Protein-glycan conjugate having multiple payloads

Abstract

A protein-glycan conjugate having multiple payloads. By means of a site-specific glycan conjugation technique, different types of payloads are conjugated to specific positions of a protein-glycan chain, so as to obtain a conjugate having multiple payloads. The conjugate exhibits good biological effects.

Description

Protein glycosyl conjugates with multiple payloads

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024116124648, filed on November 12, 2024, and Chinese Patent Application No. 2025105176747, filed on April 23, 2025. Technical Field

[0003] This application relates to the field of biomedicine, specifically to a protein glycosyl conjugate and its preparation method. Background Technology

[0004] Antibody-drug conjugates (ADCs), which link antibodies to other bioactive substances (such as toxin molecules) via linkers, can simultaneously exert the high targeting specificity of antibodies and the therapeutic effects of bioactive substances. As a representative example, more than a dozen ADCs have been approved for marketing in the treatment of cancer, with many more conjugate molecules in clinical or preclinical research stages.

[0005] Tumor heterogeneity and drug resistance are significant challenges when using ADC therapy. Existing research indicates that tumors contain a heterogeneous mixture of cell populations, each with varying levels of treatment resistance. Under treatment selection pressure, resistance can increase due to the expansion of pre-existing subclonal populations or the evolution of treatment-resistant cells. For this reason, almost all effective small-molecule chemotherapy regimens involve the co-delivery of two or more complementary drugs with different mechanisms of action. This approach not only minimizes the development of resistant cell populations but also provides additional, or even synergistic, anti-cancer effects. To date, research on combination therapies using ADCs remains limited, and many current ADC-based treatments exhibit acquired resistance, thus failing to provide durable responses.

[0006] In contrast to combination chemotherapy and antibody-drug conjugate (ADC) therapy, multiple-load ADCs (antibody-drug conjugates) – drugs that simultaneously conjugate multiple cytotoxic substances or other active ingredients – are attracting increasing attention. These molecules can ensure the precise and controllable simultaneous delivery of multiple payloads to a single cell. To counter this treatment strategy, tumor cells must simultaneously develop resistance mechanisms to multiple drugs, which is unlikely.

[0007] Several multiple payload ADC schemes have been reported in the literature. For example, Levengood et al. (Angew. Chem. Int. Ed. 2017, 56, 733-737) disclosed a method for constructing dual payload ADCs using antibody thiol groups and subsequently introduced thiol-containing side chains. The resulting ADC molecules were coupled with two different toxin molecules, MMAE and MMAF, and achieved certain results in a heterogeneous model. However, the coupling between thiol and maleimide is not stable and a reverse Michael reaction occurs in the blood, causing the toxin molecules to detach and resulting in peripheral toxicity. Patent application WO2023065137 discloses a method for constructing dual-load antibody-drug conjugates using α-1,3-fucosyltransferase. However, this method has stringent requirements on the substrate glycoform (e.g., N-acetylglucosamine as the substrate must be pre-linked with galactose or its derivative), requiring additional modification. Furthermore, the structure of the resulting product differs significantly from the native glycoform of the antibody. Moreover, in the resulting ADC, different loads attach to adjacent monosaccharides, resulting in close proximity. Given the typically strong hydrophobicity of load molecules, their tight binding leads to the concentration of hydrophobic regions on the protein, and non-covalent stacking interactions may occur between adjacent loads. This increases aggregation and consequently, the clearance rate of the ADC in vivo, ultimately reducing efficacy and safety. Therefore, it is necessary to explore new methods for constructing multi-load protein conjugates to address the issues of tumor heterogeneity and drug resistance. Summary of the Invention

[0008] In a first aspect, this disclosure relates to a protein conjugate comprising a protein and a glycan, said glycan comprising the structure of formula I:

[0009] in, The core component is N-acetylglucosamine, which is linked to proteins; It is a fucose derivative containing the first bioactive molecule (BM1); G is a distal glycosyl group containing substituted galactose.

[0010] In the protein conjugates disclosed herein, the fucose derivative and the substituted galactose can be coupled with the same or different payloads, such as toxin molecules. Experimental results show that when different payloads are coupled at these two sites, they exhibit superior in vitro and / or in vivo antitumor activity compared to a single payload.

[0011] Secondly, this disclosure also relates to a method for preparing the protein conjugate, the method comprising step (a): in the presence of a first catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of a second catalyst, react the product obtained in step (a) with... Contact; or,

[0012] The scheme includes step (a): in the presence of a second catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of the first catalyst, the product obtained in step (a) is reacted with... touch;

[0013] Nu and Nu' each independently contain ribonucleotides; It is a fucose derivative, which contains the first bioactive molecule. It is substituted galactose.

[0014] Thirdly, this disclosure relates to a pharmaceutical composition comprising the protein conjugate described in the first aspect; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0015] Fourthly, this disclosure relates to a method of treating and / or preventing tumors or cancer, comprising administering the protein conjugate described in the first aspect or the pharmaceutical composition described in the third aspect to a subject in need. This disclosure also relates to the use of the aforementioned protein conjugate or pharmaceutical composition in the preparation of medicaments for treating and / or preventing tumors or cancer.

[0016] Fifthly, this disclosure relates to a glycan fragment having formula (I) as defined in the first aspect of this disclosure, wherein the same or different payloads, such as toxin molecules, are coupled thereto by fucose derivatives and distal glycosyl groups, and having additional linking sites for further binding to biomolecules such as proteins with targeting effects.

[0017] Sixthly, this disclosure relates to a conjugate comprising a sugar chain fragment as defined in the fifth aspect of this disclosure.

[0018] In a seventh aspect, this disclosure relates to protein conjugates of the first aspect and conjugates of the sixth aspect of this disclosure for the treatment and / or prevention of tumors or cancer.

[0019] Eighthly, this disclosure relates to the use of the protein conjugate of the first aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the third aspect in the preparation of a medicament for treating and / or preventing tumors or cancer. Attached Figure Description

[0020] The specific features of the invention disclosed herein are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0021] Figure 1 shows the following sequences: (a) the flow chart for constructing ADC molecules using GalT1; (b) the flow chart for constructing ADC molecules using Fut8; (c) the flow chart for constructing dual-load ADC molecules; (d) the detection spectrum of the dual-load ADC molecule MEHDagYTE-LP6-LP1; (e) the detection spectrum of the dual-load ADC molecule MEHDagYTE-LP7-LP1-P8; (f) the detection spectrum of the dual-load ADC molecule MEHDag-LP3-LP1; (g) the detection spectrum of the dual-load ADC molecule MEHDag-LP4-LP1; and (h) the detection spectrum of the dual-load ADC molecule MEHDagYTE-LP6-LP1. The detection chromatograms of HDag-LP3-LP8, (i) the detection chromatogram of the dual-load ADC molecule Iso-IgG1ag-LP4-LP1, (j) the detection chromatogram of the dual-load ADC molecule Isoag-LP3-LP1, (k) the detection chromatogram of the dual-load ADC molecule Paniag-LP3-LP1, (l) the detection chromatogram of the dual-load ADC molecule Paniag-LP5-LP1, (m) the detection chromatogram of the dual-load ADC molecule hRS7ag-LP3-LP1, and (n) the detection chromatogram of the dual-load ADC molecule hRS7ag-LP5-LP1; among which It represents N-acetylglucosamine. Mannose, It is N-acetylgalactosamine.

[0022] Figure 2 shows the binding of the ADC drug to (a) tumor cells MDA-MB-468 and (b) tumor cells A431.

[0023] Figure 3 shows (a) the effect of ADC drugs on tumor volume in a female BALB / c nude mouse model with subcutaneous transplantation of A431 cell line, and (b) the effect of ADC drugs on mouse body weight.

[0024] Figure 4 shows the effect of ADC drugs on tumor volume in a female BALB / c nude mouse model with HCC827 cell line subcutaneously transplanted.

[0025] Figure 5 shows the release of (a) MMAE and (b) CPT2 from serum of various genus sources of ADC drugs after incubation.

[0026] Figure 6 shows the inhibitory activity of ADC drugs on the proliferation of tumor cells (a, d, e) A431, (b) BxPC-3 and (c) HCC827 / ABCB1.

[0027] Figure 7 shows the effect of ADC drugs on tumor volume in a female Balb / c nude mouse model with subcutaneous transplantation of LS513 cell line.

[0028] Figure 8 shows the effect of ADC drugs on tumor volume in a female Balb / c nude mouse model with subcutaneous transplantation of JIMT-1 cell line.

[0029] Figure 9 shows the effect of ADC drugs on tumor volume in a female NOD SCID mouse model of patient-derived tumor xenograft. Detailed Implementation

[0030] Terminology Definition

[0031] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0032] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin”, whether referring herein to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (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 (e.g., in the terms “immunoglobulin sequence,” “antibody sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more specific interpretation is required herein.

[0033] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In some embodiments, the Fc region of the present invention is derived from IgG1, IgG2, IgG3, or IgG4.

[0034] The term "conjugate," also known as a conjugate, generally refers to a protein or polypeptide (e.g., an antibody or its antigen-binding fragment) linked to one or more bioactive molecules, such as antibody-drug conjugates (ADCs). The bioactive molecule can be any small molecule drug (e.g., a cytotoxin), a radioactive isotope and its chelates, a nucleic acid, a polypeptide, or an antibody or its antigen-binding fragment. The conjugate can have any number of protein-conjugated bioactive molecules from 1 to 20, for example, it can include 2, 4, 6, or 8 drug-loaded species. In some embodiments, the conjugate is an antibody-drug conjugate (ADC).

[0035] As used herein, the term "bioactive molecule" refers to a therapeutic compound intended to treat or prevent a disease or disorder in a subject. The term "bioactive molecule" is not intended to be particularly limited and can be any beneficial therapeutic compound that targets a specific target, receptor, gene, cell, tissue, or organism. Non-limiting examples of bioactive molecules in this disclosure include small molecule drugs, radioisotopes and their chelates, nucleic acids, peptides and antibodies or their antigen-binding fragments, and combinations thereof.

[0036] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.

[0037] The term "N-glycan" refers to a glycan chain attached to asparagine (Asn) of a protein's Asn-X-Ser / Thr sequence (Ser is serine, Thr is threonine, and X is any amino acid except proline). The term "O-glycan" refers to a glycan chain covalently linked to the -OH group of a serine or threonine residue in a protein via a terminal monosaccharide residue. O-glycosylation sites do not have conserved sequences, and glycans do not have a fixed core structure; they can be composed of a monosaccharide or a polysaccharide.

[0038] The term "G0 glycoform" refers to a glycosemyform, such as Fc glycosylated sugars, that does not contain terminal galactose residues or a core fucose residue. in It is N-acetylglucosamine. It is mannitol.

[0039] The term "substituted galactose" refers to galactose in which any hydroxyl group of galactose is substituted, specifically, for example, N-acetylgalactosamine, which is further substituted with an active group as defined in this disclosure.

[0040] The term "hexose" refers to a polyhydroxy aldehyde or polyhydroxy ketone with 6 carbon atoms. Hexoses suitable for use in this invention include, but are not limited to, ribose, rhamnose, arabinose, xylose, glucose, lythose, mannose, and galactose.

[0041] The term "hexuronic acid" refers to compounds in which the primary alcohol of a hexose, as defined above, is oxidized to its carboxyl form. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0042] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0043] The term "glycosidic bond" refers to the chemical bond that connects the sugar and the other hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative through dehydration condensation of the hydroxyl group on the hemiacetal structure of a sugar.

[0044] The term "DAR" refers to the ratio of a bioactive molecule conjugated to the protein described herein to the protein itself. In some embodiments described herein, the DAR can be 1 to 16, for example 2-16, 2-10, 2-8, 2-6, or 3-4, for example 2, 3, 4, 5, or 6. The DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug portion conjugated to the ligand portion in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR or the measured DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1.0 to 16.0, for example 0.5 to 8.0, 2.0 to 16.0, 3.0 to 12.0, 4.0 to 8.0, or 5.0 to 7.0. For example, the average DAR value of the protein conjugate is approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, and approximately 8.0. The range is defined by two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the couplings of the present invention refer to a group of coupling molecules or a mixture of coupling molecules that contain coupling molecules having the same and / or different DAR values.

[0045] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0046] As used herein, the term “and / or” refers to any one of the options or two or more of the options.

[0047] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0048] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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% above or below a specified value. Unless otherwise specified, all values ​​mentioned in this application are considered to be modified by "about". In case of doubt, or if the error range for a particular value or parameter is not generally understood in the art, "about" means ±5% of that value or parameter.

[0049] "Conservative substitution" refers to the replacement of a polypeptide sequence without substantially altering its intended functional activity. For example, conservative substitution often results in an amino acid being replaced by a chemically similar amino acid. Eight exemplary groups of amino acids containing mutually conserved substitutions are listed below: 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).

[0050] The terms “pharmaceutical excipients” and “pharmaceuticalally acceptable carriers” refer to diluents, adjuvants (e.g., Freund’s adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are administered together with the active substance.

[0051] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0052] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0053] Invention Details

[0054] protein conjugates

[0055] In a first aspect, this disclosure relates to a protein conjugate comprising a protein and a glycan, said glycan comprising the structure of formula I:

[0056] in, The core component is N-acetylglucosamine, which is linked to proteins; It is a fucose derivative containing the first bioactive molecule (BM1); G is a distal glycosyl group containing substituted galactose; the wavy line indicates the linkage with the protein.

[0057] In this disclosure, the distal glycosyl group refers to the portion of the glycan chain excluding the core N-acetylglucosamine-fucose derivative.

[0058] In some embodiments, the sugar chain can be an N-glycan or an O-glycan.

[0059] In some embodiments, the protein includes an Fc region. For example, an Fc region derived from immunoglobulins such as IgG, IgA, IgD, IgM, or IgE.

[0060] In some embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. In other embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of IgG1, IgG2, IgG3, or IgG4. In other embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

[0061] In some embodiments, the glycan is an N-glycan and is connected to the CH2 domain of the Fc region; for example, the N-glycan may be connected to Asn in the CH2 domain of the Fc region.

[0062] In some implementations, the sugar chain is connected to Asn297 of the Fc region, wherein the Fc region is numbered according to the EU index of Kabat.

[0063] In some embodiments, the protein in the protein conjugate may be a fusion protein with an Fc region. The fusion protein may comprise an Fc region and a bioactive protein. For example, the bioactive protein may be a therapeutic protein. For example, the bioactive protein may be derived from a non-immunoglobulin, such as a cytokine, complement, and / or antigen or fragment thereof.

[0064] In other embodiments, the protein conjugate may contain an antigen-binding portion, for example, it may contain an Fc region and an antigen-binding portion. In some embodiments, the protein may be an antibody or an antigen-binding fragment thereof, encompassing multispecific antibodies such as bispecific antibodies or full-length antibodies.

[0065] In some embodiments, the protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to HER3 and / or EGFR. In some embodiments, the protein comprises or is a bispecific antibody or fragment thereof that specifically binds to HER3 and EGFR. As an example, the antibody may be Duligotuzumab (MEHD7945A), whose amino acid sequence is referenced from WHO Drug Information, Vol. 28, No. 3, 2014 Recommended INN: List 72, as follows:

[0066] In some embodiments, the protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to HER3 and / or EGFR, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region.

[0067] In some embodiments, the heavy chain variable region comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 9, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 12, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 13, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0068] In some embodiments, the heavy chain variable region includes CDR-H1 shown in SEQ ID NO: 9, CDR-H2 shown in SEQ ID NO: 10, and CDR-H3 shown in SEQ ID NO: 11; the light chain variable region includes CDR-L1 shown in SEQ ID NO: 12, CDR-L2 shown in SEQ ID NO: 13, and CDR-L3 shown in SEQ ID NO: 14.

[0069] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 7.

[0070] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 8.

[0071] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.

[0072] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment further comprises a constant region or a variant thereof, such as a constant region derived from mice or humans. In some embodiments, the heavy chain comprises a constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof. For example, the heavy chain comprises a constant region that may contain or consist of the amino acid sequences described in SEQ ID NO: 15-18.

[0073] In some embodiments, the antibody or its antigen-binding fragment includes a constant region, wherein the constant region includes an Fc region.

[0074] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may further contain mutations that reduce or eliminate effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutations that reduce or eliminate effector function weaken or eliminate the binding between immunoglobulins and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or C1q.

[0075] The mutation that weakens or eliminates the effector function can be any known mutation or combination of mutations in the prior art, such as Esohe EI, J Immunol 2000; 164:4178-4184; Hutchins, JT, 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, SY, Mol. Immunol. 2008, 45, 3926–3933; Sazinsky, SL, Proc. Natl. Acad. Sci. USA 2008, 105, 20167–20172; Oganesyan, V., Acta Crystallogr.Sect.D Biol.Crystallogr.2008, 64Pt 6, 700–704; An, Z., MAbs 2009, 1, 572–579; Moore, GL, Methods 2019, 154, 38–50; Schlothauer, T., Protein Eng.Des.Sel.2016, 29, 457–466; Strohl, W., US20150337053; Engelberts, PJ, EBioMedicine 2020, 52, 102625, etc.

