar chain (e.g. N-glycan) of an antibody has become a possible site for site-directed conjugation. Patent application WO2022037665 discloses a method for constructing an antibody conjugate using an a-1,3-fucosyltransferase, however, this method has strict requirements for the substrate glycoform (e.g. the N-acetylglucosamine as a substrate must be connected with galactose), needs additional modification, and the resulting product has a structure that is significantly different from the natural glycoform of the antibody. Okeley et al. (Bioconjug Chem 2013 Oct 16; 24(10): 1650-5) introduced 6-thiofucose into the N-glycan of the Fc region of an antibody by a metabolic introduction strategy, and then reacted the thiol group on the fucose derivative with a maleimide to obtain a conjugate. However, this method requires the introduction of additional reduction and oxidation steps, and the proportion of 6-thiofucose introduction is difficult to control, resulting in high heterogeneity of the conjugate and low DAR value.

[0007] Therefore, it is necessary to explore a method for efficiently introducing a biologically active substance at the core fucose position of a sugar chain to prepare a conjugate with high uniformity and high stability. SUMMARY

[0008] In a first aspect, the present disclosure relates to a method for preparing a protein conjugate, which can site-direct conjugate various substrates with different molecular weights to the core fucose position of the sugar chain of a protein through a one-step enzyme-catalyzed reaction, and the resulting conjugate has a significant biological effect.

[0009] Specifically, the present disclosure relates to a method for preparing a protein conjugate, comprising the following steps: contacting Nu-Fuc* with a protein comprising a sugar chain in the presence of a catalyst, thereby obtaining a protein conjugate;

[0010] wherein Nu comprises a ribonucleotide;

[0011] Fuc* is a fucose derivative comprising a biologically active molecule;

[0012] The sugar chain is an N-glycan lacking a core fucose;

[0013] The protein conjugate comprises a structure of Formula I:

[0014] wherein GlcNAc is a core N-acetylglucosamine, the a end of which is connected to the protein, and the b end of which is connected to the distal glycosyl of the N-glycan.

[0015] In a second aspect, the present disclosure also relates to a method for preparing another protein conjugate, which comprises the following steps:

[0016] In particular, the present disclosure provides a method for preparing a protein conjugate, which comprises the following steps:

[0017] (A) contacting Nu-Fuc** with a protein comprising a sugar chain in the presence of a catalyst, thereby obtaining a protein conjugate precursor;

[0018] (B) contacting the protein conjugate precursor with a target molecule, thereby obtaining a protein conjugate;

[0019] wherein Nu comprises a ribonucleotide; Fuc** is a reactive fucose variant comprising a chemically active group X; the sugar chain is an N-glycan chain lacking a core fucose;

[0020] the target molecule comprises a bioactive molecule and a chemically active group Y;

[0021] the protein conjugate comprises a structure of Formula I:

[0022] the protein conjugate precursor comprises a structure of Formula I’:

[0023] wherein Fuc* is a fucose derivative comprising a bioactive molecule, GlcNAc is a core N-acetylglucosamine, the a end of which is connected to the protein, and the b end of which is connected to a distal glycosyl of the N-glycan chain.

[0024] In a third aspect, the present disclosure also relates to a protein conjugate comprising a structure of Formula II-B:

[0025] wherein BM is a bioactive molecule, LU is a linking unit connecting the bioactive molecule and the fucosyl, and GlcNAc is a core N-acetylglucosamine, the a end of which is connected to the protein, and the b end of which is connected to a distal glycosyl of the N-glycan chain.

[0026] In a fourth aspect, the present disclosure also relates to a compound comprising a polypeptide, for example, a compound of Formula VI:

[0027] wherein PP is a polypeptide having GLP-1R agonistic activity; L’ is a connecting peptide at the C-terminal of PP; and LU is a linking unit connecting the bioactive molecule and the fucosyl.

[0028] In a fifth aspect, the present disclosure also provides a composition, e.g., preferably, a pharmaceutical composition, containing one or a combination of the conjugates of the present disclosure formulated together with a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF DRAWINGS

[0029] The specific features of the invention to which this disclosure pertains are set forth in the appended claims. The features and advantages of the invention to which this disclosure pertains can be better understood by reference to the following detailed description of exemplary embodiments and accompanying drawings. A brief description of the drawings is as follows:

[0030] Figures la and lb show the flow of preparing antibody-fucose-polypeptide conjugates.

[0031] Figures 2a and 2b show the mass spectrometry of antibody-fucose-P002 before (2a) and after (2b) conjugation.

[0032] Figures 3a and 3b show the mass spectrometry of antibody-fucose-Az (3a) and antibody-fucose-P001 (3b).

[0033] Figures 4a to 4c show the killing effect of MEHD-fucose-VC-PABC-MMAE on tumor cells HCC827 (4a), MDA-MB-468 (4b) and A431 (4c).

[0034] Figures 5a and 5b show the killing effect of MEHD-fucose-P4-MMAF on tumor cells HCC827 (5a) and MDA-MB-468 (5b).

[0035] Figure 6 shows the agonistic activity of antibody-fucose-P001 on GLP-1R. DETAILED DESCRIPTION

[0036] TERMS DEFINITION

[0037] Unless otherwise indicated or defined, all terms used have the ordinary meaning that would be understood by one of skill in the art, which meaning will be apparent from the context in which the terms are used. Reference is made, for example, to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nded.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nded.), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; and furthermore, all methods, procedures, techniques and manipulations not specifically detailed are performed and known in the art per se, unless otherwise stated, which will be apparent to the skilled person. Reference is also made, for example, to standard handbooks, the above-mentioned general prior art and other references cited therein.

[0038] The terms "antibody" or "immunoglobulin", which are used interchangeably unless otherwise indicated, are used herein as general terms to include both heavy chain antibodies and conventional 4-chain antibodies, whether full-length or not, their individual chains as well as all portions, domains or fragments thereof, including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively. Furthermore, the term "sequence" as used herein, for example in the terms "immunoglobulin sequence", "antibody sequence" or "protein sequence", is to be understood generally to include both the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding said sequence, unless a more restricted interpretation is required herein.

[0039] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Native immunoglobulin "Fc domains" comprise two or three constant domains, i.e., CH2 domains, CH3 domains, and optionally, CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of a heavy chain derived from an IgG, IgA, and IgD class antibody; or the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of a heavy chain derived from an IgM and IgE class antibody. Unless otherwise specified herein, amino acid residue positions in an Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as EU index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, and the heavy chain constant region CH1 and light chain constant region CL, but can in some cases include the hinge region N-terminal to the heavy chain constant region. In some embodiments, the Fc region of the present application is from IgGl, IgG2, IgG3, or IgG4.

[0040] The term "conjugate" also known as "conjugate" generally refers to a binding protein or polypeptide (e.g., an antibody or antigen-binding fragment thereof) linked to one or more biologically active molecules, such as an antibody drug conjugate (ADC). The biologically active molecule can be any small molecule drug (e.g., a cytotoxin), a radioisotope and chelates thereof, a nucleic acid, a polypeptide, and an antibody or antigen-binding fragment thereof. The conjugate can have any number of biologically active molecules conjugated to the protein from 1-20, such as can include 2, 4, 6, or 8 drug loaded species. In some embodiments, the conjugate is an antibody drug conjugate (ADC).

[0041] The term "alkyl" as used herein refers to saturated aliphatic hydrocarbon groups which are straight-chain or branched-chain groups containing from 1 to 20 carbon atoms, preferably alkyl groups containing from 1 to 12 carbon atoms, more preferably alkyl groups containing from 1 to 10 carbon atoms, most preferably alkyl groups containing from 1 to 6 carbon atoms.

[0042] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0043] The term "cycloalkylene" refers to a residue derived from the same carbon atom or two different carbon atoms removed from the parent ring of a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group.

[0044] The term "heterocyclic alkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.

[0045] The term "heterocyclic alkyl" refers to a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent ring of a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, wherein the cyclic hydrocarbon group comprises 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.

[0046] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein the definitions of alkyl or cycloalkyl are as described above.

[0047] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. The aryl group may be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, amino, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, and heterocyclic alkylthio.

[0048] The term "aryl" refers to residues derived from removing two hydrogen atoms from two different carbon atoms of a parent aromatic ring of a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring with a conjugated π-electron system.

[0049] The term "heteroaryl" refers to a heteroaromatic system comprising from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5- to 10-membered, more preferably 5- or 6-membered, e.g., furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is the heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, amino, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0050] The term "heteroarylene" refers to a residue derived from the removal of two hydrogen atoms from two different carbon atoms of a parent aromatic ring of a heteroaromatic polycyclic ring comprising from 1 to 4 heteroatoms, from 5 to 14 ring atoms.

[0051] The term "optional" or "optionally": means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, when a group or structure is "optionally substituted," the group or structure can be substituted or unsubstituted.

[0052] The term "glycosidic bond" is a chemical bond formed by the dehydration condensation of a hydroxyl group on a sugar hemiacetal structure with another hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative, wherein the chemical bond connecting the sugar and the other hydroxyl-containing compound is a glycosidic bond.

[0053] The term "DAR" refers to the ratio of biologically active molecules conjugated to the proteins described herein to the proteins. In some embodiments described herein, the DAR can be 1 to 16, such as 2-16, 2-10, 2-8, 2-6, or 3-4, such as 2, 3, 4, 5, or 6. DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the total ratio of drug moieties to ligand moieties conjugated to the ligand moieties described herein in a product as measured by detection methods, such as by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and / or HPLC, which DAR is referred to herein as the average DAR or measured DAR. In some embodiments, the average DAR value of the conjugates of the application is 1 to 16, such as 0.5 to 8.0, 0.5 to 4.0, 1.0 to 3.0, 1.5 to 2.5. For example, the average DAR value of the protein conjugates is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0. Ranges with two of these values as endpoints. It is understood that when referring to an average DAR value, the conjugate of the application refers to a population or mixture of conjugate molecules, which comprise conjugate molecules having the same and / or different DAR.

[0054] The terms "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably herein without causing contradiction depending on the context.

[0055] The term "and / or", as used in the context of this specification, refers to either or both of the items it conjoins, i.e., it represents an optional item (and can also mean the "and" that follows is not intended to mean "and / or" or "and / or").

[0056] The terms "comprising" or "including", as used herein, mean including the recited elements, integers and / or steps, but do not exclude other elements, integers or steps. In this context, the term "comprising" means that at least the recited elements, integers or steps are present, but additional elements, integers or steps can also be present.

[0057] In this application, the term "about" generally means within 0.5%-10% of a given value or range, e.g., within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value. Unless otherwise specified, numerical values referred to herein are to be understood as being modified in all instances by the term "about". If there is any question as to whether a value or parameter is to be understood as "about", the unmodified value or parameter is intended. "About" means ±5% of the value of the term it modifies, in cases where error margins for a particular value or parameter are not recognized by the ordinary understanding of the field.

[0058] A "conservative substitution" is one in which one amino acid is replaced with a chemically similar amino acid. The following groups of amino acids are exemplary of conservative substitutions: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0059] The terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, carriers, or stabilizers, etc., that are together administered with the active agent.

[0060] The term "pharmaceutical composition" refers to a composition that is in a form that is effective for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptable to the subject to whom the composition is administered.

