WO2024002330A1 - 固定化的内切糖苷酶融合蛋白及其应用 - Google Patents

固定化的内切糖苷酶融合蛋白及其应用 Download PDF

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WO2024002330A1
WO2024002330A1 PCT/CN2023/104549 CN2023104549W WO2024002330A1 WO 2024002330 A1 WO2024002330 A1 WO 2024002330A1 CN 2023104549 W CN2023104549 W CN 2023104549W WO 2024002330 A1 WO2024002330 A1 WO 2024002330A1
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antibody
endoglycosidase
oxazoline
endo
group
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PCT/CN2023/104549
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English (en)
French (fr)
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吕操
袁金铎
秦刚
熊美军
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启德医药科技（苏州）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof

Definitions

  • the present invention relates to the field of biotechnology, and specifically relates to a method for preparing drug conjugates or antibody-conjugated drugs using immobilized endoglycosidase fusion proteins with glycoside transfer activity, wherein the antibody-conjugated drugs are based on antibodies
  • the N-glycosylation site of the Fc region undergoes one-step site-specific coupling.
  • the present invention also relates to an endoglycosidase fusion protein comprising a covalently linked endoglycosidase, a Halo tag and/or a His tag, and an immobilized endoglycosidase fusion protein obtained by fixing the endoglycosidase fusion protein on a support. Endoglycosidase fusion protein.
  • Antibody-drug conjugate is a new type of targeted drug that is included in the category of bioconjugates. It organically connects highly active small molecule drugs and monoclonal antibodies through chemical links, and has the functions of small molecule drugs. The high activity and targeting of antibody drugs can better overcome the toxic side effects of small molecule toxins on the human body, and at the same time overcome the limitations of antibody therapy's limited efficacy on solid tumors, becoming a class of antibiotics with great development prospects. Oncology drugs.
  • the first generation of coupling technology mainly uses random coupling based on lysine or cysteine residues, which generally results in a highly heterogeneous mixture with random coupling sites and uneven drug/antibody ratio (DAR).
  • DAR drug/antibody ratio
  • Second-generation coupling technology usually requires the introduction of specific amino acids into antibodies or the insertion of polypeptide fragments of specific sequences through engineered mutations. On this basis, site-specific chemical coupling or enzymatic coupling is used to achieve site-specific coupling of antibodies. Union.
  • the third-generation coupling technology does not require the engineering modification of antibodies. It focuses on selecting specific amino acid sites and using specific conditions or proximity effects for site-specific coupling. Specifically, it includes the modification of inter-chain disulfide bonds and chemical selective modifications. , modification based on proximity effect and glycosyl modification.
  • glycosyl modification does not require antibody engineering, does not involve changes in the core skeleton structure of the antibody, does not affect the binding ability of the antibody's antigen-binding region, does not require fine adjustment of chemical reaction conditions, and has good coupling repeatability and uniformity.
  • the advantages have become a research hotspot in antibody conjugation technology.
  • Asparagine at position 297 in the Fc region of the antibody has highly conserved glycosylation (N-297Glycan). Through the remodeling of the sugar chain at this position, the site-specific connection of different molecules on the antibody can be achieved (Wang LX, Tong X, Li C. Glycoengineering of Antibodies for Modulating Functions, Annu Rev Biochem, 2019, 88, 433-459).
  • Site-directed coupling technologies based on antibody Fc glycosyl modification mainly include:
  • the aldehyde carbonyl group can be used to couple with small molecule toxin fragments to prepare ADC. Due to the diversity of the glycosyl structure at position N-297 of this method, not all monoclonal antibodies have glycosyl residues. Contains core fucose or sialic acid reaction sites and has certain substrate limitations;
  • the deglycosylation-transglycosylation (deglycosylation-transglycosylation) relay catalysis of tool enzymes such as endoglycosidase and glycosyltransferase is used to reshape the sugar chain and introduce bioorthogonal reactive groups.
  • tool enzymes such as endoglycosidase and glycosyltransferase
  • the targeted coupling preparation of ADC is achieved (Wang LX, Tong X, Li C, Glycoengineering of antibodies for modulating functions, Annu Rev Biochem, 2019, 88, 433-459; and Zeng Y, Tang F, Shi W et al., Recent advances in synthetic glycoengineering for biological applications, Current Opinion in Biotechnol, 2022, 74, 247-255).