[0076] In some embodiments, the Fc region may further include mutations selected from the following that reduce or eliminate effector function: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S29 8G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the Fc region may further contain mutations selected from the following that reduce or eliminate effector function: D265A / P331G (hereinafter abbreviated as AG).

[0077] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment contains a constant region comprising, or being composed of, the amino acid sequence shown in SEQ ID NO: 19.

[0078] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may further contain mutations that regulate its binding affinity to FcRn and / or prolong its half-life. For example, the Fc region variant improves binding to FcRn under acidic pH conditions without affecting binding to FcRn under neutral pH conditions, thereby achieving a longer half-life compared to the parent.

[0079] The mutation regulating the binding affinity to FcRn and / or prolonging the half-life can be any known mutation or combination of mutations in the prior art, such as those described in Dall'Acqua W, J. Immunol. 2002; 169:5171–5180; Dall'Acqua W, J. Biol. Chem. 2006b; 281:23514–23524; Hinton PR, J. Immunol. 2006; 176:346–356; Petkova, J. Exp. Med. 2006; 203:275–280; Yeung, J. Immunol. 2009; 182:7663–7671; Zalevsky, Nat. Biotechnol. 2010; 28:157–159; Monnet, MAbs. 2014; 6:422–436, etc.

[0080] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may contain mutations selected from the following that regulate binding affinity to FcRn and / or prolong half-life: M252Y / S254T / T256E, T250Q / M428L, N434A, M428L / N434S, N315D / A330V / N361D / A378V / N434Y, E294D / T307P / N434Y, V259I / N315D / N434Y, T307A / N315D / A330V / E382V / N389T / N434Y, or L234F / L235E / D265A. Preferably, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may contain mutations selected from the following that regulate the binding ability with FcRn and / or prolong the half-life: M252Y / S254T / T256E (hereinafter abbreviated as YTE).

[0081] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment contains a constant region comprising an Fc region containing a mutation that reduces or eliminates effector function, for example, the constant region comprising, or being composed of, the amino acid sequence shown in SEQ ID NO: 27.

[0082] In some embodiments, the Fc region of the heavy chain of the antibody or its antigen-binding fragment may simultaneously contain the aforementioned mutations that reduce or eliminate effector function and mutations that regulate its binding ability to FcRn. For example, in some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a constant region containing an Fc region that simultaneously contains the aforementioned mutations that reduce or eliminate effector function and mutations that regulate its binding ability to FcRn. For example, the constant region contains, or is composed of, the amino acid sequence shown in SEQ ID NO: 28.

[0083] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 20-21 or 29-30.

[0084] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof described in this disclosure further comprises a constant region or a variant thereof, for example, a constant region derived from mouse or human. In some embodiments, the light chain comprises a constant region of human Igκ or a variant thereof; for example, the light chain comprises a constant region, which may comprise or consist of the amino acid sequence described in SEQ ID NO: 22.

[0085] In some embodiments, the light chain of the antibody or its antigen-binding fragment described in this disclosure comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 23.

[0086] In some embodiments, the antibody that specifically binds to HER3 and / or EGFR according to this disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 20, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 23. In other embodiments, the antibody that specifically binds to HER3 and / or EGFR according to this disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 21, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 23. In still other embodiments, the antibody that specifically binds to HER3 and / or EGFR according to this disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 29, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 23. In other embodiments, the antibodies that specifically bind to HER3 and / or EGFR as described in this disclosure comprise a heavy chain and a light chain, the heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 30, and the light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 23.

[0087] In some embodiments, the protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to EGFR or TROP2.

[0088] In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises 1, 2, or 3 heavy chain variable region CDRs of a known EGFR-specific antibody (e.g., Panitumumab), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises 1, 2, or 3 light chain variable region CDRs of a known EGFR-specific antibody (e.g., Panitumumab), namely LCDR1, LCDR2, and LCDR3. In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises a heavy chain variable region of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises a light chain variable region of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises both a heavy chain variable region and a light chain variable region of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises the heavy chain of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises the light chain of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises both the heavy chain and the light chain of a known EGFR-specific antibody (e.g., Panitumumab). In some embodiments, the EGFR antibody or its antigen-binding fragment comprises, or is composed of, two heavy chains and two light chains of a known antibody that specifically binds to EGFR (e.g., Panitumumab).

[0089] In some embodiments, the antibody that specifically binds to EGFR or its antigen-binding fragment includes complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are identical to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Panitumumab, respectively; or the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Panitumumab. In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are identical to the amino acid sequences of the heavy chain variable region and the light chain variable region of Panitumumab, respectively; or the heavy chain variable region and the light chain variable region are respectively the heavy chain variable region and the light chain variable region of Panitumumab. In some embodiments, the EGFR-specific antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and the light chain are identical to the amino acid sequences of the heavy chain and the light chain of Panitumumab, respectively; or the heavy chain and the light chain are respectively the heavy chain and the light chain of Panitumumab. In some embodiments, the EGFR-specific antibody has the same heavy chain and the same light chain as Panitumumab. For example, the Panitumumab includes a heavy chain variable region as shown in SEQ ID NO:34 and a light chain variable region as shown in SEQ ID NO:35; or, the Panitumumab includes a heavy chain as shown in SEQ ID NO:36 and a light chain as shown in SEQ ID NO:37.

[0090] In some embodiments, the antibody that specifically binds to EGFR or its antigen-binding fragment comprises or is panitumumab.

[0091] In some embodiments, the EGFR-specific antibody or its antigen-binding fragment has a D265A / P331G mutation in the heavy chain constant region. Therefore, in some embodiments, the EGFR-specific antibody comprises or is panitumumab with the D265A / P331G mutation. For example, the EGFR-specific antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:38 and a light chain as shown in SEQ ID NO:37.

[0092] In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises 1, 2, or 3 heavy chain variable region CDRs of a known TROP2-specific antibody (e.g., Sacituzumab), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises 1, 2, or 3 light chain variable region CDRs of a known TROP2-specific antibody (e.g., Sacituzumab), namely LCDR1, LCDR2, and LCDR3. In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2-specific antibody or its antigen-binding fragment comprises a heavy chain variable region of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2-specific antibody or its antigen-binding fragment comprises a light chain variable region of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2-specific antibody or its antigen-binding fragment comprises both a heavy chain variable region and a light chain variable region of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2-specific antibody or its antigen-binding fragment comprises the heavy chain of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2-specific antibody or its antigen-binding fragment comprises both the heavy chain and light chain of a known TROP2-specific antibody (e.g., Sacituzumab). In some embodiments, the TROP2 antibody or its antigen-binding fragment comprises, or is composed of, two heavy chains and two light chains of a known antibody that specifically binds to TROP2 (e.g., Sacituzumab).

[0093] In some embodiments, the antibody that specifically binds to TROP2 or its antigen-binding fragment includes complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are identical to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Sacituzumab, respectively; or the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Sacituzumab. In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are identical to the amino acid sequences of the heavy chain variable region and the light chain variable region of Sacituzumab, respectively; or the heavy chain variable region and the light chain variable region are respectively the heavy chain variable region and the light chain variable region of Sacituzumab. In some embodiments, the antibody specifically binding to TROP2 or its antigen-binding fragment comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and the light chain are identical to the amino acid sequences of the heavy chain and the light chain of Sacituzumab, respectively; or the heavy chain and the light chain are respectively the heavy chain and the light chain of Sacituzumab. In some embodiments, the antibody specifically binding to TROP2 has the same heavy chain and the same light chain as Sacituzumab. For example, the Sacituzumab includes a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40; or, the Sacituzumab includes a heavy chain as shown in SEQ ID NO:41 and a light chain as shown in SEQ ID NO:42.

[0094] In some embodiments, the antibody that specifically binds to TROP2 or its antigen-binding fragment comprises or is Sacituzumab.

[0095] In some embodiments, the antibody that specifically binds to TROP2, or its antigen-binding fragment, has a D265A / P331G mutation in the heavy chain constant region. Therefore, in some embodiments, the antibody that specifically binds to TROP2 comprises or is Sacituzumab with the D265A / P331G mutation. For example, the antibody that heterologously binds to TROP2, or its antigen-binding fragment, comprises a heavy chain as shown in SEQ ID NO:43 and a light chain as shown in SEQ ID NO:42.

[0096] In some embodiments, the sugar chain can be an N-glycan or an O-glycan, preferably an N-glycan.

[0097] In some embodiments, the fucose derivative is linked to the core N-acetylglucosamine via an α-1,6-glycosidic bond.

[0098] The fucose moiety in the fucose derivative disclosed herein has the following structure:

[0099] Derivatization can occur at any position among positions 1, 2, 3, 4, 5, and 6, but is preferably performed at position 6 of the fucosylate.

[0100] In some embodiments, the distal glycosyl G may be selected from the following structures:

[0101] in, For substituted galactose, It is N-acetylglucosamine. It is mannitol;

[0102] Preferably, G is selected from

[0103] In some embodiments, the substituted galactose is linked to the N-acetylglucosamine thereto via a β-1,4-glycosidic bond or a β-1,3-glycosidic bond; preferably, via a β-1,4-glycosidic bond.

[0104] The galactosyl group in the substituted galactose of this disclosure has the following structure:

[0105] The substitution can be located at any position among positions 1, 2, 3, 4, 5, and 6, but is preferably made at position 2 and / or position 6 of galactose.

[0106] In some embodiments, the substituted galactose comprises a chemically active group X1 and / or a second bioactive molecule BM2.

[0107] In some embodiments, the chemically active group X1 is selected from chemical groups that can undergo click chemistry reactions.

[0108] In some embodiments, when a chemically active group X1 is included, X1 may be selected from the following structures:

[0109] The first bioactive molecule BM1 contained in the fucose derivative described herein and the second bioactive molecule BM2 contained in the substituted galactose can each be independently selected from drugs or prodrugs, diagnostic agents, proteins, peptides, amino acids, polysaccharides, lipids, vitamins, steroids, nucleotides, nucleosides, polynucleotides, RNA, or DNA.

[0110] The first bioactive molecule BM1 and the second bioactive molecule BM2 described in this disclosure can each be independently selected from small molecule drugs, radioactive isotopes and their chelates, nucleic acids, peptides and antibodies or their antigen-binding fragments.

[0111] The term "small molecule drug" as used in this disclosure refers to drug molecules with a molecular weight of less than 1,000 Daltons.

[0112] For example, the first bioactive molecule BM1 and the second bioactive molecule BM2 can be independently selected from the following small molecule drugs: topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin inhibitors, radioisotopes, metal complexes, glycopeptide antibiotics, glucocorticoids, calcineurin inhibitors, DNA alkylating agents, drugs that interfere with DNA synthesis, serine kinase inhibitors, threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, histidine kinase inhibitors, proteasome inhibitors, protease inhibitors, histone deacetylase inhibitors, angiogenesis inhibitors, cyclin inhibitors, Toll-like receptor modulators, and STING modulators.

[0113] In some embodiments, the small molecule drug may be a topoisomerase I inhibitor, such as camptothecin compounds, wherein the camptothecin derivative refers to a compound having the same 5-membered fused core structure as naturally derived camptothecin and having substitution modifications at positions 7, 9, 10 and 11, and having topoisomerase I inhibitory activity and / or antitumor activity comparable to or stronger than that of natural camptothecin.

[0114] In some embodiments, the small molecule drug may be selected from Dxd, ixotecan, SN-38, 10-hydroxycamptothecin, irinotecan, topotecan, belotetcan, Gimatecan, Lurtotecan, Rubitecan, and Namitecan.

[0115] In other embodiments, the small molecule drug may be selected from the structure shown in Formula II:

[0116] Among them, R3 is selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, and halogen, p is an integer from 0 to 5.

[0117] In some embodiments, the small molecule drug may be selected from the following structures:

[0118] In some embodiments, the small molecule drug may be a topoisomerase II inhibitor; preferably selected from anthracycline antibiotics, podophyllotoxin derivatives and anthraquinone antibiotics; more preferably selected from doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, arubicin, PNU-159682, etoposide, teniposide and mitoxantrone.

[0119] In other embodiments, the small molecule drug may be a tubulin inhibitor; preferably from salitoxin 10 and its derivatives, maytansine compounds, tubulin compounds, taxane compounds, vinblastine compounds, eribulin and its derivatives, and colchicine and its derivatives; more preferably from MMAE, MMAF, Duo5, PF-06380101, DM1, DM4, and eribulin.

[0120] In some embodiments, the first bioactive molecule BM1 and the second bioactive molecule BM2 may be the same or different. For example, the first bioactive molecule and the second bioactive molecule may be small molecule drugs of different classes; or, the first bioactive molecule and the second bioactive molecule may be small molecule drugs of the same class (e.g., both are microtubule inhibitors) but have different chemical structures; or, the first bioactive molecule and the second bioactive molecule may be the same small molecule drug.

[0121] In some embodiments, the first bioactive molecule BM1 and the second bioactive molecule BM2 may be selected from the following combinations:

[0122] (1) BM1: microtubule inhibitor, BM2: topoisomerase I inhibitor;

[0123] (2) BM1: Topoisomerase I inhibitor, BM2: Tubulin inhibitor;

[0124] (3) BM1: microtubule inhibitor, BM2: microtubule inhibitor; and

[0125] (4) BM1: Topoisomerase I inhibitor, BM2: Topoisomerase I inhibitor.

[0126] The microtubule inhibitor may be selected from MMAE, MMAF, Duo5, DM4 and eribulin, and the topoisomerase I inhibitor may be selected from Dxd, eczema, SN-38 and compounds represented by Formula II, such as CPT-1, CPT-2, CPT-3, CPT-4, CPT-5, CPT-6 and CPT-7.

[0127] In some embodiments, the tubulin inhibitor may be MMAE or MMAF, preferably MMAE; for example, the bioactive molecule may have a structure selected from the following:

[0128] In some embodiments, the topoisomerase I inhibitor may be selected from Dxd, eczema, SN-38, and CPT-2, preferably Dxd or CPT-2; for example, the bioactive molecule may have a structure selected from the following:

[0129] For example, the first bioactive molecule BM1 and the second bioactive molecule BM2 can be specifically selected from the following combinations:

[0130] In some embodiments, the aforementioned first bioactive molecule BM1 is directly or indirectly linked to the 6th position of the fucose group; the aforementioned second bioactive molecule BM2 is directly or indirectly linked to the 2nd or 6th position of the galactose group.

[0131] In the protein conjugate disclosed herein, the molar ratio of the first bioactive molecule BM1 to the second bioactive molecule BM2 is 2:1 to 1:8, preferably 1:1 to 1:4, and more preferably 1:1 to 1:2.

[0132] In some embodiments, the fucose derivative comprises a first bioactive molecule BM1, and the substituted galactose comprises a chemically active group X1, wherein the first bioactive molecule BM1 and the chemically active group X1 are as defined above.

[0133] In some embodiments, the first bioactive molecule BM1 is selected from microtubule inhibitors or topoisomerase I inhibitors, while the chemically active group X1 is selected from:

[0134] In some embodiments, the first bioactive molecule BM1 may be selected from MMAE, MMAF, Duo5, DM4, and eribulin, preferably MMAE or MMAF, while the chemically active group X1 is selected from:

[0135] In other embodiments, the first bioactive molecule BM1 may be selected from Dxd, eczema, SN-38, and CPT-2, preferably Dxd and CPT-2, while the chemically active group X1 is selected from:

[0136] In other embodiments, the fucose derivative has the structure of Formula III:

[0137] BM1 is the first bioactive molecule, and LU1 is the linking unit that connects the first bioactive molecule to the fucose group.

[0138] In some embodiments, the modified galactose has a structure of formula IV-A or IV-B:

[0139] BM2 is the second bioactive molecule, and LU2 is the linking unit that connects the second bioactive molecule to the galactose group.

[0140] In some embodiments, the modified galactose has a structure of formula IV-C or IV-D:

[0141] Wherein, the chemically active group X1 is as defined above.

[0142] In some implementations, the aforementioned connection units LU1 and LU2 each independently have the structure shown in Equation V:

[0143] Wherein, L1 is the extension group attached to the sugar 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.

[0144] In some embodiments, L1 comprises a group selected from the following:

[0145] In some embodiments, the extending group L1 comprises one or more of the following structures:

[0146] The side connected to the direction of L2 is denoted as L2, and the other side is connected to the glycosyl group.

[0147] Preferably, the extending group L1 comprises a structure selected from the following:

[0148] In some implementations, L1 is selected from:

[0149] Where m is an integer selected from 0 to 10, and n is an integer selected from 0 to 20.

[0150] Preferably, L1 is selected from:

[0151] In some implementations, L1 is selected from:

[0152] In some embodiments, the structure of the first spacer group L2 is shown in Formula VI:

[0153] Where a1 = 0 or 1, a2 = 0 or 1, a3 = an integer from 0 to 8, a4 = 0 or 1, 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 b2 and b3 is 0.

[0154] In some implementations, a1 = 1 in the structure of L2; in other implementations, a1 = 0.

[0155] In some implementations, a2 = 1; in others, a2 = 0.

[0156] In some implementations, a3 = 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0157] In some implementations, a4 = 1; in others, a4 = 0.

[0158] In some implementations, b1 = 0 or 1.