[0061] As used herein, "treatment" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0062] DETAILED DESCRIPTION

[0063] Preparation method

[0064] One-step preparation

[0065] In a first aspect, the present disclosure relates to a method for preparing a protein conjugate, comprising the step of: contacting Nu-Fuc* with a protein comprising a sugar chain in the presence of a catalyst, thereby obtaining a protein conjugate;

[0066] wherein Nu comprises a ribonucleotide;

[0067] Fuc* is a fucose derivative comprising a biologically active molecule;

[0068] The sugar chain is an N-glycan chain lacking a core fucose;

[0069] The protein conjugate comprises a structure of Formula I:

[0070] wherein GlcNAc is a core N-acetylglucosamine, the a end of which is linked to the protein and the b end of which is linked to a distal glycosyl of the N-glycan chain.

[0071] In some embodiments, the fucose derivative is linked to the core N-acetylglucosamine via an a-1,6-glycosidic bond.

[0072] In some embodiments, the catalyst is a fucosyltransferase or a functional variant or fragment thereof; for example, the catalyst is an a-1,6-fucosyltransferase or a functional variant or fragment thereof, which can be derived from different species, such as mammals, bacteria, and nematodes.

[0073] In some embodiments, the catalyst is a human-derived a-1,6-fucosyltransferase or a functional variant or fragment thereof. Further, the catalyst can be FUT8 (Uniprot ID: Q546E0) or a functional variant or fragment thereof.

[0074] In some embodiments, the catalyst comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.

[0075] In some embodiments, the catalyst comprises a functional variant or fragment of the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.

[0076] In some embodiments, the catalyst comprises or consists of (1) the amino acid sequence set forth in SEQ ID NO: 1; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 1; or (3) an amino acid sequence comprising one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 1, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the catalyst comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0077] In some embodiments, the catalyst comprises or consists of (1) the amino acid sequence set forth in SEQ ID NO: 2; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 2; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the catalyst comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0078] In some embodiments, the catalyst comprises or consists of (1) the amino acid sequence set forth in SEQ ID NO: 3; (2) an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 3; or (3) an amino acid sequence that comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 3, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the catalyst comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0079] In some embodiments, the Nu comprises a ribonucleotide diphosphate or a salt thereof; preferably, the Nu comprises a structure selected from the group consisting of guanosine diphosphate (GDP), uridine diphosphate (UDP), cytidine diphosphate (CDP), and adenosine diphosphate (ADP), or a salt thereof:

[0080] In some embodiments, the Nu comprises guanosine diphosphate (GDP) or a salt thereof.

[0081] In some embodiments, the fucose derivative Fuc* has the structure of Formula II:

[0082] wherein, BM is a biologically active molecule, and LU is a linking unit linking the biologically active molecule and the fucosyl group. The biologically active molecule is a pharmacological and / or biological molecule, i.e., a molecule having a pharmacological and / or biological activity.

[0083] In some embodiments, the Nu-Fuc* has the structure of Formula II-A:

[0084] wherein BM, LU are defined as previously described; correspondingly, the protein conjugate comprises a structure of Formula II-B:

[0085] wherein BM, LU are defined as previously described; GlcNAc is a core N-acetylglucosamine, with its a end linked to the protein and its b end linked to a distal glycosyl of the N-glycan chain. In some embodiments, the GlcNAc is linked to the fucosyl through an a-1,6-glycosidic bond.

[0086] In some embodiments, the glycan chain can comprise 2-30 monosaccharides, for example, can comprise 2-15 monosaccharides.

[0087] The methods and conjugates of the present disclosure do not have specific requirements on the particular glycoform of the N-glycan chain, as long as it has a core N-acetylglucosamine for conjugation and lacks a core fucose prior to conjugation. For example, the glycoform of the N-glycan chain can be G0, G1, G2, G2S1, G2S2, M5, M6, etc.

[0088] The “distal glycosyl” is understood in the art to mean the portion of the N-glycan chain remaining after removal of the core N-acetylglucosamine and the core fucose, which is distal to the core N-acetylglucosamine in the protein.

[0089] In some embodiments, the linking unit LU further has a structure of Formula III:

[0090] (with the side for linking to the bioactive molecule denoted as BM and the side for linking to the fucosyl denoted as fuc), wherein L1 is an extension group, L2 is a first spacer group, L3 is absent or a linker group, and L4 is absent or a second spacer group.

[0091] In some embodiments, the extension group L1 comprises one or more of the following structures:

[0092] (with the side for linking in the direction of L2 denoted as L2 and the side for linking in the direction of the fucosyl denoted as fuc).

[0093] In some embodiments, the L1 is selected from:

[0094] (with the side for linking to L2 denoted as L2 and the side for linking to the fucosyl denoted as fuc); wherein m is an integer selected from 0-10 and n is an integer selected from 0-20. In some embodiments, m is an integer selected from 1-10 and n is an integer selected from 1-12.

[0095] In some embodiments, the first spacer group L2has a structure according to Formula IV:

[0096] (the side connected to L3is designated L3, and the side connected to L1is designated L1);

[0097] wherein a1= 0 or 1, a2= 0 or 1, a3= an integer from 0 to 8, b1= 0 or 1, b2= an integer from 0 to 16, b3= an integer from 0 to 16, c= an integer from 0 to 8, and at least one of b2and b3is 0.

[0098] In some embodiments, the structure of L2has a1= 1; in other embodiments, a1= 0.

[0099] In some embodiments, a2= 1; in other embodiments, a2= 0.

[0100] In some embodiments, a3= 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0101] In some embodiments, b1= 0 or 1.

[0102] In some embodiments, b2= 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0103] In some embodiments, b3= 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0104] In some embodiments, c= 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0105] Any combination of the above alternatives for a1, a2, a3, b1, b2, b3, and c can be made, provided that at least one of b2and b3is 0.

[0106] In some specific embodiments, the structure of L2is selected from the group consisting of:

[0107] (1) a1= 0, a2= 0, a3= 0, 1, 2, 3, 4, 5, or 6, b1= 0, b2= 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b3= 0, c= 0;

[0108] (2) a1= 0, a2= 0, a3= 0, 1, 2, 3, 4, 5, or 6, b1= 1, b2= 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b3= 0, c= 0;

[0109] (3) a1 = 1, a2 = 1, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b3 = 0, c = 0;

[0110] (4) a1 = 1, a2 = 0, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0111] (5) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0112] (6) a1 = 1, a2 = 0, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b3 = 0, c = 0;

[0113] (7) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0114] (8) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5, or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, b3 = 0, c = 0;

[0115] (9) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 0, b3 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, c = 0, 1, 2, 3, 4, 5, or 6; and

[0116] (10) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0117] In some embodiments, the structure of L2is specifically selected from the group of:

[0118] (1.1) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 2, b3 = 0, c = 0;

[0119] (1.2) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 4, b3 = 0, c = 0;

[0120] (1.3) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 6, b3 = 0, c = 0;

[0121] (1.4) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 8, b3 = 0, c = 0;

[0122] (1.5) a1=0, a2=0, a3=0, b1=0, b2=10, b3=0, c=0;

[0123] (1.6) a1=0, a2=0, a3=0, b1=0, b2=12, b3=0, c=0;

[0124] (1.7) a1=0, a2=0, a3=1, b1=0, b2=2, b3=0, c=0;

[0125] (1.8) a1=0, a2=0, a3=1, b1=0, b2=4, b3=0, c=0;

[0126] (1.9) a1=0, a2=0, a3=1, b1=0, b2=6, b3=0, c=0;

[0127] (1.10) a1=0, a2=0, a3=1, b1=0, b2=8, b3=0, c=0;

[0128] (1.11) a1=0, a2=0, a3=1, b1=0, b2=10, b3=0, c=0;

[0129] (1.12) a1=0, a2=0, a3=1, b1=0, b2=12, b3=0, c=0;

[0130] (1.13) a1=0, a2=0, a3=2, b1=0, b2=2, b3=0, c=0;

[0131] (1.14) a1=0, a2=0, a3=2, b1=0, b2=4, b3=0, c=0;

[0132] (1.15) a1=0, a2=0, a3=2, b1=0, b2=6, b3=0, c=0;

[0133] (1.16) a1=0, a2=0, a3=2, b1=0, b2=8, b3=0, c=0;

[0134] (1.17) a1=0, a2=0, a3=2, b1=0, b2=10, b3=0, c=0;

[0135] (1.18) a1=0, a2=0, a3=2, b1=0, b2=12, b3=0, c=0;

[0136] (2.1) a1=0, a2=0, a3=0, b1=1, b2=2, b3=0, c=0;

[0137] (2.2) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 4, b3 = 0, c = 0;

[0138] (2.3) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 6, b3 = 0, c = 0;

[0139] (2.4) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 8, b3 = 0, c = 0;

[0140] (2.5) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 10, b3 = 0, c = 0;

[0141] (2.6) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 12, b3 = 0, c = 0;

[0142] (2.7) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 2, b3 = 0, c = 0;

[0143] (2.8) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 4, b3 = 0, c = 0;

[0144] (2.9) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 6, b3 = 0, c = 0;

[0145] (2.10) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 8, b3 = 0, c = 0;

[0146] (2.11) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 10, b3 = 0, c = 0;

[0147] (2.12) a1 = 0, a2 = 0, a3 = 1, b1 = 1, b2 = 12, b3 = 0, c = 0;

[0148] (2.13) a1 = 0, a2 = 0, a3 = 2, b1 = 1, b2 = 2, b3 = 0, c = 0;

[0149] (2.14) a1 = 0, a2 = 0, a3 = 2, b1 = 1, b2 = 4, b3 = 0, c = 0;

[0150] (2.15) a1 = 0, a2 = 0, a3 = 2, b1 = 1, b2 = 6, b3 = 0, c = 0;

[0151] (2.16) a1 = 0, a2 = 0, a3 = 2, b1 = 1, b2 = 8, b3 = 0, c = 0;

[0152] (2.17) a1=0, a2=0, a3=2, b1=1, b2=10, b3=0, c=0;

[0153] (2.18) a1=0, a2=0, a3=2, b1=1, b2=12, b3=0, c=0;

[0154] (3.1) a1=1, a2=1, a3=2, b1=0, b2=2, b3=0, c=0;

[0155] (3.2) a1=1, a2=1, a3=2, b1=0, b2=4, b3=0, c=0;

[0156] (3.3) a1=1, a2=1, a3=2, b1=0, b2=6, b3=0, c=0;

[0157] (3.4) a1=1, a2=1, a3=2, b1=0, b2=8, b3=0, c=0;

[0158] (3.5) a1=1, a2=1, a3=2, b1=0, b2=10, b3=0, c=0;

[0159] (3.6) a1=1, a2=1, a3=2, b1=0, b2=12, b3=0, c=0;

[0160] (4.1) a1=1, a2=0, a3=1, b1=0, b2=0, b3=0, c=0;

[0161] (4.2) a1=1, a2=0, a3=2, b1=0, b2=0, b3=0, c=0;

[0162] (4.3) a1=1, a2=0, a3=3, b1=0, b2=0, b3=0, c=0;

[0163] (4.4) a1=1, a2=0, a3=4, b1=0, b2=0, b3=0, c=0;

[0164] (4.5) a1=1, a2=0, a3=5, b1=0, b2=0, b3=0, c=0;

[0165] (4.6) a1=1, a2=0, a3=6, b1=0, b2=0, b3=0, c=0;

[0166] (5.1) a1=0, a2=0, a3=1, b1=0, b2=0, b3=0, c=0;

[0167] (5.2) a1=0, a2=0, a3=2, b1=0, b2=0, b3=0, c=0;

[0168] (5.3) a1=0, a2=0, a3=3, b1=0, b2=0, b3=0, c=0;

[0169] (5.4) a1=0, a2=0, a3=4, b1=0, b2=0, b3=0, c=0;

[0170] (5.5) a1=0, a2=0, a3=5, b1=0, b2=0, b3=0, c=0;

[0171] (5.6) a1=0, a2=0, a3=6, b1=0, b2=0, b3=0, c=0;

[0172] (6.1) a1=1, a2=0, a3=2, b1=0, b2=2, b3=0, c=0;

[0173] (6.2) a1=1, a2=0, a3=2, b1=0, b2=4, b3=0, c=0;

[0174] (6.3) a1=1, a2=0, a3=2, b1=0, b2=6, b3=0, c=0;

[0175] (6.4) a1=1, a2=0, a3=2, b1=0, b2=8, b3=0, c=0;

[0176] (6.5) a1=1, a2=0, a3=2, b1=0, b2=10, b3=0, c=0;

[0177] (6.6) a1=1, a2=0, a3=2, b1=0, b2=12, b3=0, c=0;

[0178] (7.1) a1=0, a2=1, a3=1, b1=0, b2=0, b3=0, c=0;

[0179] (7.2) a1=0, a2=1, a3=2, b1=0, b2=0, b3=0, c=0;

[0180] (7.3) a1=0, a2=1, a3=3, b1=0, b2=0, b3=0, c=0;

[0181] (7.4) a1=0, a2=1, a3=4, b1=0, b2=0, b3=0, c=0;

[0182] (7.5) a1=0, a2=1, a3=5, b1=0, b2=0, b3=0, c=0;

[0183] (7.6) a1=0, a2=1, a3=6, b1=0, b2=0, b3=0, c=0;

[0184] (8.1) a1=0, a2=1, a3=2, b1=0, b2=2, b3=0, c=0;

[0185] (8.2) a1=0, a2=1, a3=2, b1=0, b2=4, b3=0, c=0;

[0186] (8.3) a1=0, a2=1, a3=2, b1=0, b2=6, b3=0, c=0;

[0187] (8.4) a1=0, a2=1, a3=2, b1=0, b2=8, b3=0, c=0;

[0188] (8.5) a1=0, a2=1, a3=2, b1=0, b2=10, b3=0, c=0;

[0189] (8.6) a1=0, a2=1, a3=2, b1=0, b2=12, b3=0, c=0;

[0190] (9.1) a1=0, a2=0, a3=0, b1=0, b2=0, b3=2, c=1;

[0191] (9.2) a1=0, a2=0, a3=0, b1=0, b2=0, b3=3, c=1;

[0192] (9.3) a1=0, a2=0, a3=0, b1=0, b2=0, b3=4, c=1;

[0193] (9.4) a1=0, a2=0, a3=0, b1=0, b2=0, b3=5, c=1;

[0194] (9.5) a1=0, a2=0, a3=0, b1=0, b2=0, b3=6, c=1;

[0195] (9.6) a1=0, a2=0, a3=0, b1=0, b2=0, b3=2, c=2;

[0196] (9.7) a1=0, a2=0, a3=0, b1=0, b2=0, b3=3, c=2;

[0197] (9.8) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 0, b3 = 4, c = 2;

[0198] (9.9) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 0, b3 = 5, c = 2;

[0199] (9.10) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 0, b3 = 6, c = 2;

[0200] (10.1) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 0;

[0201] (10.2) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 1;

[0202] (10.3) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 2;

[0203] (10.4) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 3;

[0204] (10.5) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 4;

[0205] (10.6) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 5; and

[0206] (10.7) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 6.

[0207] In some embodiments, L3 is absent; while in other embodiments, L3 is a peptide linking group.

[0208] In some embodiments, L3 is selected from a peptide linking group capable of being cleaved by a cathepsin; preferably, the cathepsin can be selected from the group consisting of cathepsins A, B, C, D, E, F, G, H, K, LI, L2, O, S, W, and Z; more preferably, the cathepsin is cathepsin B.

[0209] In some embodiments, L3 is a di-amino acid peptide, a tri-amino acid peptide, or a tetra-amino acid peptide residue.

[0210] In some embodiments, L3 is selected from the group consisting of di-amino acid peptide residues of -Lys-Phe-, -Ala-Val-, -Lys-Val-, -Cit-Val-, -Lys-Ala-, -Cit-Phe-, -Cit-Leu-, -Cit-Ile-, -Arg-Phe-, -Cit-Trp-, -Gly-Gly-, -Ala-Ala-, -Val-Gly-, and -Glu-Gly-; the left side of which is connected to L4 and the right side of which is connected to L2.

[0211] In other embodiments, L3 is selected from the group consisting of tri-amino acid peptide residues of -Ala-Val-Glu-, -Cit-Val-Glu-, -Ala-Val- aGlu-, -Cit-Val-aGlu-, -Gly-Lys-Val-, and -Gly-Cit-Val-; the left side of which is connected to L4 and the right side of which is connected to L2.

[0212] In yet other embodiments, L3 is selected from the group consisting of tetra-amino acid peptide residues of -Gly-Phe-Gly-Gly- and -Gly-Gly-Phe-Gly-; the left side of which is connected to L4 and the right side of which is connected to L2.

[0213] In some embodiments, L4 is absent; while in other embodiments, L4 is selected from the group consisting of:

[0214] (BM side is designated as BM, L3 side is designated as L3); wherein R1 is independently selected from the group consisting of hydrogen, C 1-6 alkyl, hydroxyl, amino, halogen, nitro, cyano, d is an integer from 1 to 20, e is an integer from 1 to 20; R2 is selected from the group consisting of hydrogen and C 1-6 alkyl.

[0215] In some embodiments, wherein L4 is absent, or is selected from the group consisting of:

[0216] In some embodiments, the LU as a whole can be selected from the group consisting of:

[0217] wherein each of k1, k2 is independently selected from an integer from 1 to 20, and k’ is an integer from 0 to 10. In some embodiments, each of k1, k2 is independently selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k’ is selected from 0, 1, 2, 3, 4, and 5.

[0218] In some embodiments, the LU of the present disclosure is selected from the structure of:

[0219] wherein k1, k2, and k’ are as defined previously.

[0220] In some embodiments, the bioactive molecule (BM) can be a drug or prodrug, a diagnostic agent, a protein, a polypeptide, an amino acid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, a nucleoside, a polynucleotide, RNA, or DNA.

[0221] In some embodiments, the bioactive molecule can be selected from small molecule drugs (e.g., drugs with a molecular weight less than 1000 daltons), radioisotopes and chelates thereof, nucleic acids, polypeptides, and antibodies or antigen-binding fragments thereof.

[0222] In some embodiments, the bioactive molecule can be selected from the following small molecule drugs: topoisomerase inhibitors, tubulin inhibitors, radioisotopes, metal complexes, glycopeptide antibiotics, glucocorticoids, calcineurin inhibitors, DNA alkylating agents, DNA synthesis interfering drugs, serine kinase inhibitors, threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, histidine kinase inhibitors, proteasome inhibitors, protease inhibitors, histone deacetylase inhibitors, neovascularization inhibitors, cyclin inhibitors, Toll-like receptor modulators, and STING modulators.

[0223] In some embodiments, the bioactive molecule is a topoisomerase I inhibitor, preferably a camptothecin compound, which refers to a compound having the same 5-membered fused parent nucleus structure as naturally derived camptothecin, and having substitutional modification at its 7-, 9-, 10-, and 11-positions, which has equal or stronger topoisomerase I inhibitory activity than natural camptothecin.

[0224] In some embodiments, the bioactive molecule can be selected from Dxd, exatecan, SN-38, 10-hydroxycamptothecin, irinotecan, topotecan, belotecan, Gimatecan, Lurtotecan, Rubitecan, and Namitecan.

[0225] In some embodiments, the biologically active molecule is a topoisomerase II inhibitor; preferably selected from the group consisting of anthracyclines, podophyllotoxin derivatives, and anthraquinone antibiotics; more preferably selected from the group consisting of doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, PNU-159682, etoposide, teniposide, and mitoxantrone.

[0226] In other embodiments, the biologically active molecule is a tubulin inhibitor; preferably selected from the group consisting of a dolastatin 10 and its derivatives, maytansinoids, a combretastatin, a taxane, a vinblastine, eribulin and its derivatives, and colchicine and its derivatives; more preferably selected from the group consisting of MMAE, MMAF, Duo5, PF-06380101, DM1, DM4, and eribulin.

[0227] In other embodiments, the biologically active molecule can be selected from a polypeptide, for example, a peptide condensed from 5-50 amino acids.

[0228] Preferably, the biologically active molecule can comprise a polypeptide having GLP-1R agonistic activity. The “having GLP-1R agonistic activity” means having at least part of the activity of a natural ligand of GLP-1R, for example, having one or more activities of human GLP-1 protein, or having at least 10% (e.g., at least 15%, at least 20%, at least 25%, 30%, 35%, 40%, 45%, or 50% or more) of the activity of human GLP-1 protein.

[0229] The agonistic activity does not require the same level as a natural ligand of GLP-1R (e.g., human GLP-1), and can be higher, similar, or lower than the activity of human GLP-1 protein. In certain cases, it can refer to one or more selected from the group consisting of an activity of binding to GLP-1 receptor, an activity of activating GLP-1 receptor, an activity of activating adenylate cyclase, an activity of promoting an increase in intracellular cyclic adenosine monophosphate (cAMP) level, an activity of positively regulating intracellular Ca 2+ level, an activity of stimulating insulin secretion, an activity of increasing hepatic glycogen storage, an activity of delaying gastric emptying, an activity of inhibiting gastric motility, an activity of reducing appetite, an activity of inhibiting beta cell apoptosis, an activity of inhibiting postprandial glucagon secretion, an activity of relieving hypoglycemia, and an activity of reducing body weight. For example, it can be detected by detecting the ability of binding to GLP-1 receptor, the expression level of cAMP. For example, it can be detected by luciferase method to detect the level of activation of cAMP / PKA signaling pathway.

[0230] One representative of a natural ligand of GLP-1R is human GLP-1, which has the following sequence (SEQ ID NO: 4):

[0231] Another example of a polypeptide having GLP-1R agonistic activity is a human GLP-1 variant set forth in SEQ ID NO: 5:

[0232] In some embodiments, the polypeptide having GLP-1R agonistic activity comprises or consists of: (1) an amino acid sequence set forth in SEQ ID NO: 4; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0233] In some embodiments, the polypeptide having GLP-1R agonistic activity comprises or consists of: (1) an amino acid sequence set forth in SEQ ID NO: 5; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0234] In some embodiments, the polypeptide having GLP-1R agonistic activity comprises or consists of a human GLP-1 variant; in some embodiments, the human GLP-1 variant can be selected from the GLP-1 variant sequences disclosed in prior patent application WO2022143516, which is incorporated by reference in its entirety.

[0235] In some embodiments, the human GLP-1 variant can further comprise an amino acid mutation at position G8 compared to the amino acid sequence set forth in SEQ ID NO: 5.

[0236] In some embodiments, the human GLP-1 variant can further comprise an amino acid mutation at one or more of positions Y19, K26, and W31 compared to the amino acid sequence set forth in SEQ ID NO: 5.

[0237] In some embodiments, the human GLP-1 variant can further comprise an amino acid mutation selected from the following positions compared to the amino acid sequence set forth in SEQ ID NO: 5:

[0238] (1) G8; (2) Y19; (3) K26; (4) W31; (5) G8 and Y19; (6) G8 and K26; (7) G8 and W31; (8) G8, K26, and W31; and (9) G8, Y19, K26, and W31.

[0239] In some embodiments, the amino acid substitution at position G8 is G8Aib.

[0240] In some embodiments, the amino acid substitution at K26 position is K26R.