[0159] In some implementations, b2 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0160] In some implementations, b3 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0161] In some implementations, c = 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0162] The above options a1, a2, a3, b1, b2, b3 and c can be combined in any way, provided that at least one of b2 and b3 is 0.

[0163] In some specific implementations, the structure of L2 is selected from the following group:

[0164] (1) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0;

[0165] (2) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 1, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0;

[0166] (3) a1=1, a2=1, a3=0, 1, 2, 3, 4, 5 or 6, a4=0, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0;

[0167] (4) a1 = 1, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0168] (5) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0169] (6) a1=1, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=0, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0;

[0170] (7) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0171] (8) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3 = 0, c = 0;

[0172] (9) a1=0, a2=0, a3=0, a4=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;

[0173] (10) a1=1, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=1, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0;

[0174] (11) a1=0, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=1, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0;

[0175] (12) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0; and

[0176] (13) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 0, 1, 2, 3, 4, 5 or 6.

[0177] In some implementations, the structure of L2 is specifically selected from the following group:

[0178] (1.1) a1=0, a2=0, a3=0, b1=0, b2=2, b3=0, c=0;

[0179] (1.2) a1=0, a2=0, a3=0, b1=0, b2=4, b3=0, c=0;

[0180] (1.3) a1=0, a2=0, a3=0, b1=0, b2=6, b3=0, c=0;

[0181] (1.4) a1=0, a2=0, a3=0, b1=0, b2=8, b3=0, c=0;

[0182] (1.5) a1=0, a2=0, a3=0, b1=0, b2=10, b3=0, c=0;

[0183] (1.6) a1=0, a2=0, a3=0, b1=0, b2=12, b3=0, c=0;

[0184] (1.7) a1=0, a2=0, a3=1, b1=0, b2=2, b3=0, c=0;

[0185] (1.8) a1=0, a2=0, a3=1, b1=0, b2=4, b3=0, c=0;

[0186] (1.9) a1=0, a2=0, a3=1, b1=0, b2=6, b3=0, c=0;

[0187] (1.10) a1=0, a2=0, a3=1, b1=0, b2=8, b3=0, c=0;

[0188] (1.11) a1=0, a2=0, a3=1, b1=0, b2=10, b3=0, c=0;

[0189] (1.12) a1=0, a2=0, a3=1, b1=0, b2=12, b3=0, c=0;

[0190] (1.13)a1=0, a2=0, a3=2, b1=0, b2=2, b3=0, c=0;

[0191] (1.14)a1=0, a2=0, a3=2, b1=0, b2=4, b3=0, c=0;

[0192] (1.15)a1=0, a2=0, a3=2, b1=0, b2=6, b3=0, c=0;

[0193] (1.16)a1=0, a2=0, a3=2, b1=0, b2=8, b3=0, c=0;

[0194] (1.17)a1=0, a2=0, a3=2, b1=0, b2=10, b3=0, c=0;

[0195] (1.18)a1=0, a2=0, a3=2, b1=0, b2=12, b3=0, c=0;

[0196] (2.1)a1=0, a2=0, a3=0, b1=1, b2=2, b3=0, c=0;

[0197] (2.2)a1=0, a2=0, a3=0, b1=1, b2=4, b3=0, c=0;

[0198] (2.3)a1=0, a2=0, a3=0, b1=1, b2=6, b3=0, c=0;

[0199] (2.4)a1=0, a2=0, a3=0, b1=1, b2=8, b3=0, c=0;

[0200] (2.5)a1=0, a2=0, a3=0, b1=1, b2=10, b3=0, c=0;

[0201] (2.6)a1=0, a2=0, a3=0, b1=1, b2=12, b3=0, c=0;

[0202] (2.7)a1=0, a2=0, a3=1, b1=1, b2=2, b3=0, c=0;

[0203] (2.8)a1=0, a2=0, a3=1, b1=1, b2=4, b3=0, c=0;

[0204] (2.9)a1=0, a2=0, a3=1, b1=1, b2=6, b3=0, c=0;

[0205] (2.10)a1=0, a2=0, a3=1, b1=1, b2=8, b3=0, c=0;

[0206] (2.11)a1=0, a2=0, a3=1, b1=1, b2=10, b3=0, c=0;

[0207] (2.12)a1=0, a2=0, a3=1, b1=1, b2=12, b3=0, c=0;

[0208] (2.13)a1=0, a2=0, a3=2, b1=1, b2=2, b3=0, c=0;

[0209] (2.14)a1=0, a2=0, a3=2, b1=1, b2=4, b3=0, c=0;

[0210] (2.15)a1=0, a2=0, a3=2, b1=1, b2=6, b3=0, c=0;

[0211] (2.16)a1=0, a2=0, a3=2, b1=1, b2=8, b3=0, c=0;

[0212] (2.17)a1=0, a2=0, a3=2, b1=1, b2=10, b3=0, c=0;

[0213] (2.18)a1=0, a2=0, a3=2, b1=1, b2=12, b3=0, c=0;

[0214] (3.1)a1=1, a2=1, a3=2, b1=0, b2=2, b3=0, c=0;

[0215] (3.2)a1=1, a2=1, a3=2, b1=0, b2=4, b3=0, c=0;

[0216] (3.3)a1=1, a2=1, a3=2, b1=0, b2=6, b3=0, c=0;

[0217] (3.4)a1=1, a2=1, a3=2, b1=0, b2=8, b3=0, c=0;

[0218] (3.5)a1=1, a2=1, a3=2, b1=0, b2=10, b3=0, c=0;

[0219] (3.6)a1=1, a2=1, a3=2, b1=0, b2=12, b3=0, c=0;

[0220] (4.1)a1=1, a2=0, a3=1, b1=0, b2=0, b3=0, c=0;

[0221] (4.2)a1=1, a2=0, a3=2, b1=0, b2=0, b3=0, c=0;

[0222] (4.3)a1=1, a2=0, a3=3, b1=0, b2=0, b3=0, c=0;

[0223] (4.4)a1=1, a2=0, a3=4, b1=0, b2=0, b3=0, c=0;

[0224] (4.5)a1=1, a2=0, a3=5, b1=0, b2=0, b3=0, c=0;

[0225] (4.6)a1=1, a2=0, a3=6, b1=0, b2=0, b3=0, c=0;

[0226] (5.1)a1=0, a2=0, a3=1, b1=0, b2=0, b3=0, c=0;

[0227] (5.2)a1=0, a2=0, a3=2, b1=0, b2=0, b3=0, c=0;

[0228] (5.3)a1=0, a2=0, a3=3, b1=0, b2=0, b3=0, c=0;

[0229] (5.4)a1=0, a2=0, a3=4, b1=0, b2=0, b3=0, c=0;

[0230] (5.5)a1=0, a2=0, a3=5, b1=0, b2=0, b3=0, c=0;

[0231] (5.6)a1=0, a2=0, a3=6, b1=0, b2=0, b3=0, c=0;

[0232] (6.1)a1=1, a2=0, a3=2, b1=0, b2=2, b3=0, c=0;

[0233] (6.2)a1=1, a2=0, a3=2, b1=0, b2=4, b3=0, c=0;

[0234] (6.3)a1=1, a2=0, a3=2, b1=0, b2=6, b3=0, c=0;

[0235] (6.4)a1=1, a2=0, a3=2, b1=0, b2=8, b3=0, c=0;

[0236] (6.5)a1=1, a2=0, a3=2, b1=0, b2=10, b3=0, c=0;

[0237] (6.6)a1=1, a2=0, a3=2, b1=0, b2=12, b3=0, c=0;

[0238] (7.1)a1=0, a2=1, a3=1, b1=0, b2=0, b3=0, c=0;

[0239] (7.2)a1=0, a2=1, a3=2, b1=0, b2=0, b3=0, c=0;

[0240] (7.3)a1=0, a2=1, a3=3, b1=0, b2=0, b3=0, c=0;

[0241] (7.4)a1=0, a2=1, a3=4, b1=0, b2=0, b3=0, c=0;

[0242] (7.5)a1=0, a2=1, a3=5, b1=0, b2=0, b3=0, c=0;

[0243] (7.6)a1=0, a2=1, a3=6, b1=0, b2=0, b3=0, c=0;

[0244] (8.1)a1=0, a2=1, a3=2, b1=0, b2=2, b3=0, c=0;

[0245] (8.2)a1=0, a2=1, a3=2, b1=0, b2=4, b3=0, c=0;

[0246] (8.3)a1=0, a2=1, a3=2, b1=0, b2=6, b3=0, c=0;

[0247] (8.4)a1=0, a2=1, a3=2, b1=0, b2=8, b3=0, c=0;

[0248] (8.5)a1=0, a2=1, a3=2, b1=0, b2=10, b3=0, c=0;

[0249] (8.6)a1=0, a2=1, a3=2, b1=0, b2=12, b3=0, c=0;

[0250] (9.1)a1=0, a2=0, a3=0, b1=0, b2=0, b3=2, c=1;

[0251] (9.2)a1=0, a2=0, a3=0, b1=0, b2=0, b3=3, c=1;

[0252] (9.3)a1=0, a2=0, a3=0, b1=0, b2=0, b3=4, c=1;

[0253] (9.4)a1=0, a2=0, a3=0, b1=0, b2=0, b3=5, c=1;

[0254] (9.5)a1=0, a2=0, a3=0, b1=0, b2=0, b3=6, c=1;

[0255] (9.6)a1=0, a2=0, a3=0, b1=0, b2=0, b3=2, c=2;

[0256] (9.7)a1=0, a2=0, a3=0, b1=0, b2=0, b3=3, c=2;

[0257] (9.8)a1=0, a2=0, a3=0, b1=0, b2=0, b3=4, c=2;

[0258] (9.9)a1=0, a2=0, a3=0, b1=0, b2=0, b3=5, c=2;

[0259] (9.10)a1=0, a2=0, a3=0, b1=0, b2=0, b3=6, c=2;

[0260] (10.1)a1=1, a2=0, a3=1, a4=1, b1=0, b2=2, b3=0, c=0;

[0261] (10.2)a1=1, a2=0, a3=1, a4=1, b1=0, b2=4, b3=0, c=0;

[0262] (10.3)a1=1, a2=0, a3=1, a4=1, b1=0, b2=6, b3=0, c=0;

[0263] (10.4)a1=1, a2=0, a3=1, a4=1, b1=0, b2=8, b3=0, c=0;

[0264] (10.5)a1=1, a2=0, a3=2, a4=1, b1=0, b2=2, b3=0, c=0;

[0265] (10.6)a1=1, a2=0, a3=2, a4=1, b1=0, b2=4, b3=0, c=0;

[0266] (10.7)a1=1, a2=0, a3=2, a4=1, b1=0, b2=6, b3=0, c=0;

[0267] (10.8)a1=1, a2=0, a3=2, a4=1, b1=0, b2=8, b3=0, c=0;

[0268] (10.9)a1=1, a2=0, a3=3, a4=1, b1=0, b2=2, b3=0, c=0;

[0269] (10.10)a1=1, a2=0, a3=3, a4=1, b1=0, b2=4, b3=0, c=0;

[0270] (10.11)a1=1, a2=0, a3=3, a4=1, b1=0, b2=6, b3=0, c=0;

[0271] (10.12)a1=1, a2=0, a3=3, a4=1, b1=0, b2=8, b3=0, c=0;

[0272] (11.1)a1=0, a2=0, a3=1, a4=1, b1=0, b2=2, b3=0, c=0;

[0273] (11.2)a1=0, a2=0, a3=1, a4=1, b1=0, b2=4, b3=0, c=0;

[0274] (11.3)a1=0, a2=0, a3=1, a4=1, b1=0, b2=6, b3=0, c=0;

[0275] (11.4)a1=0, a2=0, a3=1, a4=1, b1=0, b2=8, b3=0, c=0;

[0276] (11.5)a1=0, a2=0, a3=2, a4=1, b1=0, b2=2, b3=0, c=0;

[0277] (11.6)a1=0, a2=0, a3=2, a4=1, b1=0, b2=4, b3=0, c=0;

[0278] (11.7)a1=0, a2=0, a3=2, a4=1, b1=0, b2=6, b3=0, c=0;

[0279] (11.8) a1 = 0, a2 = 0, a3 = 2, a4 = 1, b1 = 0, b2 = 8, b3 = 0, c = 0;

[0280] (11.9) a1 = 0, a2 = 0, a3 = 3, a4 = 1, b1 = 0, b2 = 2, b3 = 0, c = 0;

[0281] (11.10) a1 = 0, a2 = 0, a3 = 3, a4 = 1, b1 = 0, b2 = 4, b3 = 0, c = 0;

[0282] (11.11) a1 = 0, a2 = 0, a3 = 3, a4 = 1, b1 = 0, b2 = 6, b3 = 0, c = 0;

[0283] (11.12) a1 = 0, a2 = 0, a3 = 3, a4 = 1, b1 = 0, b2 = 8, b3 = 0, c = 0;

[0284] (12.1) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 0, b2 = 0, b'3 = 0, c = 0;

[0285] (13.1) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 1;

[0286] (13.2) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 2;

[0287] (13.3) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 3;

[0288] (13.4) a1 = 0, a2 = 0, a3 = 0, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 4; <\

[0289] (13.5) a1 = 0, a2 = 0, a3 = 1, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 1;

[0290] (13.2) a1 = 0, a2 = 0, a3 = 1, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 2;

[0291] (13.3) a1 = 0, a2 = 0, a3 = 1, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 3; and

[0292] (13.4) a1 = 0, a2 = 0, a3 = 1, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 4. It should be noted that in the original text, there seems to be a small error in line ID=16 where it says "b'3" which might be a typo. I translated it as "b3" in the translation. Also, in line ID=30, the original tag

[0289] seems to be a bit odd with a backslash in it. I just removed the backslash for the translation as it's likely an error in the original text. If these are not errors in the original and there's a specific meaning to these, the translation might need to be adjusted accordingly.

[0293] In some implementations, L2 is selected from the following structures:

[0294] Where a3 = integers from 0 to 8, b2 = integers from 0 to 16, b3 = integers from 0 to 16, and c = integers from 0 to 8.

[0295] In some implementations, L3 is absent; while in others, L3 is a peptide linker.

[0296] In some embodiments, L3 is selected from peptide linking groups that can be cleaved by cathepsins; preferably, the cathepsin is selected from cathepsins A, B, C, D, E, F, G, H, K, L1, L2, O, S, W and Z; more preferably, the cathepsin is cathepsin B.

[0297] In some implementations, L3 is a diamino acid peptide, triamino acid peptide, or tetraamino acid peptide residue.

[0298] In some embodiments, 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.

[0299] In other embodiments, 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.

[0300] In other embodiments, 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.

[0301] In some implementations, L4 is absent; while in others, L4 is selected from:

[0302] (The side connected to the bioactive molecule is labeled BM, and the side connected to L3 is labeled L3); where 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; Su is selected from autosaccharides and hexuronic acids.

[0303] In some implementations, Su is selected from glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0304] In some implementations, L4 is absent, or is selected from:

[0305] In some implementations, the aforementioned connection units LU1 and LU2 are each independently selected from the following structures:

[0306] Wherein, k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10. In some implementations, k1 and k2 are 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.

[0307] In some implementations, the connection units LU1 and LU2 are each independently selected from the following structures:

[0308] Where k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

[0309] In some implementations, the connection unit LU1 is selected from the following structures:

[0310] And / or, the connection unit LU2 is selected from the following structures:

[0311] Where k1, k2, and k' are as defined previously;

[0312] And / or, the connection unit LU2 is selected from the following structures:

[0313] Among them, k1 and k' are as described previously.

[0314] In some embodiments, the protein conjugates described in this disclosure have glycan chains comprising or having a structure as shown in formula VII-A or VII-B:

[0315] in The core N-acetylglucosamine, It is N-acetylglucosamine. Mannose, It is a fucose derivative. The substituted galactose is used; the fucose derivative and the substituted galactose are as defined above; q = 0.5 to 8, for example q = 1 to 6, preferably q = 1.5 to 4, more preferably q = 1.8 to 2.5.

[0316] For example, in some embodiments, the fucose derivative has the structure of Formula III:

[0317] BM1 is the first bioactive molecule, and LU1 is the linking unit that connects the first bioactive molecule to the fucose group.

[0318] In some embodiments, the modified galactose has a structure of formula IV-A or IV-B:

[0319] BM2 is the second bioactive molecule, and LU2 is the linking unit that connects the second bioactive molecule to the galactose group.

[0320] In some embodiments, the modified galactose has a structure of formula IV-C or IV-D:

[0321] The BM1, LU1, BM2, LU2, and X1 mentioned therein may be defined in accordance with the foregoing of this disclosure.

[0322] In some implementations, the BM1-LU1- may specifically be selected from the following structures:

[0323] as well as

[0324] Among them, k1, k2 and k' are as defined previously.

[0325] In some implementations, the BM2-LU2- may specifically be selected from the following structures:

[0326] as well as

[0327] Where k1 and k' are as defined previously.

[0328] In some implementations, X1 is selected from the following structures: as well as Preferred

[0329] In some implementations, q = 1-6; preferably, q = 1.5-4; more preferably, q = 1.8-2.5.