[0241] In some embodiments, the amino acid substitution at Y19 position is selected from Y19A, Y19L, Y19T, Y19F, Y19I, Y19V and Y19S.

[0242] In some embodiments, the amino acid substitution at W31 position is selected from W31Y, W31L and W31A.

[0243] In the present disclosure, the amino acid substitution "XnY" means that the residue X at the n-th position in the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 is substituted with the amino acid residue Y, wherein n is a positive integer (for GLP-1, n starts from 7), X and Y are the abbreviations of any amino acid residue respectively, and X is different from Y. For example, the amino acid substitution "W31Y" means that the amino acid residue W at the 31-st position in the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 is substituted with the amino acid residue Y.

[0244] In some embodiments, the human GLP-1 variant can comprise a mutation or a combination of mutations selected from the following, as compared to the amino acid sequence set forth in SEQ ID NO: 5:

[0245] (1) G8Aib; (2) Y19A; (3) K26R; (4) W31Y; (5) G8Aib and Y19A; (6) G8Aib and K26R; (7) G8Aib and W31Y; (8) G8Aib, K26R and W31Y; and (9) G8Aib, Y19A, K26R and W31Y.

[0246] In some embodiments, the human GLP-1 variant can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6-14.

[0247] In some embodiments, the biologically active molecule comprising the human GLP-1 variant can be linked to the LU via the amino acid side chain of the human GLP-1 variant.

[0248] In some embodiments, the biologically active molecule further comprises a linker peptide located at the C-terminal or N-terminal of the aforementioned polypeptide. In some embodiments, the aforementioned human GLP-1 variant can be linked to the LU via the linker peptide.

[0249] In some embodiments, the linker peptide has a length of 2-20 amino acids, for example, the linker peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-20.

[0250] In some embodiments, the bioactive molecule comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 21-32. The aforementioned bioactive molecule can be linked via the amino group on the side chain of the C-terminal lysine of the connecting peptide to LU.

[0251] In some embodiments, the C-terminus of the connecting peptide or bioactive molecule is a carboxylic acid or an amide.

[0252] In some embodiments, the bioactive molecule can be selected from an antibody or an antigen-binding fragment thereof.

[0253] In some embodiments, the structure of the fucose derivative Fuc* of the present disclosure is selected from:

[0254] wherein k1 is an integer selected from 1-20, and k’ is an integer selected from 0-10. In some embodiments, wherein k1 is selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k’ is selected from 0, 1, 2, 3, 4, and 5.

[0255] In some embodiments, the structure of the fucose derivative Fuc* of the present disclosure is selected from:

[0256] wherein k1 is an integer selected from 1-20, and k’ is an integer selected from 0-10. In some embodiments, wherein k1 is selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k’ is selected from 0, 1, 2, 3, 4, and 5.

[0257] In some embodiments, in the structure of Formula I and Formula II-B, the a-terminus is linked to the Fc region of a protein; preferably to the CH2 domain of the Fc region; more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc region.

[0258] The protein used for constructing the protein conjugate in the present disclosure can be selected from an antibody or an antigen-binding fragment thereof, and a fusion protein with an Fc region.

[0259] In some embodiments, the Fc region is an Fc region derived from IgG; preferably, the Fc region is an Fc region derived from IgG1, IgG2, IgG3, or IgG4. For example, the immunoglobulin Fc region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-37.

[0260] In some embodiments, the protein and / or protein conjugate of the present disclosure is a dimer, preferably a homodimer.

[0261] In some embodiments, the present disclosure also finds a protocol for improving the reaction efficiency of the catalyst and / or quickly obtaining the protein conjugate in the aforementioned method; specifically, the protein comprises an immunoglobulin Fc region variant comprising a mutation that reduces or eliminates effector function.

[0262] In some embodiments, the protein is an antigen binding protein or a fragment thereof comprising an Fc region variant; in some embodiments, the protein is an antibody or an antigen binding fragment thereof.

[0263] In some embodiments, the variant of the immunoglobulin Fc region is derived from the Fc region of human IgG1, IgG2, IgG3 or IgG4.

[0264] In some embodiments, the effector function can be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutation that reduces or eliminates effector function weakens or eliminates the binding between the immunoglobulin and FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, FcyRIIIb and / or Clq.

[0265] The mutations that attenuate or eliminate effector function can be any known mutation or combination of mutations in the art, such as described in Esohe E.I., J Immunol 2000; 164: 4178-4184; Hutchins, J.T., Proc. Natl. Acad. Sci. USA 1995, 92, 11980-11984; Xu D., Cell Immunol. 2000, 200, 16-26; Hezareh, M., J. Virol. 2001, 75, 12161-12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Chu, S.Y., Mol. Immunol. 2008, 45, 3926-3933; Sazinsky, S.L., Proc. Natl. Acad. Sci. USA 2008, 105, 20167-20172; Oganesyan, V., Acta Crystallogr. Sect. D Biol. Crystallogr. 2008, 64 Pt 6, 700-704; An, Z., MAbs 2009, 1, 572-579; Moore, G.L., Methods 2019, 154, 38-50; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Strohl, W., US20150337053; Engelberts, P.J., EBioMedicine 2020, 52, 102625, and the like.

[0266] In some embodiments, the Fc region can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P329G, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A, L234A / L235A / P329G, L234F / L235E / D265A, or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the Fc region can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of D265A / P331G (hereinafter can be abbreviated as AG), L234A / L235A (hereinafter abbreviated as AA), and L234A / L235A / P329G (hereinafter abbreviated as GAA).

[0267] In some embodiments, the protein comprises an immunoglobulin Fc region variant, which further comprises a D265A / P331G mutation based on the amino acid sequence set forth in SEQ ID NO: 33 or 34.

[0268] In some embodiments, the protein comprises an immunoglobulin Fc region variant, which further comprises a L234A / L235A mutation based on the amino acid sequence set forth in SEQ ID NO: 33 or 34.

[0269] In some embodiments, the protein comprises an immunoglobulin Fc region variant, which further comprises a L234A / L235A / P329G mutation based on the amino acid sequence set forth in SEQ ID NO: 33 or 34.

[0270] In some embodiments, the protein comprises an Fc region variant, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42 or 43.

[0271] Two-step process

[0272] In a second aspect, the present disclosure also provides a method for preparing a protein conjugate, which comprises the following steps:

[0273] (A) contacting Nu-Fuc** with a protein comprising a sugar chain in the presence of a catalyst, thereby obtaining a protein conjugate precursor;

[0274] (B) contacting the protein conjugate precursor with a target molecule, thereby obtaining a protein conjugate;

[0275] wherein Nu comprises a ribonucleotide; Fuc** is a reactive fucose variant comprising a chemically active group X; the sugar chain is an N-glycan chain lacking a core fucose;

[0276] the target molecule comprises a bioactive molecule and a chemically active group Y;

[0277] the protein conjugate comprises a structure of Formula I:

[0278] the protein conjugate precursor comprises a structure of Formula I’:

[0279] wherein Fuc* is a fucose derivative comprising a bioactive molecule, GlcNAc is a core N-acetylglucosamine, the a end of which is attached to the protein and the b end of which is attached to a distal glycosyl group of the N-glycan chain; and,

[0280] Fuc** is not:

[0281] For the protein, catalyst, Nu, sugar chain, Fuc*, bioactive molecule, etc., refer to the definitions described in the first aspect.

[0282] In some embodiments, the reactive fucose variant, fucose derivative, and core N-acetylglucosamine are linked via an a-1,6-glycosidic bond.

[0283] In some embodiments, the chemically active group X and chemically active group Y each independently comprises a structure selected from the group consisting of:

[0284] In some embodiments, the chemically active group X and chemically active group Y are selected from the group consisting of combinations of:

[0285] (1) X comprises: Y comprises:

[0286] (2) X comprises: Y comprises:

[0287] (3) X comprises: Y comprises:

[0288] (4) X comprises: Y comprises:

[0289] (5) X comprises: Y comprises:

[0290] (6) X comprises: Y comprises: and

[0291] (7) X comprises: Y comprises:

[0292] In some embodiments, the Fuc** has a structure according to Formula II’:

[0293] In some embodiments, the chemically active group X is selected from the following structures:

[0294] wherein p is an integer from 0 to 20, for example, p = 2, 3, 4, 5, 6, 8, 10, 12, 14, or 16.

[0295] In some embodiments, the Fuc** is selected from the following structures:

[0296] wherein p is an integer from 0 to 20, for example, p = 2, 3, 4, 5, 6, 8, 10, 12, 14, or 16. In some embodiments, the target molecule has a structure according to Formula V:

[0297] BM-L4-L3-L2-Y (V); wherein L2, L3, L4, and BM are as defined in the first aspect.

[0298] In some embodiments, the chemically active group Y according to the present disclosure is selected from the following structures:

[0299] In some embodiments, L4-L3-L2-Y in Formula V has a structure selected from the following:

[0300] wherein each of k1, k2 is independently selected from an integer from 1 to 20, and k' is an integer from 0 to 10. In some embodiments, wherein each of k1, k2 is independently selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k' is selected from 0, 1, 2, 3, 4, and 5.

[0301] In some embodiments, L4-L3-L2-Y in the formula V has a structure selected from:

[0302] wherein k1, k2, and k' are as defined above.

[0303] In some embodiments, the target molecule has a structure selected from:

[0304] wherein k1 is an integer from 1 to 20, and k' is an integer from 0 to 10. In some embodiments, wherein k1 is selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k' is selected from 0, 1, 2, 3, 4, and 5.

[0305] In some embodiments, the target molecule has a structure selected from:

[0306] wherein k' is an integer from 0 to 10. In some embodiments, k' is selected from 0, 1, 2, 3, 4, and 5.

[0307] In some embodiments, the protein conjugate obtained in step (B) is as defined in the first aspect.

[0308] For the one-step method of preparation according to the first aspect of the disclosure and / or the two-step method of preparation according to the second aspect, both can further comprise a step of obtaining a protein comprising a sugar chain, which is an N-glycan chain lacking a core fucose. Preferably, the sugar chain is an N-glycan chain lacking a core a-1,6-core fucose.

[0309] Methods of obtaining the protein comprising an N-glycan chain lacking a core fucose are well known to those skilled in the art. For example, in some embodiments, the method comprises a step of contacting a protein comprising a core fucose with an a-1,6-fucosidase, thereby obtaining a protein comprising an N-glycan chain lacking a core fucose.

[0310] In some embodiments, the a-1,6-fucosidase can be selected from a-L-fucosidase (Alfc), or a functional variant thereof, or any combination thereof.

[0311] In some embodiments, the Alfc comprises an amino acid sequence as set forth in SEQ ID NO: 38 or SEQ ID NO: 39.

[0312] In some embodiments, the protein comprising N-glycan lacking core fucose can also be expressed, purified by using a cell line with FUT8 gene knockout. One example of knocking out the FUT8 gene in the cell line for expression is by homologous recombination technology, which is specifically described in Biotechnology and Bioengineering, 2004, 87(5): 614-622; other examples of knocking out the FUT8 gene include using zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN), which are specifically described in Biotechnology and bioengineering, 2010, 106(5): 774-783. Biotechnology and Bioengineering, 2013, 110(3): 871-880, and the like.

[0313] Protein conjugate

[0314] In a third aspect, the present disclosure also relates to a protein conjugate comprising a structure of Formula II-B:

[0315] wherein BM is a bioactive molecule, LU is a linking unit linking the bioactive molecule and the fucosyl group, and GlcNAc is a core N-acetylglucosamine, the a end of which is linked to the protein, and the b end of which is linked to the distal glycosyl group of the N-glycan;

[0316] and the BM-LU is not a structure of:

[0317] In some embodiments, the protein conjugate is obtained according to the method of the first aspect or the second aspect.

[0318] In some embodiments, the GlcNAc and the fucosyl group are linked by an a-1,6-glycosidic bond.

[0319] In some embodiments, the linking unit LU further has a structure as set forth in Formula III:

[0320] wherein, L1is an extension group, L2is a first spacer group, L3is absent or a linker group, and L4is absent or a second spacer group. The specific definitions of L1, L2, L3and L4may refer to the description in the first aspect.

[0321] In some embodiments, the LU can be specifically selected from the following structures:

[0322] wherein, k1, k2are each independently selected from an integer from 1 to 20, and k’ is an integer from 0 to 10. In some embodiments, wherein k1, k2are each independently selected from 2, 3, 4, 5, 6, 8, 10, 12, 14 and 16, and k’ is selected from 0, 1, 2, 3, 4 and 5.

[0323] In some embodiments, the bioactive molecule can be selected from small molecule drugs, radioisotopes and chelates thereof, nucleic acids, polypeptides and antibodies or antigen-binding fragments thereof. In some embodiments, the bioactive molecule can refer to the definition in the first aspect.

[0324] In some embodiments, the protein conjugate can comprise a structure selected from the following:

[0325] wherein, k1is an integer from 1 to 20, and k’ is an integer from 0 to 10. In some embodiments, wherein k1is selected from 2, 3, 4, 5, 6, 8, 10, 12, 14 and 16, and k’ is selected from 0, 1, 2, 3, 4 and 5.

[0326] In some embodiments, the GlcNAc and the fucosyl group are connected through an α-1, 6-glycosidic bond.

[0327] In some embodiments, in the protein conjugate, the a-terminus is connected to the Fc region of the protein; preferably to the CH2 domain of the Fc region; more preferably to Asn297 (numbered according to the EU index of Kabat) of the Fc region.

[0328] In some embodiments, the protein can be selected from antibodies or antigen-binding fragments thereof, and fusion proteins with Fc regions.

[0329] In some embodiments, the Fc region is an Fc region derived from IgG; preferably, the Fc region is an Fc region derived from IgGl, IgG2, IgG3, or IgG4. For example, the immunoglobulin Fc region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-37.

[0330] In some embodiments, the protein and / or protein conjugate of the present disclosure is a dimer, preferably a homodimer.

[0331] In some embodiments, the average DAR value of the protein conjugate is about 0.5 to 8.0, preferably about 0.5 to 4.0, more preferably about 1.0 to 3.0, even more preferably about 1.5 to 2.5. For example, the average DAR value of the protein conjugate is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0.

[0332] Compound

[0333] In a fourth aspect, the present disclosure also provides a compound as shown in formula VI:

[0334] wherein, PP is a polypeptide having GLP-1R agonistic activity; L’ is a linker peptide, which is located at the C-terminus of PP; and LU is a linking unit as defined in the first aspect.

[0335] Further, the polypeptide having GLP-1R agonistic activity and the linker peptide can refer to the definitions in the first aspect of the present disclosure.

[0336] For example, the PP can comprise or consist of a human GLP-1 variant. In some embodiments, the PP can comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs: 6-14.

[0337] In some embodiments, the linker peptide L’ can comprise or consist of an amino acid sequence as set forth in any one of SEQ ID NOs: 15-20.

[0338] In some embodiments, the aforementioned PP-L’ comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 21-32. The aforementioned PP-L’ can be connected to LU via the amino group on the side chain of the lysine at the C-terminus of the linker peptide.

[0339] In some embodiments, the C-terminus of the aforementioned linker peptide or PP-L’ is carboxylic acid or amide.

[0340] In some embodiments, the compound of Formula VI is selected from:

[0341] wherein, k1 is an integer from 1 to 20, and k' is an integer from 0 to 10. In some embodiments, wherein k1 is selected from 2, 3, 4, 5, 6, 8, 10, 12, 14, and 16, and k' is selected from 0, 1, 2, 3, 4, and 5.

[0342] Compositions

[0343] In a fifth aspect, the present disclosure provides a composition, e.g., preferably a pharmaceutical composition, containing one or a combination of the conjugates of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0344] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers and isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active compound, i.e., the conjugate of the present disclosure, can be coated with a material to protect the conjugate from the action of acids and other natural conditions that can inactivate the conjugate.

[0345] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent (100%), this amount will range from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, in combination with a pharmaceutically acceptable carrier.

[0346] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular conjugate employed or salt thereof, the

[0347] In some embodiments, the DAR value of the protein conjugate compositions of the present disclosure is about 0.5 to 8.0, preferably about 0.5 to 4.0, more preferably about 1.0 to 3.0, even more preferably about 1.5 to 2.5. For example, the DAR value of the protein conjugate compositions is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0.

[0348] The compositions of the present disclosure can be administered using one or more methods known in the art by one or more routes of administration. Those of skill in the art will appreciate that the route and / or mode of administration depends on the desired results. Preferred routes of administration of the conjugates of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes, such as by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0349] The present disclosure also provides a pharmaceutical combination or combination product or kit comprising a conjugate of the present disclosure or a pharmaceutical composition thereof, and one or more other therapeutic agents, such as any therapeutic agent effective in treating and / or preventing a tumor or cancer, such as a cytotoxic agent, a chemotherapeutic agent, a small molecule compound, a vascular inhibiting agent, other immunotherapeutic agents such as other antibodies, or other conjugates, etc.

[0350] The present disclosure refers to a "pharmaceutical combination" or "combination product" including, but not limited to, a kit or pharmaceutical composition. A pharmaceutical combination encompasses both a non-fixed combination and a fixed combination. The term "non-fixed combination" means that the active ingredients are presented in a separate entity and are administered simultaneously, either at the same time or separately without specific time limitations in any order. The term "fixed combination" means that the two or more active ingredients are administered as a single entity. When referring to a "pharmaceutical combination" or "combination product" it also encompasses the case where two active ingredients are present in a separate entity but are packaged or presented together in a single package or kit, for example, in a single vial or syringe.

[0351] DETAILED DESCRIPTION

[0352] The following examples are illustrative of the application and are not limiting thereof. The data (e.g., amounts, temperatures, etc.) given are those found to be the most preferred, but other data (e.g., amounts, temperatures, etc.) can also be used. All temperatures are in degrees Celsius, and all pressures are at or near atmospheric pressure, unless otherwise designated. Except for synthetic intermediates, all reagents and starting materials used in the present application are available from commercial suppliers.

[0353] List of abbreviations used in the following examples: ACN acetonitrile DBCO dibenzocyclooctyne DCC dicyclohexylcarbodiimide DCM dichloromethane DIEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide ESI-MS electrospray ionization mass spectrometry h hour HATU 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HFIP hexafluoroisopropanol HPLC high performance liquid chromatography H2O water HOBt 1-hydroxybenzotriazole HRMS high resolution mass spectrometry LC-MS liquid chromatography-mass spectrometry MeOH methanol mg milligram mL milliliter mM millimolar MMAE monomethyl auristatin E MMAF monomethyl auristatin F mmol millimole MTBE methyl tert-butyl ether NHS N-hydroxysuccinimide PBS phosphate buffered saline RP-HPLC reverse phase high performance liquid chromatography TFA trifluoroacetic acid THPTA tris(3-hydroxypropyltriazolylmethyl)amine Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride TSTU 2-succinimidyl-l, l,3,3-tetramethyluronium tetrafluoroborate μM micromolar

[0354] Synthesis of coupling substrates

[0355] 1.1 GDP-Fuc-PEG4-MMAE (1)

[0356] 1.1.1 Synthesis of Int 1

[0357] Carboxylic acid-tetramethylene glycol-tert-butyl propionate (CAS: 1835759-85-7) (900 mg, 2.57 mmol) and HATU (977 mg, 2.57 mmol) were dissolved in DMF (9 mL), after the solution was stirred, it was cooled to -15 °C, DIEA (663 mg, 5.14 mmol) was added, followed by the addition of propargylamine (CAS: 2450-71-7) (283 mg, 5.14 mmol), and then slowly raised to 10 °C, stirred for 16 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (purification conditions: Xtimate C18, 21.2*250mm, 5μm, 0.1% TFA, ACN in H2O from 55% to 60%), and the preparation liquid was freeze-dried to obtain 1.0 g of colorless oily product Int 1, yield: about 100%. ESI-MS (+) m / z = 388.3 [M+H].

[0358] 1.1.2 Synthesis of Int 2

[0359] Compound Int 1 (1.0 g, 2.57 mmol) was added to DCM (6 mL), and then TFA (2 mL) was added, and then stirred at 10 °C for 16 hours. The reaction solution was purified by high performance liquid chromatography (purification conditions: Xtimate C18, 21.2*250mm, 5μm, 0.1% TFA, ACN in H2O from 25% to 35%), and the preparation liquid was freeze-dried to obtain 760 mg of colorless oily product Int 2, yield: 89.4%. ESI-MS (+) m / z = 332.3 [M+H].

[0360] 1.1.3 Synthesis of Int 3

[0361] Compound Int 2 (100 mg, 0.302 mmol) and HATU (115 mg, 0.302 mmol) were dissolved in DMF (5 mL), after the solution was stirred, DIEA (78 mg, 0.604 mmol) was added, followed by the addition of MMAE (CAS: 474645-27-7) (217 mg, 0.302 mmol), and the reaction was slowly raised to 12 °C, and stirred for 4 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (purification conditions: Ultimate XB-C18, 50*250 mm, 10 μm, 0.1% TFA, ACN in H2O from 35% to 65%), and the preparation liquid was lyophilized to obtain 190 mg of white solid product, yield: 61%. ESI-MS (+) m / z = 1031.6 [M+H].

[0362] 1 H NMR (400 MHz, DMSO) δ 8.60 (t, J = 9.9 Hz, 1H), 8.30 (t, J = 5.2 Hz, 1H), 7.95 - 7.58 (m, 1H), 7.26 (ddd, J = 32.9, 9.5, 4.9 Hz, 5H), 5.39 (dd, J = 28.4, 5.0 Hz, 1H), 4.83 - 4.60 (m, 1H), 4.58 - 4.39 (m, 2H), 4.10 - 3.93 (m, 3H), 3.87 (dd, J = 5.4, 2.5 Hz, 2H), 3.80 (d, J = 9.5 Hz, 1H), 3.71 - 3.58 (m, 5H), 3.57 - 3.42 (m, 13H), 3.33 (s, 1H), 3.26 (d, J = 7.4 Hz, 4H), 3.21 (dd, J = 9.6, 3.6 Hz, 3H), 3.10 (ddd, J = 19.5, 11.8, 5.3 Hz, 3H), 2.98 (dd, J = 11.9, 8.0 Hz, 2H), 2.88 (s, 1H), 2.85 - 2.76 (m, 1H), 2.72 - 2.53 (m, 2H), 2.44 (d, J = 16.6 Hz, 1H), 2.31 (dt, J = 28.0, 7.9 Hz, 3H), 2.16 (dt, J = 21.0, 10.2 Hz, 2H), 2.08 - 1.91 (m, 1H), 1.78 (dd, J = 25.1, 9.9 Hz, 3H), 1.64 - 1.42 (m, 2H), 1.33 (s, 1H), 1.03 (ddd, J = 14.8, 12.0, 6.6 Hz, 7H), 0.95 - 0.73 (m, 18H).