[0330] In some embodiments, this disclosure relates to a protein conjugate comprising a protein and a glycan, said glycan comprising a structure of formula IA or formula IB:

[0331] in, The core is N-acetylglucosamine, which is linked to the protein; the wavy line indicates the linkage bond with the protein. It is a fucose derivative that contains the first bioactive molecule BM1; It is a substituted galactose that contains a second bioactive molecule, BM2. It is N-acetylglucosamine. It is mannitol;

[0332] The fucose derivative has the structure of Formula III:

[0333] The substituted galactose has a structure of formula IV-A or IV-B:

[0334] Among them, BM1-LU1- is selected from the following structures:

[0335] BM2-LU2- is selected from the following structures:

[0336] Where k1 is independently selected from integers from 1 to 20, and k' is independently selected from integers from 0 to 10.

[0337] Based on the above disclosure, this disclosure also provides a sugar chain fragment having the structure of Formula I as defined above: in, The core is N-acetylglucosamine; It is a fucose derivative containing a first bioactive molecule BM1; G is a distal glycosyl group containing substituted galactose; the wavy line indicates the linkage; wherein the fucose derivative, the first bioactive molecule BM1, G, and the substituted galactose are as generally or specifically defined above.

[0338] Based on the above disclosure, this disclosure also provides a conjugate comprising a sugar chain fragment of formula (I) as generally or specifically defined in this disclosure.

[0339] Preparation method

[0340] Secondly, this disclosure also relates to a method for preparing the aforementioned protein conjugate, the method comprising step (a): in the presence of a first catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of a second catalyst, react the product obtained in step (a) with... Contact; or,

[0341] The scheme includes step (a): in the presence of a second catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of the first catalyst, the product obtained in step (a) is reacted with... touch;

[0342] Nu and Nu' each independently contain ribonucleotides; It is a fucose derivative, which contains the first bioactive molecule. It is substituted galactose.

[0343] In some embodiments, the fucose derivative or substituted galactose is as defined in the first aspect.

[0344] In some embodiments, the starting glycan lacks core fucose. In other embodiments, the terminal end of the starting glycan (i.e., the end not connected to the protein) lacks galactose or a derivative thereof.

[0345] In some embodiments, the starting sugar chain is of the G0 sugar form.

[0346] The starting glycan lacking the core fucose, terminal galactose or its derivatives and / or GO glycoform described in this disclosure, and the protein containing the starting glycan, can be obtained by methods well known to those skilled in the art.

[0347] For example, a protein containing a core fucose can be contacted with an α-1,6-fucosidase to obtain a protein containing a starting glycan lacking a core fucose. In some embodiments, the α-1,6-fucosidase may be selected from α-L-fucosidase (Alfc), its functional variants, or any combination thereof. In some embodiments, the Alfc comprises the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 25.

[0348] For example, a protein containing terminal galactose in its glycan chain can be contacted with β-1,4-galactosidase to remove any terminal galactose residues, leaving terminal N-acetylglucosamine residues, thereby obtaining a starting glycan chain lacking terminal galactose. In some embodiments, the β-1,4-galactosidase is derived from human or Streptococcus pneumoniae, such as the protein with uniprot accession number P16278, or the protein with CAS number 9031-11-2, or a functional variant thereof, or any combination thereof. In some embodiments, the β-1,4-galactosidase comprises the amino acid sequence shown in SEQ ID NO: 26. Other methods for obtaining proteins with terminal galactose in their glycan chains can be found in patent document WO2017132298, which is incorporated herein by reference in its entirety.

[0349] Those skilled in the art also know that the starting glycan with the G0 glycoform can be obtained by sequentially contacting a protein containing a glycan with the aforementioned α-1,6-fucosidase and β-1,4-galactosidase; alternatively, the starting glycan with the G0 glycoform can be obtained by adding α-1,6-fucosidase and β-1,4-galactosidase together to the reaction system in a one-pot reaction.

[0350] In other embodiments, the starting glycans lacking core fucose, terminal galactose or its derivatives and / or G0 glycoform described herein can also be obtained by expression and purification using FUT8 or / and B4GALT1 gene knockout cell lines. One example of knocking out the FUT8 and / or B4GALT1 gene in cell lines is through homologous recombination, as described in Biotechnology and Bioengineering, 2004, 87(5):614-622; other examples of knocking out the FUT8 and / or B4GALT1 gene include the use of zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), as described in Biotechnology and Bioengineering, 2010, 106(5):774-783. Biotechnology and Bioengineering, 2013, 110(3):871-880. Nature Biotechnology, volume 33, pages 842-844 (2015). Int. J. Mol. Sci. 2015, 16(10), 23849-23866, etc.

[0351] In the preparation method disclosed herein, the first catalyst is a fucosyltransferase or a functional variant or fragment thereof. In some embodiments, the first catalyst is an α-1,6-fucosyltransferase or a functional variant or fragment thereof, which may be derived from different species, such as mammals, bacteria, and nematodes.

[0352] In some embodiments, the first catalyst is a human-derived α-1,6-fucosyltransferase or a functional variant or fragment thereof. Further, the first catalyst may be FUT8 (Uniprot ID: Q546E0) or a functional variant or fragment thereof.

[0353] In some embodiments, the first catalyst comprises the amino acid sequence shown in any one of SEQ ID NO: 1-3.

[0354] In some embodiments, the first catalyst comprises a functional variant or fragment of the amino acid sequence shown in any one of SEQ ID NO: 1-3.

[0355] In some embodiments, the first catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 1, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the first catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0356] In some embodiments, the first catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 2; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the first catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0357] In some embodiments, the first catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 3; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 3, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the first catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0358] In the preparation method disclosed herein, the second catalyst is a galactosyltransferase or a functional variant or fragment thereof. In some embodiments, the second catalyst is a β-1,4-galactosyltransferase or a functional variant or fragment thereof. The β-1,4-galactosyltransferase may be derived from different species, such as mammals, for example, from humans, cattle, and pigs.

[0359] In some embodiments, the second catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase, or a functional variant or fragment thereof. For example, the second catalyst may be B4GALT1 (Uniprot ID: P15291 or P08037) or a functional variant or fragment thereof.

[0360] In some embodiments, the second catalyst has acetylgalactose transfer function.

[0361] In some embodiments, the second catalyst comprises the amino acid sequence shown in any one of SEQ ID NO: 4-6 or 33.

[0362] In some embodiments, the second catalyst comprises a functional variant or fragment of the amino acid sequence shown in any one of SEQ ID NO: 4-6 or 33.

[0363] Those skilled in the art should understand that the galactosyltransferase shown in SEQ ID NO:4 is obtained by adding a short GS peptide to the N-terminus of the sequence shown in SEQ ID NO:33, and the catalytic ability and catalytic activity of the two are comparable with no significant difference.

[0364] In some embodiments, the second catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 4; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 4, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the second catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0365] In some embodiments, the second catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 5; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 5, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the second catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0366] In some embodiments, the second catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 6; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 6; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 6, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the second catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0367] In some embodiments, the second catalyst comprises (1) the amino acid sequence shown in SEQ ID NO: 33; (2) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 33; or (3) an amino acid sequence containing one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 33, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, or consisting thereof. In some embodiments, the second catalyst comprises or consists of the amino acid sequence shown in SEQ ID NO: 33.

[0368] In the preparation method disclosed herein, Nu and Nu' each independently comprise ribonucleotide diphosphate or a salt thereof; preferably, Nu and Nu' each independently comprise a structure selected from guanosine diphosphate (GDP), uridine diphosphate (UDP), cytidine diphosphate (CDP), and adenosine diphosphate (ADP), or a salt thereof:

[0369] In some embodiments, Nu comprises guanosine diphosphate (GDP) or a salt thereof, and Nu' comprises uridine diphosphate (UDP) or a salt thereof.

[0370] In some embodiments, Nu is guanosine diphosphate (GDP) or a salt thereof, and Nu' is uridine diphosphate (UDP) or a salt thereof.

[0371] In some implementations, the It has the structure shown in Formula VIII-A, or is a salt thereof:

[0372] Among them, BM1 and LU1 are as defined in the first aspect.

[0373] In some implementations, the It has the structure shown in formula VIII-B or VIII-C, or a salt thereof:

[0374] Among them, BM2 and LU2 are as defined in the first aspect.

[0375] In other implementations, the It has the structure shown in formula VIII-D or VIII-E, or is a salt thereof:

[0376] Wherein, X1 is a chemically active group X1, as defined in the first aspect.

[0377] When the When the product has the structure shown in Formula VIII-D or VIII-E, or a salt thereof, the preparation method of this disclosure may further include step (c): contacting the product obtained in step (b) with the compound shown in Formula IX.

[0378] BM2-L4-L3-L2-Y1(IX), where Y1 is a chemically active group selected from the following structures: L2, L3, L4 and BM2 are as defined in the first aspect.

[0379] In some embodiments, the chemically active groups X1 and Y1 can be coupled through a click chemical reaction.

[0380] In some embodiments, the chemically active groups X1 and Y1 may be selected from the following combinations:

[0381] (1) X1 includes: Y1 includes:

[0382] (2) X1 includes: Y1 includes:

[0383] (3) X1 includes: Y1 includes:

[0384] (4) X1 includes: Y1 includes:

[0385] (5)X1 includes: Y1 includes:

[0386] (6)X1 includes: Y1 includes: as well as

[0387] (7)X1 includes: Y1 includes:

[0388] In some implementations, -L4-L3-L2-Y1 in Formula IX can be selected from the following structures:

[0389] as well as

[0390] Where k1 is selected from an integer from 1 to 20, and k' is an integer from 0 to 10. In some implementations, 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.

[0391] In some implementations, BM2-L4-L3-L2-Y1 can be selected from the following structures:

[0392] as well as

[0393] Where k1 and k' are as defined previously.

[0394] Composition

[0395] Thirdly, this disclosure provides a composition, for example, preferably, a pharmaceutical composition, comprising one or a combination of conjugates of this disclosure formulated together with a pharmaceutically acceptable carrier.

[0396] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the conjugate of this disclosure, may be encapsulated in a material to protect the conjugate from acids and other natural conditions that could inactivate it.

[0397] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, expressed as 100%, ranges 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, combined with a pharmaceutically acceptable carrier.

[0398] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific conjugate of this disclosure or its salt applied, the route of administration, the time of administration, the excretion rate of the specific conjugate applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific conjugate applied, the age, sex, weight, condition, general health status, and medical history of the patient being treated, and similar factors known in the medical field.

[0399] In some embodiments, the DAR value of the protein conjugate composition disclosed herein can be divided into two parts: firstly, the DAR value of the bioactive molecule linked to the fucosylation group is about 0.5 to 8.0, preferably about 0.5 to 4.0, more preferably about 1.0 to 3.0, and even more preferably about 1.5 to 2.5. For example, in the protein conjugate composition, the DAR value of the bioactive molecule linked to the fucosylation group 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, or about 3.0. Furthermore, the DAR value of the bioactive molecule linked to the galactose group is about 1.0 to 12.0, preferably about 2.0 to 8.0, more preferably about 3.0 to 6.0, and even more preferably about 3.5 to 4.5. For example, in the protein conjugate composition, the DAR value of the bioactive molecule linked to the galactose group is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0.

[0400] Based on the above two parts, the overall DAR value of the protein conjugate composition disclosed herein is about 2.0 to 16.0, preferably about 3.0 to 12.0, more preferably about 4.0 to 8.0, and even more preferably about 5.0 to 7.0. For example, the overall DAR value of the protein conjugate composition is approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, and approximately 8.0.

[0401] The compositions of this disclosure can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the conjugates of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteric and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0402] This disclosure also provides pharmaceutical combinations or combination products or kits comprising conjugates of this disclosure or pharmaceutical compositions thereof, and one or more other therapeutic agents, such as any therapeutic agent effective for treating and / or preventing tumors or cancers, such as cytotoxic agents, chemotherapeutic agents, small molecule compounds, angiogenic inhibitors, other immunotherapeutic agents such as other antibodies, or other conjugates, etc.

[0403] When this disclosure refers to "drug combination" or "combination product," it includes, but is not limited to, a pillbox or a pharmaceutical composition. Drug combination encompasses both non-fixed and fixed combinations. The term "non-fixed combination" refers to the simultaneous, unspecified, or sequential administration of active ingredients in separate entities at the same or different time intervals. The term "fixed combination" means that two or more active agents are administered simultaneously to a patient in the form of a single entity. When referring to "drug combination" or "combination product," it also covers situations where two active ingredients are present in different formulations but are administered in combination.

[0404] This disclosure also provides pharmaceutical compositions or combinations thereof described above for the treatment and / or prevention of tumors or cancer.

[0405] Treatment methods and uses

[0406] Fourthly, this disclosure also provides a method for treating and / or preventing tumors or cancers, such as solid tumors or non-solid tumors, comprising administering the conjugate of this disclosure or a pharmaceutical composition thereof to a patient in need. This application also provides a method for treating and / or preventing tumors or cancers, such as advanced or metastatic solid malignancies, comprising administering the conjugate of this disclosure or a pharmaceutical composition thereof to a patient in need.

[0407] In some embodiments, the conjugates or pharmaceutical compositions thereof disclosed herein may also be administered in combination with one or more therapies. Therapies may be any treatment or prevention effective in tumors or cancers, such as treatments (e.g., radiation therapy or surgery), or other therapeutic agents such as cytotoxic agents, chemotherapeutic agents, small molecule compounds, angiogenic inhibitors, other immunotherapeutic agents such as other antibodies, or other conjugates.

[0408] The descriptions of "administering a combination of drugs" or "combining drugs" in this disclosure include both cases of simultaneous administration of multiple drugs and cases of sequential or separate administration of multiple drugs. When administered sequentially, the interval between administrations of the multiple drugs shall not exceed 24 hours, for example, not exceeding 18 hours, not exceeding 15 hours, not exceeding 12 hours, not exceeding 10 hours, not exceeding 8 hours, not exceeding 5 hours, not exceeding 3 hours, not exceeding 2 hours, not exceeding 1 hour, or not exceeding 0.5 hours.

[0409] On the other hand, this disclosure also relates to the use of the conjugate in the preparation of a medicament for the treatment and / or prevention of tumors. In some embodiments, the tumor includes solid tumors and / or non-solid tumors.

[0410] In some embodiments, the tumor is cancer. In some embodiments, the cancer is in the early, middle, or late stage, or it is metastatic.

[0411] In some embodiments, the tumor or cancer is a tumor-associated antigen (TAA) positive and / or mediated by it. For example, the TAA may be selected from EGFR and / or HER3.

[0412] In some embodiments, the TAA-positive tumor or cancer refers to an individual suffering from the tumor or cancer that contains abnormally expressed TAA or abnormal TAA activity. In some embodiments, the individual has elevated levels of TAA-encoding nucleic acid or elevated levels of TAA protein expression or activity compared to a control, for example, the control being the level of TAA-encoding nucleic acid or the protein expression or activity of TAA in a healthy individual or healthy tissue or cells. In some embodiments, the individual's biological sample (e.g., tumor cells or tumor tissue) contains (e.g., elevated levels, such as nucleic acid or protein levels or activity) TAA (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to TAA in adjacent healthy tissue or cells of the subject).

[0413] In some implementations, the tumor or cancer is selected from lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, stomach cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial cancer, and osteosarcoma, etc.

[0414] With regard to the use of the protein conjugates of this disclosure, this disclosure also provides the use of the protein conjugates, conjugates or pharmaceutical compositions of this disclosure in the preparation of medicaments for treating and / or preventing tumors or cancers, such as the aforementioned tumors or cancers, wherein the protein conjugates are optionally administered in combination with one or more of the aforementioned therapies.

[0415] Example

[0416] Example 1: Preparation of the compound

[0417] 1.1 Synthesis of LP1(DBCO-PEG4-VA-AM-CPT2)

[0418] 1.1.1 Synthesis of CPT2

[0419] 3 g (7.65 mmol, 1.0 eq) of 10,11-methylenedioxycamptothecin (CAS: 135415-73-5) was dissolved in methanol (80 mL) and water (70 mL). The reaction solution was cooled to 0 °C, and 60 mL of 75% H₂SO₄ was added, followed by 6.38 g (22.95 mmol, 3.0 eq) of FeSO₄·7H₂O. 15 mL of 30% H₂O₂ was slowly added dropwise at 0 °C, and the reaction solution was stirred at room temperature for 16 h. LC-MS analysis showed that the reaction was complete, and the reaction was stopped. The reaction solution was poured directly into ice water, filtered, and the filter cake was dried to obtain 3.1 g of crude product, a yellowish-brown solid. The crude product was slurried with DMF to obtain 1.2 g of yellow solid compound CPT₂, with a yield of 37%. ESI-MS (+) m / z = 423.1 [M+H] + .