[0363] 1.1.4 Synthesis of GDP-Fuc-PEG4-MMAE (1)

[0364] Compound Int 3 (100 mg, 0.097 mmol) was dissolved in MeOH-H2O (1 :5, 10 mL) mixed solvent, and GDP-6-N3-fucose (100 mM, 1.2 mL) was added. To the reaction mixture, a solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added, and the reaction was incubated at 37 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated under vacuum, and the residue was purified by HPLC to obtain 105 mg of the target compound 1, with a yield of 65.2%. HRMS (ESI): m / z calcd for C 70 H 113 N 14 O 28 P2[M-H]-1659.7329; found 1659.7331.

[0365] 1 H NMR (600 MHz, D2O) δ 8.09 (s, 1H), 7.98 - 7.93 (m, 1H), 7.43 - 7.22 (m, 5H), 5.89 (d, J = 5.7 Hz, 1H), 4.93 - 4.88 (m, 1H), 4.69 - 4.63 (m, 2H), 4.61 - 4.44 (m, 7H), 4.39 - 4.31 (m, 2H), 4.27 - 4.07 (m, 4H), 4.02 - 3.96 (m, 1H), 3.86 - 3.76 (m, 6H), 3.72 - 3.58 (m, 16H), 3.51 (dd, J = 15.2, 7.4 Hz, 1H), 3.46 - 3.31 (m, 7H), 3.25 - 3.22 (m, 1H), 3.17 - 2.97 (m, 5H), 2.83 - 2.73 (m, 2H), 2.59 - 2.48 (m, 3H), 2.38 - 2.02 (m, 4H), 1.90 - 1.78 (m, 2H), 1.68 - 1.50 (m, 2H), 1.42 - 1.22 (m, 5H), 1.20 - 1.09 (m, 3H), 1.02 - 0.79 (m, 18H).

[0366] 1.2 GDP-Fuc-PEG4-MMAF (2)

[0367] 1.2.1 Synthesis of Int 4

[0368] Compound Int 2 (400 mg, 1.21 mmol) and TSTU (364 mg, 1.21 mmol) were dissolved in DMF (6 mL), after the solution was stirred, DIEA (75 mg, 0.585 mmol) was added, and the reaction was stirred at 15 °C for 2 h. After the reaction was completed, the reaction solution was directly used for the next step without purification. ESI-MS (+) m / z = 429.2 [M+H].

[0369] 1.2.2 Synthesis of Int 5

[0370] To the reaction mixture obtained in the previous step, MMAF (CAS: 745017-94-1) (442 mg, 0.605 mmol) was added, and heated to 50 °C for 24 h. LC-MS showed that the starting material remained, and about 30% of the product was generated. The reaction was stopped, and the reaction solution was purified by high performance liquid chromatography (purification conditions: Ultimate XB-C18, 50*250 mm, 10 μm, 0.1% TFA, ACN in H2O from 40% to 70%), and the preparation liquid was lyophilized to obtain 107 mg of light yellow solid product, yield: 8.6%. ESI-MS (+) m / z = 1045.6 [M+H].

[0371] 1 H NMR (400 MHz, DMSO) δ 8.45 (dt, J = 19.8, 8.0 Hz, 1H), 8.28 (t, J = 5.1 Hz, 1H), 8.16 - 7.62 (m, 1H), 7.29 - 7.07 (m, 5H), 4.81 - 4.36 (m, 4H), 4.01 - 3.98 (m, 1H), 3.85 (dd, J = 5.4, 2.5 Hz, 2H), 3.73 (t, J = 10.6 Hz, 1H), 3.67 - 3.55 (m, 4H), 3.54 - 3.36 (m, 13H), 3.36 - 3.13 (m, 8H), 3.13 - 2.90 (m, 3H), 2.88 - 2.74 (m, 3H), 2.66 (td, J = 12.5, 5.1 Hz, 1H), 2.60 - 2.51 (m, 1H), 2.45 - 2.28 (m, 3H), 2.29 - 1.93 (m, 4H), 1.87 - 1.55 (m, 3H), 1.51 - 1.15 (m, 3H), 1.04 (dd, J = 13.5, 6.7 Hz, 3H), 0.99 - 0.61 (m, 19H).

[0372] 1.2.3 Synthesis of GDP-Fuc-PEG4-MMAF (2)

[0373] Compound Int 5 (100 mg, 0.096 mmol) was dissolved in a MeOH-H2O (1:5, 10 mL) mixed solution, and GDP-6-N3-fucose (100 mM, 1.2 mL) was added. To this reaction mixture, a solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added, and the reaction was incubated at 37 °C for 3 hours. After the reaction was completed, the reaction was concentrated under vacuum, and the residue was purified by HPLC. 103 mg of the target compound 2 was obtained, with a yield of 64.1%. HRMS (ESI): m / z calcd for C 70 H 111 N 14 O 29 P2[M-H]-1673.7122; found 1673.7123.

[0374] 1 H NMR (600 MHz, D20) δ 8.14 - 7.92 (m, 2H), 7.37 - 7.17 (m, 5H), 5.89 (d, J = 5.9 Hz, 1H), 4.92 - 4.87 (m, 1H), 4.73 - 4.63 (m, 4H), 4.61 - 4.50 (m, 4H), 4.48 - 4.44 (m, 2H), 4.36 - 4.31 (m, 1H), 4.24 - 4.16 (m, 2H), 4.13 - 4.07 (m, 1H), 3.99 (dd, J = 7.2, 4.9 Hz, 1H), 3.85 - 3.75 (m, 6H), 3.73 (dd, J = 9.6, 1.5 Hz, 1H), 3.69 - 3.63 (m, 14H), 3.58 (t, J = 6.1 Hz, 1H), 3.50 (dt, J = 9.8, 6.9 Hz, 1H), 3.43 - 3.30 (m, 8H), 3.20 - 3.12 (m, 3H), 3.09 (d, J = 10.5 Hz, 3H), 2.92 - 2.72 (m, 3H), 2.60 - 2.47 (m, 3H), 2.35 - 2.04 (m, 5H), 1.91 - 1.79 (m, 2H), 1.76 - 1.55 (m, 2H), 1.51 - 1.28 (m, 2H), 1.17 (dd, J = 30.3, 6.7 Hz, 3H), 1.03 - 0.83 (m, 18H).

[0375] 1.3 GDP-Fuc-PEG4-GGFG-Dxd (3)

[0376] 1.3.1 Synthesis of Int 6

[0377] To a 50 mL three-necked flask was added propionic acid-tetraglycol-succinimidyl ester (CAS: 2639395-41-6) (200 mg, 0.51 mmol) at room temperature, after dissolved in DMF (15 mL), GGFG-Dxd (450 mg, 0.51 mmol) was added. The reaction was cooled to 0 °C, DIEA (131.8 mg, 1.02 mmol) was added slowly dropwise, the reaction was stirred at 0 °C for 0.5 h, LC-MS showed the reaction was complete. After that, the reaction solution was added acetic acid (0.5 mL) and purified by high performance liquid chromatography (purification conditions: Xtimate C18, 50*250 mm, 10 um, 0.1% TFA, CAN in H2O form 25% to 55%), the preparation liquid was lyophilized to give 242 mg of yellow solid compound Int 6, yield 42.53%. ESI-MS (+) m / z = 1117.3 [M+H].

[0378] 1 H NMR (400 MHz, DMSO) δ 8.65 (t, J = 6.4 Hz, 1H), 8.52 (d, J = 8.8 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.18 (t, J = 5.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.02 (t, J = 5.6 Hz, 1H), 7.77 (d, J = 11.2 Hz, 1H), 7.32 (s, 1H), 7.29 - 7.18 (m, 5H), 5.61 (dd, J = 13.6, 5.6 Hz, 1H), 5.48 - 5.38 (m, 2H), 5.25 - 5.13 (m, 2H), 4.66 (d, J = 6.4 Hz, 2H), 4.49 (td, J = 8.8, 4.4 Hz, 1H), 4.04 (s, 2H), 3.80 - 3.69 (m, 6H), 3.66 - 3.57 (m, 8H), 3.29 - 2.99 (m, 4H), 2.79 (dd, J = 13.6, 9.6 Hz, 1H), 2.45 (t, J = 6.4 Hz, 2H), 2.42 - 2.36 (m, 5H), 2.30 - 2.21 (m, 2H), 1.95 - 1.79 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).

[0379] 1.3.2 Synthesis of Int 7

[0380] Compound Int 6 (48.0 mg, 0.043 mmol) was dissolved in DCM, and NHS (1.20 eq) and DCC (1.20 eq) were added. The mixture was stirred at room temperature under N2atmosphere for 30 min, then propargylamine (1.05 eq) was added. The resulting mixture was stirred at room temperature for another 12 h, and concentrated under reduced pressure. The residue was purified by reverse phase column chromatography to give 30.0 mg of Int 7 as yellow oil in 60.5% yield. The product was used directly for the next step.

[0381] 1.3.3 Synthesis of GDP-Fuc-PEG4-GGFG-Dxd (3)

[0382] The target compound 3 was synthesized by copper-catalyzed click reaction of Int 7 (30.0 mg, 0.026 mmol) with GDP-6-N3-fucose (17.0 mg, 0.027 mmol) in the presence of CuS04 / BTTP solution (1 mM / 2 mM in H20, 1 mL) and sodium ascorbate solution (2 mM in H20, 1 mL). After mixing with HEPES buffer (1 mL) at room temperature for 6 h, the reaction was characterized by MS. The reaction solution was directly purified by reverse phase column chromatography, then by preparative HPLC to give the target compound 3 1.0 mg in 2.2% yield. ESI-MS (+) m / z = 1783.4 [M+H].

[0383] 1.4 GDP-Fuc-VC-PABc-MMAE (4)

[0384] Compound Int 8 was prepared according to the method in the published patent WO2012153193.

[0385] Int 8 (50 mg, 0.038 mmol) was dissolved in a mixture of methanol-water (1 :3, 4 mL), and GDP-6-N3-fucose (100 nM, 0.46 mL) was added. To this reaction mixture was added Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) solution, and reacted at 37 °C for 4 h. After completion of the reaction, it was concentrated under vacuum, and the residue was purified by P2 column and C18 column to give 9.2 mg of the target compound 4 in 12.5% yield. HRMS (ESI): m / z calcd for C 83 H 132 N 18 O 31 P2[M+2H] 2+ 969.4385; found 969.4387.

[0386] 1 H NMR (400 MHz, D20) δ 8.13 - 8.03 (m, 2H), 7.48 - 7.20 (m, 9H), 5.86 (d, J = 5.9 Hz, 1H), 5.32 - 5.16 (m, 1H), 5.09 - 4.99 (m, 1H), 4.90 (t, J = 7.0 Hz, 1H), 4.71 - 4.29 (m, 12H), 4.25 - 4.05 (m, 8H), 4.03 - 3.97 (m, 1H), 3.81 - 3.62 (m, 12H), 3.52 - 3.04 (m, 14H), 2.98 - 2.89 (m, 3H), 2.57 - 2.30 (m, 2H), 2.22 - 2.02 (m, 3H), 1.90 - 1.77 (m, 4H), 1.68 - 1.48 (m, 4H), 1.31 (d, J = 6.4 Hz, 3H), 1.18 (t, J = 7.6 Hz, 3H), 1.12 - 0.78 (m, 24H), 0.69 (dd, J = 15.9, 6.3 Hz, 2H), 0.53 - 0.46 (m, 1H).