[0420] 1.1.2 Synthesis of Int1

[0421] CPT2 (500 mg, 1.184 mmol, 1.0 eq) and (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecane-11-ylacetate (CAS: 2505045-86-1) (683 mg, 1.421 mmol, 1.2 eq) were dissolved in 10 mL DMSO, and BF3Et2O (500 mg, 3.552 mmol, 3.0 eq) was added. The reaction mixture was allowed to react overnight at room temperature. The reaction solution was quenched in an ice-cold aqueous solution of NaHCO3, resulting in the formation of a large amount of solid. The mixture was filtered, the filter cake was washed with water, and the filter cake was lyophilized to obtain 500 mg of crude yellow solid compound Int1. The crude product was used directly in the next reaction. ESI-MS (+) m / z = 844.2 [M+H] + .

[0422] 1.1.3 Synthesis of Int2

[0423] Compound Int1 (3.0 g, 3.55 mmol, 1.0 eq) was dissolved in DMF (6 mL), and triethylenediamine (3.99 g, 35.55 mmol, 10 eq) was added. After addition, the mixture was stirred at room temperature for 3 h. LC-MS showed that the reaction was complete. The reaction solution was directly and rapidly separated by RP flash chromatography (ACN in H2O 30%, 0.1% TFA). The solution was lyophilized to give 220 mg of yellow solid compound A2. The overall yield of the two steps was 29%. ESI-MS(+) m / z = 622.2 [M+H] + .

[0424] 1.1.4 Synthesis of LP1

[0425] Compound Int2 (100 mg, 94.15 μmol, 1.0 eq) was dissolved in DMF (6 mL). DBCO-PEG4-COOH (CAS: 1537170-85-6) (35 mg, 0.053 mmol, 1.2 eq), DIEA (61 mg, 0.47 mmol, 5 eq), and HATU (47 mg, 0.122 mmol, 1.3 eq) were added under ice bath conditions. The reaction was allowed to proceed at room temperature for 1 h. LC-MS showed the reaction was complete. The reaction solution was filtered and purified by high-performance liquid chromatography (Ultimate XB-C18, 50*250 mm, 10 μm, 0.1% FA, ACN in H2O 35%–65%). The prepared solution was lyophilized to obtain a yellow solid compound LP1 (55 mg), yield 50%, purity 98%. ESI-MS (+) m / z = 1156.4 [M+H] + .

[0426] 1 H NMR (400MHz, DMSO): δ8.83(t,J=6.3Hz,1H),8.13(d,J=6.5Hz,1H),7.80(d,J=8.9Hz,1H),7.74(t,J=5.9Hz,1H),7.69–7.64(m,1H),7.60(d,J=6.7Hz,1 H),7.52(s,1H),7.48(d,J=6.2Hz,2H),7.46–7.43(m,1H),7.39–7.30(m,2H ),7.30–7.26(m,1H),7.25(s,1H),6.48(s,1H),6.29(s,2H),5.42(s,2H),5 .29(d,J=5.2Hz,2H),5.10–4.97(m,3H),4.82–4.72(m,2H),4.24–4.15(m,2 H),3.61–3.54(m,3H),3.50–3.40(m,12H),3.27(d,J=6.2Hz,1H),3.12–2.9 7(m,2H),2.62–2.52(m,2H),2.46–2.34(m,2H),2.27–2.17(m,1H),2.08–1. 59(m,5H),1.18(d,J=7.2Hz,3H),0.90–0.82(m,6H),0.79(d,J=6.8Hz,3H).

[0427] Using a similar method as described above, LP1-P8(DBCO-PEG8-VA-AM-CPT2) was prepared:

[0428] ESI-MS(+)m / z = 1332.5 [M+H].

[0429] 1H NMR (400MHz, DMSO) δ8.85(t,J=6.7Hz,1H),8.16(d,J=6.9Hz,1H),7.83(d,J=8.8Hz,1H),7.77(t,J=5.7Hz,1H),7.70-7.68(m,1H),7.63(d,J= 6.4Hz,1H),7.54(s,1H),7.53–7.44(m,4H),7.21-7.30(m,3H),7.27(s ,1H),6.51(s,1H),6.31(s,2H),5.44(s,2H),5.40–5.23(m,2H),5.13–4 .98(m,3H),4.83-4.76(m,2H),4.25-4.20(m,2H),3.63-3.58(m,3H),3 .52–3.44(m,28H),3.15–3.05(m,2H),2.65-2.55(m,1H),2.48-2.43(m, 1H),2.43-2.33(m,2H),2.29-2.21(m,1H),2.06–1.93(m,2H),1.92-1. 84(m,2H),1.82-1.73(m,1H),1.20(d,J=7.1Hz,3H),0.92–0.79(m,9H).

[0430] 1.2 Synthesis of LP2 (Fucose-C1-VC-PABc-MMAE)

[0431] 1.2.1 Synthesis of Int3

[0432] 3-Butynedic acid (CAS: 2345-51-9) (25 mg, 1.0 eq) was added to DCM (15 mL), and after cooling to 0 °C, EEDQ (8.2 mg, 1.25 eq) was added. The reaction was stirred at 0 °C for 15 minutes, and then Val-Cit-PAB-MMAE (purchased from Haoyuan Pharmaceutical, CAS: 644981-35-1) (267.3 mg, 0.8 eq) was added. The reaction was stirred at 0 °C for 2 hours to complete. The reaction solution was concentrated and directly sent to a reverse-phase reactor for purification (TFA, Ultimate XB-C18, 50*200 mm, 10 μm, 0.1% TFA, ACN in H2O from 40%-70%). Lyophilization yielded a light brown solid compound, Int3130 mg, with a yield of 43%. ESI-MS (+) m / z = 1189.6 [M+H] + .

[0433] 1H NMR (400MHz, DMSO) δ10.00(s,1H),8.19(t,J=44.0Hz,2H),7.95–7.54(m,4H),7.37–7.10(m,7H),6.17–5.86(m,1H),5.03(dd,J=24.0,12.6Hz,2H), 4.73(s,1H),4.64(s,1H),4.48(d,J=5.8Hz,1H),4.40(dd,J=20.9,6.2Hz, 2H),4.26(dd,J=13.9,9.7Hz,2H),3.99(d,J=6.6Hz,3H),3.56(d,J=23.4H z,2H),3.47(s,1H),3.31(d,J=10.4Hz,1H),3.22(dd,J=12.2,5.4Hz,7H), 3.14(d,J=23.9Hz,3H), 3.00(dq,J=14.8,10.0Hz,5H), 2.86(d,J=14.5Hz, 3H), 2.41 (d, J = 16.2Hz, 1H), 2.28 (d, J = 9.3Hz, 1H), 2.04 (ddd, J = 20.1, 15. 0,6.7Hz,4H),1.88–1.24(m,10H),1.06–0.96(m,6H),0.92–0.69(m,24H).

[0434] 1.2.2 Synthesis of LP2

[0435] Int3 (56 mg, 0.047 mmol) was dissolved in MeOH-H2O (1:4, 5 mL), and GDP-6-N3-fucose (100 mM, 0.56 mL) was added. A solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added to the reaction mixture, and the reaction was incubated at 37 °C for 3 hours. After completion, the reaction mixture was concentrated under vacuum, and the residue was purified by C18 column chromatography to give the target product LP2 8 mg, in a yield of 9.4%.

[0436] HRMS(ESI): m / z calcd for C 78 H 122 N 18 O 28 P2[M+2H] 2+ 910.4070; found 910.4071.

[0437] 1H NMR(400MHz,D2O)δ8.07(s,1H),7.97(d,J=3.4Hz,1H),7.49–7.30(m,9H),7.24(t,J=6.9Hz,1H),5.87(d,J=6.2Hz,1H),5.36-5.18(m,1H),5. 12-5.02(m,1H),4.88(m,1H),4.66–4.55(m,2H),4.53-4.29(m,7H),4. 25–4.04(m,6H),3.97-3.90(m,1H),3.85–3.59(m,6H),3.52–3.27(m,8 H),3.19(s,1H),3.12–3.01(m,4H),2.96(dd,J=14.7,7.0Hz,3H),2.59–2.31(m,2H),2.25–1.99(m,3H),1.98–1.74(m,5H),1.69–1.44(m,4H) ,1.31(d,J=6.4Hz,3H),1.18(t,J=7.4Hz,2H),1.09(d,J=6.7Hz,2H),1.04–0.77(m,24H),0.68(dd,J=13.7,6.5Hz,2H),0.50(t,J=5.9Hz,1H)

[0438] 1.3 Synthesis of LP3 (Fucose-C5-VC-PABc-MMAE)

[0439] 1.3.1 Synthesis of Int4

[0440] Under nitrogen protection, 7-(tert-butoxy)-7-oxoheptanoic acid (CAS: 1469894-57-2) (196.3 mg, 1.0 eq), HATU (414.2 mg, 1.2 eq), and DIEA (234.7 mg, 2.0 eq) were added sequentially to DMF (5 mL). The reaction mixture was stirred at room temperature for 5 minutes, followed by the addition of propyneamine (CAS: 2450-71-7) (50 mg, 1 eq). The reaction mixture was stirred overnight at room temperature. The reaction solution was washed and separated with saline (50 mL) and MTBE (20 mL * 3). The organic phase was dried over anhydrous sodium sulfate, concentrated, and the crude product was loaded onto a column using a wet chromatography method. Column chromatography (PE / EA = 2 / 1) yielded a light brown oily substance, Int 465 mg, with a yield of 28%. MS-ESI: m / z 529.3 [2M + Na] +

[0441] 1.3.2 Synthesis of Int5

[0442] Int4 (65 mg) was added to formic acid (5 mL) at room temperature, and the reaction was completed after stirring at room temperature for 3 h. The solution was then concentrated under reduced pressure at 55 °C using a direct oil pump to obtain a white crude product, Int5, 51 mg. MS-ESI: m / z 198.1 [M+H] +

[0443] 1.3.3 Synthesis of Int6

[0444] Int5 (45 mg, 1 eq) was added to 15 mL of DCM, and after cooling to 0 °C, EEDQ (67.7 mg, 1.2 eq) was added. The reaction mixture was stirred at 0 °C for 15 minutes, and then Val-Cit-PAB-MMAE (267.3 mg, 1 eq) was added. The reaction mixture was stirred at 0 °C for 3 hours to complete the reaction. The reaction solution was concentrated and directly sent to a reverse-phase reactor for purification (TFA, Ultimate XB-C18, 50*200 mm, 10 μm, 0.1% TFA, ACN in H2O from 40%-70%). Lyophilization yielded 170 mg of a light brown solid, Int6, with a yield of 57%. MS-ESI: m / z 652.1 [M / 2+H] +

[0445] 1 H NMR(400MHz, DMSO)δ9.98(s,1H),8.38–7.99(m,3H),7.72(ddd,J=27.9,27.2,8.3Hz,4H),7.38–7.11(m,7H),6.02(s,1H),5.11–4.94(m, 2H),4.68(d,J=37.4Hz,1H),4.48(d,J=5.8Hz,1H),4.44–4.16(m,5H),3.97(dd,J=18.4,7.4Hz,3H),3.82(dd,J=5.4,2.5Hz,3H),3.78(s, 2H),3.27(dd,J=30.8,8.2Hz,5H),3.21–3.08(m,5H),3.02(ddd,J=30.8,14.6,7.2Hz,5H),2.86(d,J=14.7Hz,3H),2.41(d,J=15.6Hz,1H ),2.31–1.91(m,9H),1.86–1.64(m,4H),1.64–1.27(m,10H),1.25–1.15(m,2H),1.07–0.97(m,6H),0.80(ddd,J=26.2,12.6,7.0Hz,24H).

[0446] 1.3.4 Synthesis of LP3

[0447] Int6 (56 mg, 0.043 mmol) was dissolved in MeOH-H2O (1:4, 5 mL), and GDP-6-N3-fucose (100 mM, 0.56 mL) was added. A solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added to the reaction mixture, and the reaction was incubated at 37 °C for 3 hours. After completion, the reaction mixture was concentrated under vacuum, and the residue was purified by C18 column chromatography to give the target product LP3 46 mg, with a yield of 55.4%.

[0448] HRMS(ESI): m / z calcd for C 84 H 133 N 19 O 29 P2[M+2H] 2+ 966.9490; found 966.9493.

[0449] 1 H NMR(400MHz,D2O)δ8.12(s,1H),7.97(s,1H),7.50–7.29(m,9H),7.24(t,J=7.2Hz,1H),5.88(d,J=5.8Hz,1H),5.34–5.17(m,1H),5.11–5.0 1(m,1H),4.92–4.85(m,1H),4.67–4.30(m,10H),4.26–4.04(m,6H),3.97(dd,J=7.6,4.6Hz,1H),3.84(s,1H),3.74-3.61(m,3H),3.52-3.2 8(m,8H),3.19(s,1H),3.14–3.06(m,4H),2.95(dd,J=15.6,5.1Hz,3H),2.58-2.45(m,1H),2.30–2.13(m,5H),2.09–1.98(m,2H),1.93–1.7 4(m,5H),1.70-1.45(m,9H),1.35-1.16(m,8H),1.09(d,J=6.6Hz,2H),1.02-0.77(m,24H),0.69(dd,J=13.6,6.3Hz,2H),0.53-0.47(m,1H).

[0450] 1.4 Synthesis of LP4 (Fucose-PEG4-VC-PABc-MMAE)

[0451] 1.4.1 Synthesis of Int7

[0452] Under nitrogen protection, Acid-PEG4-C2-Boc (CAS: 1835759-85-7) (286.3 mg, 0.9 eq), HATU (414.2 mg, 1.2 eq), and DIEA (234.7 mg, 2.0 eq) were added sequentially to DMF (15 mL). The reaction mixture was stirred at room temperature for 5 minutes, followed by the addition of propyneamine (50 mg, 1 eq), and the reaction was stirred overnight at room temperature. The reaction solution was washed and separated with 50 mL of saline and MTBE (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by wet column chromatography (PE / EA = 1 / 3) to obtain a light brown oily substance, Int7, 150 mg, with a yield of 42%. MS-ESI: m / z 388.2 [M+H] +

[0453] 1.4.2 Synthesis of Int8

[0454] 150 mg of Int7 was added to 10 mL of formic acid at room temperature, and the reaction was completed after stirring at room temperature for 3 hours. The solution was then concentrated under reduced pressure using a direct oil pump at 45 °C to obtain 110 mg of off-white crude Int8. MS-ESI: m / z 332.2 [M+H]

[0455] 1.4.3 Synthesis of Int9

[0456] Int8 (110 mg, 1 eq) was added to 15 mL of DCM, and after cooling to 0 °C, EEDQ (98.5 mg, 1.2 eq) was added. The reaction mixture was stirred at room temperature for 30 minutes, and then Val-Cit-PAB-MMAE (372.9 mg, 1 eq) was added. The reaction mixture was stirred at room temperature overnight. The reaction solution was concentrated and directly purified by reverse-phase chromatography (Kinetex EVO C18, 30*250 mm, 5 μm, ACN in H2O from 30%-60%). Lyophilization yielded 88 mg of off-white solid Int9, with a yield of 26%. MS-ESI: m / z 719.0 [M / 2+H] +

[0457] 1H NMR(400MHz, DMSO)δ9.98(s,1H),8.14(dd,J=48.5,40.9Hz,3H),7.91–7.54(m,4H),7.24(ddd,J=34.4,14.8,7.1Hz,7H),5.96(s,1H),5.3 7(d,J=24.0Hz,3H),5.02(dd,J=37.8,13.7Hz,2H),4.68(d,J=41.2Hz,1H),4.41(dd,J=28.7,15.3Hz,3H),4.31–4.18(m,2H),3.88(tdd,J= 31.1,23.1,8.3Hz,5H),3.58(t,J=6.2Hz,5H),3.48(s,12H),3.30(s,4H),3.15(ddd,J=15.0,11.3,4.2Hz,9H),2.94(ddd,J=33.7,23.9,10 .6Hz,6H),2.47–2.21(m,5H),2.18–1.89(m,4H),1.70(s,4H),1.47(ddd,J=42.8,28.6,9.4Hz,6H),1.07–0.96(m,6H),0.96–0.68(m,24H).

[0458] 1.4.4 Synthesis of LP4

[0459] Int9 (53 mg, 0.037 mmol) was dissolved in H2O (5 mL), and GDP-6-N3-fucose (100 mM, 0.45 mL) was added. A solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added to the reaction mixture, and the reaction was incubated at 37 °C for 3 hours. After completion, the reaction mixture was concentrated under vacuum, and the residue was purified by C18 column chromatography to give the target product LP4 51 mg, with a yield of 66.7%.

[0460] HRMS(ESI): m / z calcd for C 89 H 143 N 19 O 33 P2[M+2H] 2+ 1033.9780; found 1033.9783.