[0387] Example 2 Preparation of antibody-fucose-toxin molecular conjugates

[0388] 2.1 ADC preparation

[0389] Alfc pretreated antibody MEHD (Duligotuzumab amino acid sequence published in Recommended INN: List 72, WHO Drug Information, Vol. 28, No. 3, 2014), conjugation substrate Cpd1-4, Fut8 (SEQ ID NO: 3), MnCl2 were mixed into Tris-HCl buffer solution, pH value between 7.50±0.1, reacted overnight at 37°C, to obtain the corresponding ADC samples MEHD-fucose-P4-MMAE, MEHD-fucose-P4-MMAF, MEHD-fucose-P4-GGFG-Dxd and MEHD-fucose-VC-PABc-MMAE.

[0390] 2.2 ADC characterization

[0391] SEC detection: after diluting the sample with phosphate solution, the sample was separated by isocratic separation with TOSOH G3000 SWxl column, and the sample purity was calculated by peak area normalization method at 280 nm wavelength;

[0392] DAR value detection: the sample was diluted with ultrapure water, separated by a waters / ACQUITY UPLC Protein BEH C4 column, and the molecular weight was detected by retention time and peak area at a wavelength of 280 nm. The number of conjugations was calculated, the percentage content of each component was calculated according to the percentage content formula, and the DAR value was calculated according to the content of each component and the number of conjugations;

[0393] The SEC purity and DAR value results are shown in Table 1. The ADC sample purity is above 97%, and the DAR value is above 1.6.

[0394] Table 1. SEC and DAR value results

[0395] Example 3 Synthesis of GLP-1 variant-DBCO conjugate

[0396] The following GLP-1 variant was prepared using solid phase synthesis method:

[0397] HAibEGTFTSDVSSALEEQAAREFIAYLVKGGGGGGGSGGGGSGGGGSK-NH2 (hereinafter referred to as P001)

[0398] (1) Weigh the Rink Amide Resin into the synthesis column, wash twice with DMF, then add DCM to swell for 30 min, then remove the DCM by suction filtration and wash twice with DMF.

[0399] (2) Add 20% piperidine / DMF solution to deprotect, then wash the resin with DMF.

[0400] (3) After removing the DMF by suction filtration, add Fmoc-Lys(Mtt)-OH / DIC / HOBT mixed DMF solution for condensation reaction, and monitor the reaction by ninhydrin colorimetry until the reaction endpoint.

[0401] (4) After DMF washing, suction filtration, the resin containing protected amino acids is obtained.

[0402] (5) Repeat steps (2)-(4), condense amino acids from the C-terminal end in order until all amino acids are condensed. The raw materials corresponding to each amino acid are as follows:

[0403] (6) After washing the resin with DMF, add HFIP / DCM mixed solution to remove the C-terminal Lys side chain protecting group. After washing with DMF, mix the DBCO-carboxylic acid (CAS: 1353016-70-2) activated with condensation reagent NHS / DCC in advance with the resin, and after the reaction is complete, wash with DMF / DCM.

[0404] (7) After the resin is dried, pre-chilled lysis reagent containing TFA is added, and stirring is performed for 2-3 hours.

[0405] (8) After the reaction is completed, the resin is filtered out, and cold MTBE is added to the solution to perform precipitation, and a crude product is obtained by suction filtration.

[0406] (9) The crude product is purified by RP-HPLC and lyophilized to obtain a final product. The product has a purity of 98.4%, a theoretical molecular weight of 4615.92±2.0, and a measured molecular weight of 4614.8 (ESI-MS (+) m / z = 1154.7 [M+4H]4+).

[0407] Using a similar method, the following GLP-1 variants were prepared:

[0408] HAibEGTFTSDVSSALEEQAAREFIAYLVKGGGGGGGQGGGGQGGGGQK-NH2 (hereinafter referred to as P002). The product has a purity of 95.2%, a theoretical molecular weight of 4739.18±2.0, and a measured molecular weight of 4738.0 (ESI-MS (+) m / z = 1185.5 [M+4H]4+).

[0409] Example 4 Preparation of antibody-fucose-polypeptide conjugate

[0410] An antibody-fucose-polypeptide conjugate was prepared using the flowchart shown in Method A (Fig. la) or Method B (Fig. lb).

[0411] Method A

[0412] (A1) Synthesis of GDP-fucose-AM-PEG4-N3

[0413] A solution of GDP-6NH2-fucose (200 mM, 650 μL) in 2 mL of H2O was dissolved with NaHCO3buffer (200 mM, 500 μL), and NHS-PEG4-N3 (100 mM, 2 mL) was added. The reaction mixture was stirred at room temperature for 5 hours and was monitored by TLC. After the reaction was completed, the solution was concentrated under reduced pressure, and the crude product was concentrated and purified using a P2 column and an ion exchange column, and finally 41 mg of GDP-fucose-AM-PEG4-N3 was obtained, with a yield of 60%. 1H NMR (600 MHz, D20) δ 8.13 (s, 1H), 5.94 (d, J = 6.2 Hz, 1H), 4.94 (t, J = 7.9 Hz, 1H), 4.83 (m, 1H), 4.55 (dd, J = 4.7, 3.5 Hz, 1H), 4.36 (s, 1H), 4.26 - 4.21 (m, 2H), 3.88 (d, J = 2.8 Hz, 1H), 3.78 (t, J = 6.2 Hz, 2H), 3.74 - 3.66 (m, 16H), 3.64 - 3.57 (m, 2H), 3.53 - 3.48 (m, 2H), 3.31 (dd, J = 14.0, 8.6 Hz, 1H), 2.57 (t, J = 6.2 Hz, 2H); HRMS (ESI): m / z calcd for C 27 H 46 N9O 20 P2[M+H] + 878.2329; found 878.2328.

[0414] (A2) Synthesis of GDP-fucose-AM-PEG4-DBCO-polypeptide (5)

[0415] To 312 μL of P002 polypeptide-DBCO conjugate (0.74 mg, dissolved in PBS to 500 μM) was added GDP-fucose-AM-PEG4-N3 (0.55 mg, dissolved in water to 7.1 mM). The reaction was incubated at 25 °C, protected from light, with shaking for about 16 h, after which the reaction was exchanged into 25 mM Tris-HCl, pH 7.5 using a 3KD ultrafiltration tube, and the excess substrate was removed by multiple exchanges to give GDP-fucose-AM-PEG4-DBCO-P002 0.59 mg. ESI-MS (+) m / z = 5616.69 [M+H]).

[0416] (A3) Preparation of antibody-fucose-P002 conjugate

[0417] Full-length antibody A (1 mg) pre-treated with Alfc, which has a heavy chain constant region sequence shown in SEQ ID NO: 41, was incubated with GDP-fucose-AM-PEG4-DBCO-P002 (0.2 mg), and FUT8 (SEQ ID NO: 3, 0.04 mg) in 25 mM Tris-HCl (pH 7.5) buffer containing 10 mM MnCl2at 30°C for about 16 h in the dark. The reaction mixture was purified by using protein A resin, and the antibody-fucose-poly peptide conjugate was obtained by buffer exchange. Mass spectrometry analysis showed that the average DAR of all compositions of antibody-fucose-P002 conjugate was 1.9-2.0 (Fig. 2a and Fig. 2b), and more than 90% of the conjugate had a DAR of 2.

[0418] Method B

[0419] (B1) Preparation of antibody-fucose-Az conjugate

[0420] To full-length antibody A (3.23 mg) pre-treated with Alfc, GDP-fucose-N3 (0.11 mg, dissolved in 50 mM water for injection) and FUT8 (0.12 mg) were added, and incubated in 25 mM Tris-HCl (pH 7.5) buffer containing 10 mM MnCl2at 30°C for about 16 h in the dark. The reaction mixture was purified by using protein A resin, and the antibody-fucose-Az conjugate was obtained by buffer exchange. Mass spectrometry analysis showed that more than 90% of antibody A had been converted into antibody-fucose-Az conjugate (Fig. 3a).

[0421] (B2) Preparation of antibody-fucose-poly peptide conjugate

[0422] P001 poly peptide (0.46 mg, dissolved in 500 μM PBS) was incubated with antibody-fucose-Az conjugate (1.94 mg, dissolved in 68 μM PBS) obtained from (B1) in PBS (pH 7.4) buffer at 25°C for about 16 h in the dark with shaking. The excess poly peptide was removed by using 30KD ultrafiltration tube for buffer exchange, and the antibody-fucose-P001 conjugate was obtained. Mass spectrometry analysis showed that the average DAR of all compositions of antibody-poly peptide conjugate was 1.9-2.0 (Fig. 3b), and more than 90% of the conjugate had a DAR of 2.

[0423] Example 5 Biological activity detection

[0424] (1) Killing effect of antibody-fucose-toxin molecule conjugate on tumor cells

[0425] Three types of tumor cells—HCC827 (purchased from Nanjing Kebai Biotechnology Co., Ltd., growth medium: RPMI-1640 + 10% FBS), MDA-MB-468 (purchased from Beina Chuanglian Biotechnology Co., Ltd., growth medium: Leibovitz's L-15 + 10% FBS), and A431 (purchased from Beina Chuanglian Biotechnology Co., Ltd., growth medium: DMEM + 10% FBS)—were digested with trypsin. Cells were collected in centrifuge tubes, resuspended in the corresponding growth medium, and 2000–5000 cells / well were added to 96-well plates for overnight culture. The next day, MEHD-fucose-VC-PABC-MMAE or antibody were serially diluted with the corresponding growth medium, and 50 μL of each diluted sample was added to the 96-well cell culture plate. The plates were incubated at 37°C and 5% CO2 for 96 hours. The cell culture plates were then removed, and cells were added to each well. The Luminescent Cell Viability Assay (Promega, G7571) reagent (50 μL) was used to detect fluorescence intensity and calculate the killing ability of ADC or antibody against tumor cells. The results are shown in Table 2 and Figures 4a-4c. MEHD-fucose-VC-PABC-MMAE showed a significant killing effect on tumor cells.

[0426] Table 2. Killing effect of cleavable ADCs on tumor cells

[0427] The cytotoxic effects of the ADC MEHD-fucose-P4-MMAF on HCC827 and MDA-MB-468 were detected using a similar method, with the unconjugated antibody MEHD and the substrate GDP-Fuc-PEG4-MMAF (Cpd 2) serving as controls. The results are shown in Table 3 and Figures 5a-5b, indicating that MEHD-fucose-P4-MMAF exhibited significant antitumor activity.

[0428] Table 3. Killing effect of non-cleavage ADCs on tumor cells

[0429] *ND indicates that no data was fitted.

[0430] (2) GLP-1R agonistic activity of antibody-fucose-peptide conjugate

[0431] The samples were prepared with experimental medium, 5-fold dilution, 8 concentrations, 50 μL of the above-mentioned drug-containing medium was added to each well of a 96-well white plate. The HEK293-GLP1R-CREB cells (HEK293 cells expressing human GLP1R protein on the cell membrane, with CREB response element protein) were collected, the cell density was adjusted to 5*10e5 cells / ml, 50 μL of cell solution was added to each well, and the cells were cultured at 37°C for 6±0.5 h. 50 μL of Luciferase substrate was added to each well, and the RLU value was immediately detected on the enzyme marker. The positive control used a GLP-1-Fc fusion protein (SEQ ID NO: 40) with a similar mutation site as P001, and the results are shown in FIG. 6. The fucose-polypeptide conjugate of the present disclosure has better GLP-1R agonist activity.

[0432] Example 6 Influence of wild-type Fc and its variants on conjugation efficiency

[0433] To confirm whether different Fc would affect the Fut8 conjugation efficiency, we used a method similar to Example 2 to conjugate compound 2 with antibody B (binding to tumor antigen, IgG1 subtype, Fc region with the amino acid sequence shown in SEQ ID NO: 34, pre-treated with Alfc) or its Fc variants (AG / AA / GAA), and compared the conjugation efficiency of wild-type Fc and its variants by determining the average DAR value and distribution of the conjugation product. The results are shown in Table 4.