[0461] 1H NMR(400MHz,D2O)δ8.08(s,1H),7.96(s,1H),7.50-7.30(m,9H),7.24(t,J=7.2Hz,1H),5.87(d,J=6.2Hz,1H),5.35–5.17(m,1H),5.13-5.02(m ,1H),4.91-4.84(m,1H),4.67–4.31(m,10H),4.24–4.06(m,5H),3.97( dd,J=7.7,4.9Hz,1H),3.82(s,1H),3.79–3.59(m,21H),3.54-3.27(m,8 H),3.20(s,1H),3.15–3.06(m,4H),2.96(dd,J=15.8,7.1Hz,3H),2.67 –2.45(m,5H),2.24–2.00(m,3H),1.97–1.75(m,5H),1.71–1.47(m,4H), 1.31(d,J=6.5Hz,3H),1.19(dd,J=11.6,4.8Hz,2H),1.09(d,J=6.7Hz,2H),1.03–0.78(m,24H),0.70(dd,J=14.5,6.6Hz,2H),0.55–0.48(m,1H)

[0462] 1.5 Synthesis of LP5 (Fucose-PEG4-β-glucuronide-PABc-MMAE)

[0463] 1.5.1 Synthesis of Int10

[0464] Bis-PEG4-acid (CAS: 31127-85-2) (5g, 16.9mmol, 1.0eq) was added to a 250mL four-necked flask. Under nitrogen protection, DCM (50mL) was added, and stirring was started. EDCI (3.91g, 20.3mmol, 1.2eq), HOBt (459mg, 3.40mmol, 0.2eq), and NMM (8.59g, 84.9mmol, 5.0eq) were added to the reaction system in one batch. Then, propyneamine (935mg, 16.9mmol, 0.9eq) was dissolved in DCM (5mL) and added dropwise to the above reaction system at room temperature (20°C). After the addition was complete, the reaction system was stirred at room temperature (20°C) for 12 hours. TLC monitoring of the reaction system (DCM:MeOH = 10:1) showed that the starting materials had reacted completely. The reaction system was then directly purified by wet chromatography onto a silica gel column to obtain compound Int10 2.1 g, with a yield of 37.5%.

[0465] 1.5.2 Synthesis of Int11

[0466] Int10 (1.85 g, 5.58 mmol, 1.0 eq) was added to a 250 mL four-necked flask. Under nitrogen protection, 20 mL of DCM was added, and stirring was started. MAC glucuronide linker-2 (CAS: 229977-57-5) (0.933 g, 2.05 mmol, 0.37 eq) and EEDQ (1.04 g, 4.19 mmol, 0.75 eq) were added to the reaction system in a single batch. The reaction system was stirred at room temperature (20 °C) for 12 hours. TLC monitoring (DCM:MeOH = 10:1) showed that the starting material had reacted completely, and product spot formation was observed. The reaction system was directly purified by wet column chromatography onto a silica gel column to obtain 1.1 g of compound Int11, with a yield of 25.8%.

[0467] 1.5.3 Synthesis of Int12

[0468] Int11 (0.75 g, 975 μmol, 1.0 eq) was added to a 100 mL four-necked flask. Under nitrogen protection, THF (7.5 mL) was added, and stirring was started. Di(p-nitrobenzene) carbonate (CAS: 5070-13-3) (593 mg, 1.95 mmol, 2.0 eq) and DIEA (189 mg, 1.46 mmol, 1.5 eq) were added to the reaction system in a single addition. The reaction system was stirred at room temperature (20 °C) for 12 hours. TLC monitoring (DCM:MeOH = 10:1) showed that the starting material had reacted completely, and product spot formation was observed. The reaction system was directly purified by wet column chromatography using silica gel to obtain compound Int120.71 g, with a yield of 58.6%.

[0469] 1.5.4 Synthesis of Int13

[0470] Int12 (0.1 g, 107 μmol, 1.0 eq) was added to a 100 mL four-necked flask. Under nitrogen protection, DMF (5 mL) was added, and stirring was started. MMAE (84.5 mg, 117.7 μmol, 1.1 eq) and DIEA (41.5 mg, 321 μmol, 3.0 eq) were added to the reaction system in a single batch. The reaction system was stirred at 20 °C for 2 hours. TLC monitoring (DCM:MeOH = 20:1) showed that the starting materials had reacted completely, and product spot formation was observed. The reaction system was directly purified by fractional lyophilization to obtain compound Int13 (0.12 g), with a yield of 75.1%.

[0471] 1.5.5 Synthesis of Int14

[0472] Int13 (150 mg, 99.1 μmol, 1.0 eq) was added to a 100 mL four-necked flask. Under nitrogen protection, THF (4.5 mL) was added, and stirring was started. An aqueous solution of lithium hydroxide (5.0 eq LiOH in 1.05 mL water) was added to the reaction system in one go. The reaction system was stirred at room temperature (20 °C) for 12 hours. TLC monitoring of the reaction system (DCM:MeOH = 10:1) showed that the starting material had reacted completely and product spots had formed. The reaction system was directly reversed for preparation. The fraction was lyophilized to obtain compound Int14 77 mg, with a yield of 57.1%. MS-ESI: m / z 1373.0 [M+H] +

[0473] 1 H NMR(400MHz,Chloroform-d)δ9.00(s,1H),8.34(s,1H),7.33(m,3H),7.23(m,1H),7.15(m,1H),7.03(m,1H),6.73(m,1H),6.3 8(m,1H),5.22(d,J=12Hz,1H),5.09–4.93(m,1H),4.91(m,1H),4.63(m,2H),4.34–3.93(m,6H),3.88(m,1H),3.77(m,4H),3.7 0(m,2H),3.61(m,6.6Hz,14H),3.50(m,6H),3.38(m,3H),3.31(m,3H),3.22–2.78(m,6H),2.72–2.14(m,8H),2.01(m,2H),1.8 5(m,3H),1.54(d,J=8Hz,1H),1.39–1.28(m,4H),1.25–1.15(m,4H),1.01-0.95(m,5H),0.91–0.76(m,10H),0.71-0.68(m,2H).

[0474] 1.5.6 Synthesis of LP5

[0475] Int14 (77 mg, 0.056 mmol) was dissolved in H₂O (5 mL), and GDP-6-N₃-fucose (100 mM, 0.67 mL) was added. A solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added to the reaction mixture, and the reaction was incubated at 37 °C for 3 hours. After completion, the reaction was concentrated under vacuum, and the residue was purified by liquid chromatography using a C18 column. The target product LP₅ was given in a yield of 58 mg (51.7%).

[0476] HRMS(ESI): m / z calcd for C84 H 131 N 15 O 37 P2[M+2H] 2+ 1001.9147; found 1001.9148.

[0477] 1 H NMR(400MHz,D2O)δ8.28–8.04(m,2H),7.96(s,1H),7.94–7.83(m,1H),7.42–7.2 9(m,4H),7.28-7.16(m,3H),5.89(d,J=5.7Hz,1H),5.30–5.15(m,1H),5.12-4.96 (m,2H),4.91-4.85(m,1H),4.76(m,1H),4.70–4.61(m,2H),4.59–4.31(m,7H),4 .26–4.06(m,4H),4.01–3.85(m,4H),3.81(s,1H),3.74–3.43(m,22H),3.38–3.20 (m,8H),3.11(d,J=6.4Hz,1H),3.00-2.89(m,3H),2.79-2.71(m,2H),2.58–2.33 (m,4H),2.24–2.00(m,2H),1.91–1.78(m,2H),1.74-1.47(m,2H),1.31(d,J=6.5H z,3H),1.17(d,J=6.6Hz,2H),1.09(d,J=6.7Hz,2H),0.97(dd,J=21.4,6.5Hz,3H ),0.91–0.79(m,12H),0.72(dd,J=20.4,6.5Hz,2H),0.53(dd,J=25.7,6.4Hz,1H)

[0478] 1.6 Synthesis of LP6 (Fucose-PEG3-VC-PABc-MMAE)

[0479] Compound Int15 was prepared by referring to the method in published patent WO2012153193.

[0480] Int15 (50 mg, 0.038 mmol) was dissolved in a methanol-water mixture (1:3, 4 mL), and GDP-6-N3-fucose (100 nM, 0.46 mL) was added. Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) solution were added to this reaction mixture, and the reaction was carried out at 37 °C for 4 hours. After the reaction was complete, the mixture was concentrated under vacuum, and the residue was purified by P2 and C18 column chromatography to give 9.2 mg of compound LP6, 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.

[0481] 1 H NMR(400MHz,D2O)δ8.13-8.03(m,2H),7.48–7.20(m,9H),5.86(d,J=5.9Hz,1H),5.32–5.16(m,1H),5.09–4.99(m ,1H),4.90(t,J=7.0Hz,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=6.4Hz,3H),1.18(t,J=7.6Hz,3H),1.12-0.78(m,24H),0.69(dd,J=15.9,6.3Hz,2H),0.53-0.46(m,1H).

[0482] 1.7 Synthesis of LP7 (Fucose-PEG4-MMAE)

[0483] 1.7.1 Synthesis of Int16

[0484] Int8 (100 mg, 0.302 mmol) and HATU (115 mg, 0.302 mmol) were dissolved in DMF (5 mL). After stirring until dissolved, DIEA (78 mg, 0.604 mmol) was added, followed by MMAE (217 mg, 0.302 mmol). After the addition was complete, the temperature 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%). The prepared solution was lyophilized to obtain 190 mg of white solid product Int16, yield: 61%. ESI-MS(+) m / z = 1031.6 [M+H].

[0485] 1 H NMR (400MHz, DMSO) δ8.60(t,J=9.9Hz,1H),8.30(t,J=5.2Hz,1H),7.95–7.58(m,1H),7. 26(ddd,J=32.9,9.5,4.9Hz,5H),5.39(dd,J=28.4,5.0Hz,1H),4.83–4.60(m,1H),4.58– 4.39(m,2H),4.10–3.93(m,3H),3.87(dd,J=5.4,2.5Hz,2H),3.80(d,J=9.5Hz,1H),3.71 –3.58(m,5H),3.57–3.42(m,13H),3.33(s,1H),3.26(d,J=7.4Hz,4H),3.21(dd,J=9.6,3 .6Hz,3H),3.10(ddd,J=19.5,11.8,5.3Hz,3H),2.98(dd,J=11.9,8.0Hz,2H),2.88(s,1 H),2.85–2.76(m,1H),2.72–2.53(m,2H),2.44(d,J=16.6Hz,1H),2.31(dt,J=28.0,7.9H z,3H),2.16(dt,J=21.0,10.2Hz,2H),2.08–1.91(m,1H),1.78(dd,J=25.1,9.9Hz,3H),1 .64–1.42(m,2H),1.33(s,1H),1.03(ddd,J=14.8,12.0,6.6Hz,7H),0.95–0.73(m,18H).

[0486] 1.7.2 Synthesis of LP7

[0487] Int16 (100 mg, 0.097 mmol) was dissolved in a MeOH-H2O (1:5, 10 mL) mixture, and GDP-6-N3-fucose (100 mM, 1.2 mL) was added. A solution of Cu / THPTA (1 / 2, 2 mM) and sodium ascorbate (4 mM) was added to the reaction mixture, and the reaction was incubated at 37 °C for 3 hours. After the reaction was complete, the reaction mixture was concentrated under vacuum, and the residue was purified by HPLC to give 105 mg of target compound 1, with a yield of 65.2%. HRMS (ESI): m / z calcd for C 70 H 113 N 14 O 28 P2[MH]-1659.7329; found 1659.7331.

[0488] 1 H NMR(600MHz,D2O)δ8.09(s,1H),7.98–7.93(m,1H),7.43–7.22(m,5H),5.89(d,J=5.7Hz,1H),4.93-4.88(m,1H),4.69–4.6 3(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,16 H),3.51(dd,J=15.2,7.4Hz,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).

[0489] 1.8 Synthesis of LP8(DBCO-PEG4-GGFG-AM-DXd)

[0490] The following linker-toxin (LP8) is prepared according to the process described in patent application CN113264983A (the contents of which are incorporated herein by reference):

[0491] Example 2, Antibody Expression

[0492] 2.1 Construction of Recombinant Plasmids

[0493] Referring to the Duligotuzumab amino acid sequence published in WHO Drug Information, Vol. 28, No. 3, 2014 Recommended INN: List 72, the VH and VL genes were synthesized to obtain nucleotide sequences expressing the heavy chain variable region and the light chain variable region, named MEHD VH and MEHD VL, respectively. MEHD VH was cloned into the plasmid pcDNA3.4-hIgG1 containing the human IgG1 constant region, resulting in the plasmid pcDNA3.4-MEHDVH-IgG1CH expressing the MEHD antibody heavy chain; the MEHD VL gene was cloned into the plasmid pcDNA3.4-KappaCL containing the human Kappa light chain constant region, resulting in the plasmid pcDNA3.4-MEHDVL-KappaCL expressing the MEHD antibody light chain. Sequencing confirmed sequence correctness. Using a similar method, recombinant antibody plasmids with AG and / or YTE mutations in the Fc region were prepared.

[0494] 2.2 Instantaneous Transition Expression

[0495] The MEHD heavy and light chain expression plasmids were transiently transfected into CHO cells. The transfection method was as follows: In a 100 ml transient transfection system, the cell density was first adjusted to 6 × 10⁶ cells / mL using expression medium (BalanCD Transfectory CHO, purchased from Fujifilm Biosciences). 6 Cells / mL were prepared for use. Then, PEI and plasmid (150ug) were mixed at a mass ratio of 10:1 and allowed to stand at room temperature for 5 minutes to form a complex. The complex was then added to the prepared cell suspension and incubated at 36.5±0.5℃, 7%±3% CO2, and 100±10rpm for 4 hours. The incubation speed was then adjusted to 130±10rpm and the incubation was continued. After 24 hours, the temperature was lowered to 32℃ and the incubation was continued for 10 days, with supplemental feeding added as needed.

[0496] 2.3 Purification of recombinant antibodies

[0497] After transient cell culture, the cells were centrifuged at 4000 rpm for 10 min at 4℃, and the cell supernatant was collected. The supernatant particles were removed by filtration through a 0.22 μm membrane. The treated supernatant was loaded onto a Protein A affinity column (AT Protein A Diamond) equilibrated with equilibration buffer (10 mM PB, pH 6.0). Unadsorbed impurities were removed by rinsing with the same buffer, and weakly adsorbed impurities were washed off the column with a high-salt buffer (25 mM PB, 500 mM NaCl, pH 7.0). The target protein was eluted with elution buffer (20 mM citrate buffer, pH 3.6), and the pH of the eluted protein was adjusted to 6.0 with neutralization buffer (2 M Tris-HCl, pH 9.5). The target antibody was obtained.

[0498] Referring to the amino acid sequences of izalontamab published in WHO Drug Information, Vol. 38, No. 1, 2024 Recommended INN: List 91 and patritumab published in WHO Drug Information, Vol. 34, No. 1, 2020 Recommended INN: List 83, recombinant plasmids were constructed, transiently expressed, and purified using similar methods to those described above, and izalontamab and patritumab were prepared. Referring to the amino acid sequences of panitumumab published in WHO Drug Information, Vol. 18, No. 2, 2004 Proposed INN: List 91 and sacituzumab published in WHO Drug Information, Vol. 31, No. 1, 2017 Recommended INN: List 77, and introducing the D265A / P331G mutation into their Fc regions, recombinant plasmids were constructed, transiently expressed, and purified using methods similar to those described above, resulting in the preparation of Panitumumab-ag (hereinafter also referred to as Paniag) and Sacituzumab-ag (hereinafter also referred to as hRS7ag). Similarly, following the aforementioned methods, the negative control antibody against egg lysozyme (HEL), ISO-IgG1ag (or ISOag), was prepared, with the heavy and light chain amino acid sequences shown in SEQ ID NO: 31 and 32, respectively.

[0499] Example 3: Preparation and Characterization of ADC Molecules

[0500] 3.1 Constructing ADC molecules using GalT1

[0501] The antibody MEHDagYTE (containing the heavy chain shown in SEQ ID NO: 30 and the light chain shown in SEQ ID NO: 23), UDP-GalNAz (purchased from Qingdao Tangzhi Pharmaceutical Technology Co., Ltd.), GalT1 (obtained by adding a His tag to the N-terminus of the sequence SEQ ID NO: 4 and then purified by treatment with β-1,4-galactosidase and α-1,6-fucosidase and purified by Protein A affinity column), and MnCl2 were mixed in Tris-HCl buffer at pH 7.0 and reacted overnight at 25-30℃ to obtain the intermediate product MEHDagYTE-(N3)4. This intermediate product was then mixed with DMSO and LP1 and reacted overnight at pH 5.1±0.2 at 25-30℃. After ultrafiltration and buffer replacement, the ADC sample MEHDagYTE-LP1 was obtained. Purity was determined by SEC, and DAR value was determined by LC-MS. The preparation process is shown in Figure 1a.

[0502] Using a similar method, the ADC sample MEHDag-LP1 (antibody MEHDag has the heavy chain shown in SEQ ID NO: 21 and the light chain shown in SEQ ID NO: 23) was prepared.

[0503] 3.2 Constructing ADC molecules using Fut8

[0504] The aforementioned antibodies MEHDagYTE, LP6, Fut8 (purified according to SEQ ID NO: 3 sequence), and MnCl2, treated with the same enzymes, were mixed in a reaction buffer (25 mM Tris-HCl, 150 mM NaCl), pH 7.50, and reacted overnight at 30-40°C. The buffer was then replaced by ultrafiltration to obtain MEHDagYTE-LP6. Purity was determined by SEC, and DAR value was determined by LC-MS. The preparation process is shown in Figure 1b. MEHDagYTE-LP7, MEHDag-LP3, and MEHDag-LP4 were prepared using a similar method.