[0434] Table 4. Conjugation efficiency of wild-type Fc and its variants

[0435] It can be seen that when the conjugation reaction time is short, the conjugation product with wild-type Fc or its AA / GAA variant contains a higher proportion of unreacted DAR1 molecules, resulting in a lower overall DAR value, while for the AG variant, the proportion of DAR2 molecules and the overall DAR value are both higher, indicating that its conjugation efficiency is superior to other groups. When the reaction time is extended to overnight, the DAR values of various conjugation products with wild-type Fc or its variants are all higher, indicating that they can all be fully conjugated.

[0436] Sequence Listing

Claims

1. A method for preparing a protein conjugate, comprising the steps of: contacting Nu-Fuc* with a protein containing a glycan chain in the presence of a catalyst to obtain a protein conjugate; in, Nu contains ribonucleotides; Fuc* is a fucose derivative that contains bioactive molecules; The sugar chain is an N-glycan lacking a core fucose; The protein conjugate comprises the structure of formula I: GlcNAc is the core N-acetylglucosamine, with its a-terminus linked to the protein and its b-terminus linked to the distal glycosyl group of the N-glycan chain.

2. The method according to claim 1, wherein the catalyst is a fucosylation enzyme or a functional variant or fragment thereof.

3. The method according to claim 1 or 2, wherein the catalyst is α-1,6-fucosyltransferase or a functional variant or fragment thereof; preferably, the catalyst is human-derived α-1,6-fucosyltransferase or a functional variant or fragment thereof.

4. The method according to any one of claims 1-3, wherein the catalyst is FUT8 or a functional variant or fragment thereof.

5. The method according to any one of claims 1-4, wherein the catalyst comprises the amino acid sequence shown in any one of SEQ ID NO: 1-3.

6. The method according to any one of claims 1-5, wherein the Nu comprises ribonucleotide diphosphate or a salt thereof; preferably, the Nu comprises a structure selected from guanosine diphosphate (GDP), uridine diphosphate (UDP), cytidine diphosphate (CDP) and adenosine diphosphate (ADP), or a salt thereof; more preferably, the Nu comprises guanosine diphosphate (GDP) or a salt thereof.

7. The method according to any one of claims 1-6, wherein the Fuc* has the structure of formula II: in, BM is a bioactive molecule, and LU is a linker unit that connects the bioactive molecule to the fucose group.

8. The method according to any one of claims 1-7, wherein the Nu-Fuc* has a structure of formula II-A: in, BM and LU are as defined in claim 7.

9. The method according to any one of claims 1-8, wherein the protein conjugate comprises a structure of formula II-B: in, BM and LU are as defined in claim 7; GlcNAc is a core N-acetylglucosamine, with its a-terminus linked to a protein and its b-terminus linked to the distal glycosyl group of the N-glycan chain.

10. The method according to claim 9, wherein, GlcNAc is linked to fucose via an α-1,6-glycosidic bond.

11. The method according to any one of claims 7-10, wherein the connecting unit LU further has the structure shown in Formula III: in, L1 is the extension group, L2 is the first spacer group, L3 is absent or is a linking group, and L4 is absent or is the second spacer group.

12. The method of claim 11, wherein L1 is selected from: in, m is an integer selected from 0 to 10, and n is an integer selected from 0 to 20.

13. The method according to any one of claims 11-12, wherein the structure of L2 is as shown in Formula IV: in, a1 = 0 or 1, a2 = 0 or 1, a3 = integers from 0 to 8, b1 = 0 or 1, b2 = integers from 0 to 16, b3 = integers from 0 to 16, c = integers from 0 to 8, and at least one of b2 and b3 is 0.

14. The method according to claim 13, wherein: (1) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0; (2) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 1, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0; (3) a1 = 1, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0; (4) a1 = 1, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0; (5) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0; (6) a1 = 1, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0; (7) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0; (8) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0; (9) a1 = 0, a2 = 0, a3 = 0, b1 = 0, b2 = 0, b3 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, c = 0, 1, 2, 3, 4, 5 or 6; and (10) a1 = 0, a2 = 0, a3 = 0, b1 = 1, b2 = 0, b3 = 0, c = 0, 1, 2, 3, 4, 5, 6, 7 or 8.

15. The method according to any one of claims 11-14, wherein L3 is absent or is a peptide linker.

16. The method according to any one of claims 11-15, wherein L3 is selected from peptide linkers capable of being cleaved by cathepsins.

17. The method according to any one of claims 11-16, wherein L3 is a diamino acid peptide, a triamino acid peptide, or a tetraamino acid peptide residue.

18. The method of claim 17, wherein L3 is selected from the following diamino acid peptide residues: -Lys-Phe-, -Ala-Val-, -Lys-Val-, -Cit-Val-, -Lys-Ala-, -Cit-Phe-, -Cit-Leu-, -Cit-Ile-, -Arg-Phe-, -Cit-Trp-, -Gly-Gly-, -Ala-Ala-, -Val-Gly-, and -Glu-Gly-; the left side of the diamino acid peptide residue is connected to L4, and the right side is connected to L2.

19. The method of claim 17, wherein L3 is selected from the following triamino acid peptide residues: -Ala-Val-Glu-, -Cit-Val-Glu-, -Ala-Val-αGlu-, -Cit-Val-αGlu-, -Gly-Lys-Val-, and -Gly-Cit-Val-; the left side of the triamino acid peptide residue is connected to L4, and the right side is connected to L2.

20. The method of claim 17, wherein L3 is selected from the following four amino acid peptide residues: -Gly-Phe-Gly-Gly- and -Gly-Gly-Phe-Gly-; the left side of the four amino acid peptide residue is connected to L4 and the right side is connected to L2.

21. The method according to any one of claims 11-20, wherein L4 is absent, or is selected from: in, R1 is independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, halogen, nitro, cyano d is an integer from 1 to 20, e is an integer from 1 to 20; R2 is selected from hydrogen and C. 1-6 alkyl.

22. The method according to any one of claims 11-21, wherein L4 is absent, or is selected from:

23. The method according to any one of claims 7-22, wherein LU is selected from: in, k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10.

24. The method according to any one of claims 1-23, wherein the bioactive molecule is selected from small molecule drugs, radioactive isotopes and their chelates, nucleic acids, polypeptides and antibodies or their antigen-binding fragments.

25. The method of claim 24, wherein the bioactive molecule is selected from small molecule drugs, for example, drugs with a molecular weight of less than 1,000 Daltons.

26. The method according to claim 24, wherein the bioactive molecule is selected from polypeptides, for example, peptides selected from the condensation of 5-50 amino acids.

27. The method according to any one of claims 1-24, wherein the structure of Fuc* is selected from: in, k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

28. The method according to any one of claims 1-24, wherein the structure of Fuc* is selected from: in, k1 is an integer from 1 to 20, and k' is an integer from 0 to 10.

29. The method according to any one of claims 1-25, wherein, In the structures of Formula I and Formula II-B, the a-terminus is connected to the Fc region of the protein; preferably, it is connected to the CH2 domain of the Fc region; more preferably, it is connected to Asn297 of the Fc region (according to the EU index number of Kabat).

30. A method for preparing protein conjugates, comprising the following steps: (A) In the presence of a catalyst, Nu-Fuc** is contacted with a protein containing glycans to obtain a protein conjugate precursor; (B) The protein conjugate precursor is contacted with the target molecule to obtain the protein conjugate; in, Nu contains ribonucleotides; Fuc** is a reactive fucose variant containing a chemically active group X; the glycan is an N-glycan lacking a core fucose. The target molecule comprises a bioactive molecule and a chemically active group Y; The protein conjugate comprises the structure of formula I: The protein conjugate precursor includes the structure of formula I': Among them, Fuc* is a fucose derivative containing bioactive molecules, and GlcNAc is the core N-acetylglucosamine, whose a-terminus is linked to the protein and its b-terminus is linked to the distal glycosyl group of the N-glycan chain; and, Fuc** is not:

31. The method of claim 30, wherein the catalyst is as defined in any one of claims 2-5, the Nu is as defined in claim 6, and the Fuc* and protein conjugate are as defined in any one of claims 7 and 9-28.

32. The method according to claim 30 or 31, wherein the structure of the Fuc** is as shown in formula II':

33. The method according to any one of claims 30-32, wherein the chemically active group X is selected from the following structures: in, p is an integer between 0 and 20.

34. The method according to any one of claims 30-33, wherein the Fuc** is selected from the following structures: in, p is an integer between 0 and 20.

35. The method according to any one of claims 30-34, wherein the target molecule has the structure shown in formula V: BM-L4-L3-L2-Y(V); where, L2 is the first spacer group, L3 is absent or is a linker group, L4 is absent or is the second spacer group, and BM is a bioactive molecule. Preferably, L2 is the first spacer group of claim 13 or 14, L3 is absent or is the linking group of any one of claims 15-20, L4 is absent or is the second spacer group of claim 21 or 22, and BM is the bioactive molecule of any one of claims 24-26.

36. The method according to any one of claims 30-35, wherein the chemically active group Y is selected from the following structures:

37. The method according to claim 35 or 36, wherein L4-L3-L2-Y in formula V has a structure selected from the following: in, k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10.

38. The method according to any one of claims 30-37, wherein the target molecule is selected from the following structures: in, k1 is an integer from 1 to 20, and k' is an integer from 0 to 10.

39. The method according to any one of claims 30-37, wherein the target molecule is selected from the following structures: in, k' is an integer from 0 to 10.

40. Protein conjugates containing the structure of formula II-B: in, BM is a bioactive molecule, LU is a linker unit that connects the bioactive molecule to the fucose group, and GlcNAc is the core N-acetylglucosamine, whose a-terminus is linked to the protein and its b-terminus is linked to the distal glycosyl group of the N-glycan chain. Furthermore, the BM-LU does not have the following structure:

41. The protein conjugate according to claim 40, wherein, GlcNAc is linked to fucose via an α-1,6-glycosidic bond.

42. The protein conjugate according to claim 40 or 41, wherein the linker LU further has the structure shown in Formula III: in, L1 is the extension group as described in claim 12, L2 is the first spacer group as described in claim 13 or 14, L3 is absent or is the linking group as described in any one of claims 15-20, and L4 is absent or is the second spacer group as described in claim 21 or 22.

43. The protein conjugate according to any one of claims 40-42, wherein LU is selected from: in, k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10.

44. The protein conjugate according to any one of claims 40-43, wherein the bioactive molecule is selected from small molecule drugs, radioactive isotopes and their chelates, nucleic acids, polypeptides and antibodies or their antigen-binding fragments.

45. The protein conjugate according to any one of claims 40-44, comprising a structure selected from: in, k1 is an integer from 1 to 20, and k' is an integer from 0 to 10.

46. ​​The protein conjugate according to any one of claims 40-45, wherein, The a-terminus is attached to the Fc region of the protein; preferably to the CH2 domain of the Fc region; more preferably to Asn297 of the Fc region (according to the Kabat EU index number).

47. The protein conjugate according to any one of claims 40-46, wherein the protein comprises an antibody or an antigen-binding fragment thereof.

48. A pharmaceutical composition comprising the protein conjugate of any one of claims 40-47; preferably, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

49. The pharmaceutical composition according to claim 48, wherein the DAR value of the protein conjugate in the pharmaceutical composition is about 0.5-4.0, preferably about 1.0-3.0, and more preferably about 1.5-2.5.

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