[0505] 3.3 Construction of dual-load ADC molecules

[0506] The obtained MEHDagYTE-LP6 was mixed with UDP-GalNAz, GalT1, and MnCl2 in Tris-HCl buffer at pH 7.0 and reacted overnight at 25-30°C to obtain the intermediate product MEHDagYTE-LP6-(N3)4. Then, it was mixed with DMSO and LP1 at pH 5.1 and reacted overnight at 25-30°C. The solution was then replaced by ultrafiltration to obtain the ADC sample MEHDagYTE-LP6-LP1. Purity was determined by SEC, and DAR value was determined by LC-MS. The preparation process is shown in Figure 1c. MEHDagYTE-LP7-LP1-P8, MEHDag-LP3-LP1, and MEHDag-LP4-LP1 were prepared using a similar method.

[0507] 3.4 Construction of thiol-coupled ADC molecules

[0508] The antibody Patritumab or Izalontamab was replaced with 20 mM Tris-HCl, pH 7.0 buffer, with an antibody concentration of approximately 2.5 mg / mL. TCEP with a molar ratio of 20 times the antibody equivalent was added, and the mixture was reduced at room temperature for 3 h. Then, Deruxtecan or Brengitecan (both purchased from MCE) with a molar ratio of 15 times the antibody equivalent was added, and the mixture was reacted at room temperature for 1.5 h. After the reaction, the mixture was ultrafiltered and replaced with 20 mM His-HCl buffer at pH 5.0 for storage, yielding positive controls Patritumab Deruxtecan (HER3-targeting ADC, hereinafter also referred to as Patri-ss-DXd) and Izalontamab Brengitecan (EGFR / HER3-targeting ADC, hereinafter also referred to as Izalontamab-ss-ED04).

[0509] 3.5 Characterization of ADC Samples

[0510] SEC testing: After diluting the sample with phosphate solution, it was isocratically separated using a TOSOH G3000 SWxl column. The purity of the sample was calculated by peak area normalization at a wavelength of 280 nm.

[0511] DAR value detection: After the sample was diluted with ultrapure water, it was separated using a waters / ACQUITY UPLC Protein BEH C4 column. At a wavelength of 280 nm, the molecular weight was detected by retention time and peak area, and the number of couplings could be calculated. According to the percentage content calculation formula, the percentage content of each component was calculated by response, and then the DAR value was calculated based on the content of each component and the number of couplings.

[0512] The results for SEC purity and DAR values ​​are shown in Table 1. The detection spectra of the multi-load ADC samples are shown in Figure 1d-1n.

[0513] Table 1. Detection results of SEC and DAR values ​​of ADC samples

[0514] Example 4: Binding of ADC drugs to tumor cells

[0515] MDA-MB-468 cells were digested with trypsin, and the cells were collected in centrifuge tubes, counted with FACS Buffer (3% BSA / DPBS), and the cell density was adjusted to 1.0 × 10⁶ cells / mL. 6 Cells / mL, then 100 μL per well was added to a 96-well U-plate and kept on ice. The test ADC drug was diluted with FACS Buffer, starting at a final concentration of 200 nM, and serially diluted 3-fold for a total of 9 concentration points. An isotype control was also set at 200 nM with one concentration point. 100 μL of the diluted sample was added to each well of the 96-well U-plate containing cells, and incubated at 2–8 °C for 30 min, followed by two washes with FACS Buffer. 100 μL of APC anti-human IgG FC Antibody (purchased from Biolegend, catalog number 366906) diluted 100-fold with FACS Buffer was added to each well, and incubated at 2–8 °C for 30 min, followed by two washes with FACS Buffer. After centrifugation, 100 μL of DPBS was added to each well to resuspend the cells. The median fluorescence intensity of the tested ADC samples was detected using a CytoFlex flow cytometer (Bechman), and the binding EC50 of the tested ADC samples was calculated using GraphPad Prism. 50 value.

[0516] Using a similar method, the binding ability of ADC drugs to A431 tumor cells was tested.

[0517] As shown in Figures 2a-2b, the binding ability of the ADC molecule conjugated with dual toxins to tumor cells was not affected compared with the naked antibody.

[0518] Example 5: Pharmacodynamic study of ADC drugs in a female BALB / c nude mouse model subcutaneously transplanted with A431 cell line.

[0519] SPF-grade female BALB / c nude mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with A431 cells (human epidermal carcinoma cells) in the right axilla, 2 × 10⁻⁶ cells per cell line.6 One per tumor. When the average tumor volume reaches ~100mm. 3 The animals were divided into 5 groups of 6 each. The specific grouping and administration regimens are shown in the table below.

[0520] Table 2. Dosing regimen information for A431 model

[0521] Day 1 was the day of grouping, and the drug was administered intraperitoneally on the same day. Tumor volume was measured twice a week, and mice were weighed and data recorded. The tumor-inhibiting efficacy of the test drug was evaluated using TGI (%), which reflects the tumor growth inhibition rate. The calculation of TGI (%) is as follows: TGI (%) = [1 - (mean tumor volume at the end of treatment in a certain treatment group - mean tumor volume at the beginning of treatment in that treatment group) / (mean tumor volume at the end of treatment in the negative control group - mean tumor volume at the beginning of treatment in the negative control group)] × 100%.

[0522] The effects of each test drug on tumor volume and mouse body weight are shown in Figure 3. All animals in both the control and test drug groups survived on day 19 after the first administration, and no mice in any group had tumor volumes exceeding 2000 mm². 3 On Day 19, the test drugs MEHDagYTE-LP1 (DAR4, G3), MEHDagYTE-LP6 (DAR2, G4), and MEHDagYTE-LP6-LP1 (DAR4+2, G5) at a test dose of 5 mg / kg showed tumor inhibition rates of 90.3%, 79.1%, and 88.9% respectively compared to the negative control group ISOagYTE-LP1, all of which significantly inhibited tumor growth.

[0523] On Day 22 and Day 27, the negative control group and the naked anti-MEHD-hIgG1agYTE control mice showed tumor volumes exceeding 2000 mmHg. 3 The tumor volume exceeds 2000 mm. 3 Mice were euthanized. The remaining mice were fed normally. At Day 50, because the tumors in both the negative control and naked anti-drug control mice exceeded 2000 mm³, only data from the groups using test drugs G3, G4, and G5 were analyzed. At Day 50, at a test dose of 5 mg / kg, test drugs G4 and G5 showed tumor inhibition rates of 35.4% and 54.3%, respectively, compared to G3. Compared to single-loaded ADC molecules, dual-loaded ADC molecules exhibited more potent and durable antitumor activity.

[0524] Example 6: Pharmacodynamic study of ADC drugs in a female Balb / c nude mouse model subcutaneously transplanted with HCC827 cell line.

[0525] HCC827 cells (human non-small cell lung cancer) were subcutaneously inoculated into the right back of SPF-grade female BALB / c nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.), at a dose of 2 × 10⁻⁶ cells. 6 One per animal. Monitor tumor growth regularly after inoculation, until the tumor reaches an average volume of approximately 108 mm. 3 Tumors were randomly assigned to groups based on tumor size, and the specific grouping and dosing regimens are shown in the table below.

[0526] Table 3. Dosing regimen information for the HCC827 model

[0527] By day 46 after the first administration, no abnormal decrease in body weight was observed in any group of mice, and no animal deaths were found in any group; the effect of each test drug on tumor volume is shown in Figure 4. On Day 46, the antibody drug MEHDag at a dose of 0.9 mpk showed a tumor inhibition rate of -0.58% compared to the PBS group, indicating no significant inhibitory effect on tumor growth compared to the PBS group. MEHDag-LP1 (DAR4) at a dose of 1 mg / kg showed a tumor inhibition rate of 47.99% compared to the PBS group, significantly inhibiting tumor growth. MEHDag-LP3 (DAR2) at a dose of 1 mpk showed a tumor inhibition rate of 5.88% compared to the PBS group. MEHDag-LP3-LP1 (DAR4+2) at a dose of 1 mpk showed a tumor inhibition rate of 61.24% compared to the PBS group, significantly inhibiting tumor growth and demonstrating a stronger anti-tumor effect than single-load ADCs.

[0528] Example 7: Serum stability study of ADC drugs

[0529] Mouse, rat, cynomolgus monkey, and human serum, as well as phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), were mixed with MEHDag-LP3-LP1 and incubated at 37°C for 0, 1, and 3 days, respectively. The incubation concentration of MEHDag-LP3-LP1 was 100 μg / mL. The release rates of CPT2 and MMAE were determined by LC-MS / MS for all analytes.

[0530] As shown in Figures 5a-5b, after incubation in serums of different species for 3 days, MEHDag-LP3-LP1 released only trace amounts of CPT2 and MMAE, demonstrating its excellent stability.

[0531] Example 8: Inhibitory activity of ADC drugs on tumor cell proliferation

[0532] A431 cells in logarithmic growth phase (purchased from Beina Biotechnology, medium: 90% DMEM-H + 10% FBS) were cultured in T75 culture flasks. The medium was discarded, and the cells were washed twice with DPBS to remove all liquid. 2 mL of trypsin was added for digestion, and the cells were incubated at 37°C for 4–8 minutes. The digestion solution was transferred to centrifuge tubes, 4 mL of the appropriate medium was added to terminate digestion, and the cells were pipetted into a single-cell suspension and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 4 mL of medium. 20 μL of the cell suspension was mixed with 20 μL of 0.2% trypan blue for cell counting. The cell density was adjusted to 2 x 10⁻⁶ cells / cells using culture medium. 4 Cells / mL, then seeded at 100 μL / well and incubated overnight at 37°C.

[0533] Starting with the highest working concentration of 150 nM, the test samples were serially diluted 6-fold with culture medium to prepare a total of 8 concentrations. A control group (negative control) was also set up by directly adding culture medium, with two replicates for each sample. 50 μL of each of the different concentrations of test sample was added to each well, and the 96-well plate was returned to the incubator. After incubation at 37°C for 4 days, 50 μL of Cell Counting Lite 2.0 (purchased from Novizan, Cat No. DD1101-02) was added to each well. After thorough mixing, 100 μL of the mixture was transferred to a 96-well white plate, fluorescence intensity was measured, and cytotoxicity of different drugs to cells was calculated. The data were imported into GraphPad Prism 7.0, and the results were analyzed using four-parameter logistic fitting.

[0534] As shown in Figure 6a, the anti-tumor cell proliferation activity of the dual-load ADC disclosed herein was superior to that of the positive control molecule izalontamab-ss-Ed04. A similar method was used to test the inhibitory activity of the ADC on the proliferation of human pancreatic cancer cells BxPC-3 (purchased from Beina Biotechnology) and human lung cancer cells HCC827 / ABCB1 (ABCB1-overexpressing HCC827 cells: based on wild-type HCC827 cells (purchased from Nanjing Kebai Biotechnology), an ABCB1-overexpressing pCDNA3.4 plasmid was constructed, and the plasmid was stably transfected into HCC827 cells using Lipo3000 transfection reagent. Single clones were obtained through limiting dilution, and libraries were constructed after flow cytometry identification). The results are shown in Figures 6b and 6c, where the anti-tumor cell proliferation activity of the dual-load ADC disclosed herein was superior to that of the positive control molecule izalontamab-ss-Ed04.

[0535] Furthermore, the inhibitory activity of the anti-EGFR and anti-Trop2 dual-load ADC on the proliferation of A431 cells was tested using a similar method. The results are shown in Figures 6d and 6e. The dual-load ADC showed better anti-tumor cell proliferation ability than the single-load drug.

[0536] Example 9: Pharmacodynamic study of ADC drugs in a female Balb / c nude mouse model subcutaneously transplanted with LS513 cell line.

[0537] LS513 cells (human colorectal cancer cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were subcutaneously inoculated into the right back of SPF-grade female Balb / c nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.), at a dose of 5*10. 6 One per animal. Monitor tumor growth regularly after inoculation, until the tumor reaches an average volume of 211 mm. 3 At that time, mice were randomly grouped and administered drugs according to tumor size and body weight. The specific grouping and administration regimens are detailed in Table 4 below.

[0538] Table 4. Dosing regimen information for the LS513 model

[0539] On day 31 after the first administration, no abnormal decrease in body weight was observed in any group of mice, and no experimental animals died in any group. The antitumor effects of each test drug are shown in Figure 7. On Day 28, the mean tumor volume in the PBS group was 2364.74 mm. 3 Animals were euthanized to meet the criteria, and tumor inhibition rate analysis was performed based on this time point. The tumor inhibition rates of the antibody MEHDag group (dose: 5 mpk) and the negative control group Isoag-LP3-LP1 (dose: 5 mpk) were 36.67% and 36.23%, respectively, showing no significant tumor growth inhibition compared to the PBS group. The tumor inhibition rate of the MEHDag-LP3 group (dose: 5 mpk) was 45.35%, which inhibited tumor growth compared to the PBS group. The tumor inhibition rate of MEHDag-LP3-LP1 (dose: 5 mpk) was 95.92%, which significantly inhibited tumor growth compared to the PBS group and showed a stronger anti-tumor effect than single-load ADCs.

[0540] Example 10: Pharmacodynamic study of ADC drugs in a female Balb / c nude mouse model subcutaneously transplanted with JIMT-1 cell line.

[0541] JIMT-1 cells (human breast cancer cells, purchased from Beina Biotechnology) were subcutaneously inoculated into the right back of SPF-grade female Balb / c nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.), at a dose of 5*10. 6 Each animal was inoculated individually, and tumor growth was monitored regularly after inoculation until the tumor reached an average volume of 202 mm². 3 At that time, mice were randomly grouped and administered drugs according to tumor size and body weight. The specific grouping and administration regimens are detailed in Table 5 below.

[0542] Table 5. Dosing regimen information for the JIMT-1 model

[0543] On day 24 after the first administration, no abnormal decrease in body weight was observed in any group of mice, and no experimental animals died in any group. The antitumor effects of each test drug are shown in Figure 8. On Day 24, the mean tumor volume in the PBS group was 730.92 mm. 3 Based on this time point, tumor inhibition rate analysis was performed. The antibody MEHDag group (dose: 3 mpk) showed a tumor inhibition rate of 32.41%, which was not significantly inhibited compared to the PBS group; the Isoag-LP3-LP1 group (dose: 3 mpk) showed a tumor inhibition rate of 2.26%, which was not inhibited compared to the PBS group; the Patri-ss-Dxd group (dose: 3 mpk) showed a tumor inhibition rate of 7.71%, which was not inhibited compared to the PBS group; the izalontamab-ss-Ed04 group (dose: 4 mpk) showed a tumor inhibition rate of 66.45%, which significantly inhibited tumor growth compared to the PBS group; the MEHDag-LP3-LP1 group (dose: 3 mpk) showed a tumor inhibition rate of 86.14%, which significantly inhibited tumor growth compared to the PBS group and showed a stronger anti-tumor effect than the positive control izalontamab-ss-Ed04.

[0544] Example 11: Pharmacodynamic study of ADC drugs in a female NOD SCID mouse model of patient-derived tumor xenograft.

[0545] Tumor tissue (provided by Crown Bioscience, HN9501) was collected from tumor-bearing mice using the HuPrime head and neck squamous cell carcinoma xenograft model. Tumor blocks with a diameter of 2-3 mm were cut and subcutaneously inoculated into the right anterior scapula of NOD SCID mice. Tumor growth was observed daily. The tumors reached an average size of approximately 150 mm. 3 The model uses randomized grouping, and the specific grouping and dosing regimens are shown in Table 6 below. Table 6: Dosing Regimen Information for PDX Model

[0546] Day 0 was the day of grouping. The drug was administered via tail vein injection on days 0, 7, and 14, for a total of three administrations. Tumor volume and mouse body weight were measured twice weekly during the experiment. Tumor volume data were used to assess efficacy, while safety was assessed based on changes in animal body weight and survival status.

[0547] By day 34 after the first administration, no abnormal decrease in body weight was observed in any group of mice, and no animal deaths were found due to factors other than euthanasia. The effect of each test drug on tumor volume is shown in Figure 9. On day 28, the antibody drug MEHDag at a dose of 3 mg / kg showed a tumor inhibition rate of 2.69% compared to the PBS group, but no significant effect on inhibiting tumor growth compared to the PBS group. MEHDag-LP3-LP1 at doses of 1 mg / kg and 3 mg / kg showed tumor inhibition rates of 97.14% and 97.30% compared to the PBS group, respectively, both doses significantly inhibiting tumor growth. The positive control izalontamab-ss-Ed04 at a dose of 4 mg / kg showed a tumor inhibition rate of 93.54% compared to the PBS group. On day 34, the mean tumor volume of the MEHDag-LP3-LP1 (1 mg / kg), MEHDag-LP3-LP1 (3 mg / kg), and izalontamab-ss-Ed04 (4 mg / kg) groups was 25.38 mm. 3 13.68mm 3 and 151.57mm 3 At the same molar dose, MEHDag-LP3-LP1 (3 mg / kg) showed significantly stronger tumor inhibition than izalontamab-ss-Ed04 (4 mg / kg), p < 0.05 (One-way ANOVA).

[0548] SEQ ID NO: 1 (FUT8 FL)

[0549] SEQ ID NO: 2(FUT8 FR)

[0550] SEQ ID NO: 3(FUT8 FR His)

[0551] SEQ ID NO: 4 (Mutant GalT1)

[0552] SEQ ID NO: 5(human GalT1 Y285L)

[0553] SEQ ID NO: 6(bovine GalT1 Y289L)

[0554] SEQ ID NO: 7(MEHD7945A VH)

[0555] SEQ ID NO: 8(MEHD7945A VL)

[0556] SEQ ID NO: 9(CDR-H1 Kabat)

[0557] SEQ ID NO: 10(CDR-H2 Kabat)

[0558] SEQ ID NO: 11(CDR-H3 Kabat)

[0559] SEQ ID NO: 12(CDR-L1 Kabat)

[0560] SEQ ID NO: 13(CDR-L2 Kabat)

[0561] SEQ ID NO: 14(CDR-L3 Kabat)

[0562] SEQ ID NO: 15(IgG1 CH)

[0563] SEQ ID NO: 16(IgG2 CH)

[0564] SEQ ID NO: 17(IgG3 CH)

[0565] SEQ ID NO: 18(IgG4 CH)

[0566] SEQ ID NO: 19(IgG1 CH AG)

[0567] SEQ ID NO: 20(MEHD7945A HC)

[0568] SEQ ID NO:21(MEHD7945A HC AG)

[0569] SEQ ID NO: 22(IgκCL)

[0570] SEQ ID NO:23(MEHD7945A LC)

[0571] SEQ ID NO:24(Alfc-1)

[0572] SEQ ID NO:25(Alfc-2)

[0573] SEQ ID NO:26(Beta-galactosidase)

[0574] SEQ ID NO:27(IgG1 CH YTE)

[0575] SEQ ID NO:28(IgG1 CH AG YTE)

[0576] SEQ ID NO:29(MEHD7945A HC YTE)

[0577] SEQ ID NO:30(MEHD7945A HC AG YTE)

[0578] SEQ ID NO:31(ISO-IgG1ag HC)

[0579] SEQ ID NO:32(ISO-IgG1ag LC)

[0580] SEQ ID NO:33(Mutant GalT1)

[0581] SEQ ID NO:34(Pani VH)

[0582] SEQ ID NO:35(Pani VL)

[0583] SEQ ID NO:36(Pani HC)

[0584] SEQ ID NO:37(Pani LC)

[0585] SEQ ID NO:38(Pani HCag)

[0586] SEQ ID NO:39(hRS7 VH)

[0587] SEQ ID NO:40(hRS7 VL)

[0588] SEQ ID NO:41(hRS7 HC)

[0589] SEQ ID NO:42(hRS7 LC)

[0590] SEQ ID NO:43(hRS7 HCag)

Claims

1. A protein conjugate comprising a protein and a glycan, said glycan comprising a structure of formula I: in, for the core N-acetylglucosamine, which is linked to the protein; It is a fucose derivative containing the first bioactive molecule (BM1); G is a distal glycosyl group containing substituted galactose; the wavy line indicates the linkage with the protein.

2. The protein conjugate according to claim 1, wherein the glycan is an N-glycan.

3. The protein conjugate according to claim 1 or 2, wherein the protein comprises an antigen-binding portion; preferably, the protein comprises an antibody or an antigen-binding fragment thereof.

4. The protein conjugate according to any one of claims 1-3, wherein the fucose derivative is linked to the core N-acetylglucosamine via an α-1,6-glycosidic bond.

5. The protein conjugate according to any one of claims 1-4, wherein G is selected from the following structures: in, substituted galactose, It is N-acetylglucosamine. It is mannitol; Preferably, G is 6. The protein conjugate according to any one of claims 1-5, wherein the substituted galactose is linked to the N-acetylglucosamine thereto via a β-1,4-glycosidic bond or a β-1,3-glycosidic bond.

7. The protein conjugate according to any one of claims 1-6, wherein the substituted galactose comprises a chemically active group X1 and / or a second bioactive molecule (BM2).

8. The protein conjugate according to claim 7, wherein the chemically active group X1 is selected from the following structures:

9. The protein conjugate according to any one of claims 1-8, wherein the first bioactive molecule BM1 and the second bioactive molecule BM2 are each independently selected from small molecule drugs, radioactive isotopes and their chelates, nucleic acids, peptides and antibodies or their antigen-binding fragments.

10. The protein conjugate according to any one of claims 1-9, wherein the first bioactive molecule BM1 and the second bioactive molecule BM2 are each independently selected from small molecule drugs, for example, drugs with a molecular weight of less than 1000 Daltons.

11. The protein conjugate according to any one of claims 1-10, wherein the first bioactive molecule BM1 and the second bioactive molecule BM2 are each independently selected from the following small molecule drugs: topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin inhibitors, radioisotopes, metal complexes, glycopeptide antibiotics, glucocorticoids, calcineurin inhibitors, DNA alkylating agents, drugs that interfere with DNA synthesis, serine kinase inhibitors, threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, histidine kinase inhibitors, proteasome inhibitors, protease inhibitors, histone deacetylase inhibitors, angiogenesis inhibitors, cyclin inhibitors, Toll-like receptor modulators, and STING modulators.

12. The protein conjugate of claim 11, wherein the first bioactive molecule BM1 and the second bioactive molecule BM2 are selected from a combination of: (1) BM1: microtubule inhibitor, BM2: topoisomerase I inhibitor; (2) BM1: Topoisomerase I inhibitor, BM2: Tubulin inhibitor; (3) BM1: microtubule inhibitor, BM2: microtubule inhibitor; and (4) BM1: Topoisomerase I inhibitor, BM2: Topoisomerase I inhibitor.

13. The protein conjugate according to claim 11 or 12, wherein the tubulin inhibitor is selected from MMAE, MMAF, Duo5, DM4 and eribulin.

14. The protein conjugate according to any one of claims 11-13, wherein the topoisomerase I inhibitor is selected from Dxd, eczema, and SN-38.

15. The protein conjugate according to any one of claims 11-13, wherein the topoisomerase I inhibitor is selected from compounds represented by formula II: in, R3is selected from the group consisting of hydrogen, C 1-6 alkyl, hydroxyl, amino and halogen, p is an integer from 0 to 5.

16. The protein conjugate of claim 15, wherein the topoisomerase I inhibitor is selected from the following structures:

17. The protein conjugate according to any one of claims 1-16, wherein the first bioactive molecule BM1 and the second bioactive molecule BM2 are selected from the combination of:

18. The protein conjugate according to any one of claims 1-17, wherein the molar ratio of the first bioactive molecule BM1 to the second bioactive molecule BM2 is 2:1 to 1:8, preferably 1:1 to 1:4, and more preferably 1:1 to 1:

2.

19. The protein conjugate according to any one of claims 1-8, wherein the fucose derivative comprises a first bioactive molecule BM1, and the substituted galactose comprises a chemically active group X1, wherein, The first bioactive molecule BM1 is selected from microtubule inhibitors or topoisomerase I inhibitors, while the chemically active group X1 is selected from:

20. The protein conjugate of claim 19, wherein the tubulin inhibitor and the topoisomerase I inhibitor are as defined in any one of claims 13-16.

21. The protein conjugate according to any one of claims 1-20, wherein the fucose derivative has the structure of formula III: in, BM1 is the first bioactive molecule, and LU1 is the linking unit that connects the first bioactive molecule to the fucose group.

22. The protein conjugate according to any one of claims 1-21, wherein the modified galactose has a structure of formula IV-A or IV-B: in, BM2 is the second bioactive molecule, and LU2 is the linking unit that connects the second bioactive molecule to the galactose group.

23. The protein conjugate according to claim 21 or 22, wherein the connecting units LU1 and LU2 each independently have the structure shown in Formula V: in, L1 is the extension group attached to the sugar 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.

24. The protein conjugate of claim 23, wherein L1 comprises one or more groups selected from:

25. The protein conjugate according to claim 23 or 24, wherein L1 comprises one or more of the following structures:

26. The protein conjugate according to any one of claims 23-25, 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.

27. The protein conjugate according to any one of claims 23-25, wherein L1 is selected from:

28. The protein conjugate according to any one of claims 23-27, wherein the structure of L2 is as shown in Formula VI: in, a1 = 0 or 1, a2 = 0 or 1, a3 = integers from 0 to 8, a4 = 0 or 1, 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.

29. The protein conjugate according to claim 28, wherein, L2 is selected from structures that have the following possible values: (1) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, 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, a4 = 0, 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, a4=0, 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, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0; (5) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0; (6) a1=1, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=0, 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, a4 = 0, b1 = 0, b2 = 0, b3 = 0, c = 0; (8) a1 = 0, a2 = 1, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, 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, a4=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; (10) a1=1, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=1, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0; (11) a1=0, a2=0, a3=0, 1, 2, 3, 4, 5 or 6, a4=1, b1=0, b2=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, b3=0, c=0; (12) a1=0, a2=0, a3=0, a4=0, b1=0, b2=0, b3=0, c=0; as well as (13) a1 = 0, a2 = 0, a3 = 0, 1, 2, 3, 4, 5 or 6, a4 = 0, b1 = 1, b2 = 0, b3 = 0, c = 0, 1, 2, 3, 4, 5 or 6.

30. The protein conjugate according to any one of claims 23-29, wherein L3 is absent or is a peptide linker.

31. The protein conjugate according to any one of claims 23-30, wherein L3 is selected from peptide linkers capable of being cleaved by cathepsins.

32. The protein conjugate according to any one of claims 23-29, wherein L3 is a diamino acid peptide, a triamino acid peptide, or a tetraamino acid peptide residue.

33. The protein conjugate according to claim 32, 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.

34. The protein conjugate according to claim 32, 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.

35. The protein conjugate of claim 32, 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.

36. The protein conjugate according to any one of claims 23-35, 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; R2is selected from the group consisting of hydrogen and C 1-6 alkyl; Su is selected from the group consisting of hexoses and hexuronic acids.

37. The protein conjugate according to any one of claims 23-35, wherein L4 is absent, or is selected from:

38. The protein conjugate according to any one of claims 21-37, wherein, The connecting units LU1 and LU2 are each independently selected from the following structures: Where k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10.

39. The protein conjugate according to any one of claims 21-37, wherein, The connecting units LU1 and LU2 are each independently selected from the following structures: Where k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

40. The protein conjugate according to any one of claims 21-39, wherein the BM1-LU1- is selected from the following structures: as well as in, k1 and k2 are each independently selected from integers from 1 to 20, and k' is an integer from 0 to 10.

41. The protein conjugate according to any one of claims 21-40, wherein the BM2-LU2- is selected from the following structures: as well as in, k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

42. The protein conjugate according to any one of claims 1-41, comprising a protein and a glycan, said glycan comprising a structure of formula IA or formula IB: in, The core is N-acetylglucosamine, which is linked to the protein; the wavy line indicates the linkage bond with the protein. It is a fucose derivative that contains the first bioactive molecule BM1; It is a substituted galactose that contains a second bioactive molecule, BM2. It is N-acetylglucosamine. It is mannitol; The fucose derivative has the structure of Formula III: The substituted galactose has a structure of formula IV-A or IV-B: Among them, BM1-LU1- is selected from the following structures: BM2-LU2- is selected from the following structures: Where k1 is independently selected from integers from 1 to 20, and k' is independently selected from integers from 0 to 10.

43. The protein conjugate according to any one of claims 1-21, wherein the modified galactose has a structure of formula IV-C or IV-D: in, The chemically active group X1 is selected from the following structures:

44. A method for preparing the protein conjugate according to any one of claims 1-43, the method comprising step (a): in the presence of a first catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of a second catalyst, react the product obtained in step (a) with... Contact; or, The scheme includes step (a): in the presence of a second catalyst, to Contact with a protein containing the initiating glycan chain; Step (b): In the presence of the first catalyst, the product obtained in step (a) is reacted with... touch; in, Nu and Nu' each contain ribonucleotides independently; It is a fucose derivative, which contains the first bioactive molecule. It is substituted galactose; Preferably, the fucose derivative or substituted galactose is as defined in any one of claims 1-43.

45. The method according to claim 44, wherein, The starting sugar chain lacks core fucose, and / or the starting sugar chain lacks galactose at its end.

46. ​​The method according to claim 44 or 45, wherein, The starting sugar chain is of the G0 sugar form.

47. The method according to any one of claims 44-46, wherein the first catalyst is a fucosyltransferase or a functional variant or fragment thereof.

48. The method of claim 47, wherein the first catalyst is α-1,6-fucosyltransferase or a functional variant or fragment thereof; preferably, the first catalyst is human-derived α-1,6-fucosyltransferase or a functional variant or fragment thereof.

49. The method according to any one of claims 44-48, wherein the first catalyst is FUT8 or a functional variant or fragment thereof.

50. The method of claim 49, wherein the first catalyst comprises the amino acid sequence shown in any one of SEQ ID NO: 1-3.

51. The method according to any one of claims 44-50, wherein the second catalyst is a galactosyltransferase or a functional variant or fragment thereof.

52. The method of claim 51, wherein the second catalyst is β-1,4-galactosyltransferase or a functional variant or fragment thereof.

53. The method according to any one of claims 44-52, wherein the second catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase or a functional variant or fragment thereof.

54. The method of claim 53, wherein the second catalyst comprises the amino acid sequence shown in any one of SEQ ID NO: 4-6 or 33.

55. The method according to any one of claims 44-54, wherein Nu and Nu' each independently comprise ribonucleotide diphosphate or a salt thereof; preferably, Nu and Nu' each independently comprise a structure selected from guanosine diphosphate (GDP), uridine diphosphate (UDP), cytidine diphosphate (CDP), and adenosine diphosphate (ADP), or a salt thereof.

56. The method of claim 55, wherein Nu comprises guanosine diphosphate (GDP) or a salt thereof, and Nu' comprises uridine diphosphate (UDP) or a salt thereof.

57. The method according to any one of claims 44-56, wherein... It has the structure shown in Formula VIII-A, or is a salt thereof: in, BM1 and LU1 are as defined in claims 21-40.

58. The method according to any one of claims 44-57, wherein... It has the structure shown in formula VIII-B or VIII-C, or a salt thereof: in, BM2 and LU2 are as defined in claims 21-41.

59. The method according to any one of claims 44-57, wherein... It has the structure shown in formula VIII-D or VIII-E, or is a salt thereof: in, X1 is a chemically active group X1, as defined in claim 43.

60. The method of claim 59, further comprising step (c): contacting the product obtained in step (b) with the compound represented by formula IX: BM2-L4-L3-L2-Y1(IX), where, Y1 is a chemically active group selected from the following structures: L2, L3, L4 and BM2 are as defined in the respective claims above.

61. The method according to claim 60, wherein the chemically active groups X1 and Y1 are selected from the following combinations: (1) X1 includes: Y1 includes: (2) X1 includes: Y1 includes: (3) X1 includes: Y1 includes: (4) X1 includes: Y1 includes: (5)X1 includes: Y1 includes: (6)X1 includes: Y1 includes: as well as (7)X1 includes: Y1 includes:

62. The method according to claim 60 or 61, wherein -L4-L3-L2-Y1 is selected from the following structures: as well as in, k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

63. The method according to any one of claims 60-62, wherein BM2-L4-L3-L2-Y1 is selected from the following structures: as well as in, k1 is selected from integers from 1 to 20, and k' is an integer from 0 to 10.

64. The protein conjugate according to any one of claims 1-43, for the treatment and / or prevention of tumors or cancer.

65. The protein conjugate according to any one of claims 1-43 and 64, wherein the DAR value of the protein conjugate is about 2.0 to 16.0, preferably about 3.0 to 12.0, more preferably about 4.0 to 8.0, and even more preferably 5.0 to 7.

0.

66. A pharmaceutical composition comprising the protein conjugate of any one of claims 1-43; preferably, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

67. The pharmaceutical composition according to claim 66, wherein the DAR value of the protein conjugate in the pharmaceutical composition is about 2.0 to 16.0, preferably about 3.0 to 12.0, more preferably about 4.0 to 8.0, and even more preferably 5.0 to 7.

0.

68. A method of treating and / or preventing tumors or cancer, comprising administering to a subject in need the protein conjugate of any one of claims 1-43 or the pharmaceutical composition of claim 66 or 67.

69. Use of the protein conjugate of any one of claims 1-43 and 64-65 or the pharmaceutical composition of claim 66 or 67 in the preparation of a medicament for treating and / or preventing tumors or cancer.

70. The protein conjugate according to any one of claims 1-7, wherein the glycan comprises or is: in The core N-acetylglucosamine, It is N-acetylglucosamine. Mannose, It is a fucose derivative. The substituted galactose is used; the fucose derivative and the substituted galactose are as defined in the preceding corresponding claims; q = 0.5 to 8, for example q = 1 to 6, preferably q = 1.5 to 4, more preferably q = 1.8 to 2.

5.

71. A sugar chain fragment having the structure of Formula I: in, The core is N-acetylglucosamine; It is a fucose derivative comprising a first bioactive molecule BM1; G is a distal glycosyl group comprising substituted galactose; the wavy line indicates the linkage; wherein the fucose derivative, the first bioactive molecule BM1, G, and the substituted galactose are as defined in the preceding claims.

72. A coupling compound comprising a sugar chain segment as defined in claim 71.

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