  • ⁇ -1,4-galactosyltransferase ⁇ -1,4-Gal-T1
  • ⁇ -1,4-Gal-T1Y289L ⁇ -1,4-galactosyltransferase
  • Gal-UDP uridine galactose diphosphate
  • Galc-NAc acetylglucosamine
  • Synaffix developed a three-step ADC synthesis of deglycosylation-transglycosylation-bioorthogonal reaction using a combination of endoglycosidase Endo S and ⁇ -1,4-Gal-T1Y289L tool enzyme relay catalysis.
  • Method Van Geel R, Wijdeven MA, Heesbeen R et al., Chemoenzymatic conjugation of toxic payloads to the globally conserved N- Glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates, Bioconjugate Chem., 2015, 26, 2233-2242).
  • liquid-phase enzyme catalysis requires a large proportion of endoglycosidase catalytic equivalents and requires high enzyme purity (complex processes, preparation High cost), and it is easy to introduce a large number of enzyme-related impurities (such as host proteins and nucleic acids) into the reaction system, which brings great challenges to the subsequent removal of impurities downstream of conjugated drugs.
  • the current sugar chain remodeling reaction avoids the problem of engineering antibodies required by other site-specific coupling technologies, but there are still very large limitations, such as lengthy coupling steps, cumbersome operations, and at least two enzyme steps. Promoting the reaction plus one step of chemical reaction, that is, at least three steps of reaction and three complete purifications are required to obtain ADC.
  • most of the existing sugar chain remodeling is based on liquid-phase enzyme-catalyzed reactions, which have problems such as difficulty in amplification, difficulty in separation and removal of glycosidases, and great industrialization challenges. According to literature reports, even trace amounts of tools remain in ADC products.
  • Enzymes may cause deglycosylation of ADC products and cause the shedding of toxin molecules (Li T, Li C, Quan DN, etc., Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies, Carbohydrate Research 2018, 458-459,77 -84), may cause serious toxic reactions, bring major challenges to drug development and production, and also bring major hidden dangers to the safety of ADC drugs.
  • the present invention aims to solve these problems.
  • Antibody-conjugated drugs can be conjugated based on the site-specific coupling technology of sugar chain remodeling. Asparagine at position 297 in the Fc region of the antibody has highly conserved glycosylation. Through sugar chain remodeling at this site, different molecules can be combined in the antibody. Fixed point coupling on. Proteins containing the Fc region (such as Fc fusion proteins) can also realize site-specific coupling of different molecules based on the asparagine at position 297 of the Fc region of the antibody; the asparagine at position 297 in the protein containing the Fc region is positioned based on the amino acid sequence of the antibody. .
  • This application uses an immobilized endoglycosidase with glycosyltransferase activity to reshape the Fc-terminal sugar chain of the antibody, and convert the linker-loaded substance into an efficient and specific Sexually coupled to Fc-containing On the protein or antibody, the antibody conjugated drug with good uniformity can be obtained in one step.
  • the present invention provides a method for preparing a conjugated drug.
  • the conjugated drug is site-directed conjugated based on the N-glycosylation site of the Fc region.
  • the method includes the following steps:
  • the Fc region-containing protein is an antibody or Fc fusion protein.
  • the invention provides a method for preparing an antibody-conjugated drug, wherein the antibody-conjugated drug is site-directed conjugated based on the N-glycosylation site of the antibody Fc region, and the method includes the following steps: ( 1) Provide an oxazoline oligosaccharide-containing donor, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase with glycoside transfer activity; (2) catalyze the activated endoglycosidase The oxazoline oligosaccharide-containing donor is covalently linked to the GlcNAc motif-containing antibody.
  • the oxazoline oligosaccharide-containing donor further contains a load.
  • the payload is selected from the group consisting of small molecule compounds, agonists, nucleic acids, nucleic acid analogs, fluorescent molecules, radionuclides, and immunomodulatory proteins (eg, interleukins).
  • the load is selected from small molecule compounds (for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs , small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine, etc.; small molecule targeted drugs, such as ethyl mesylate matinib, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, Chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptoids and proteins.
  • small molecule compounds for example, small molecule drugs with various mechanisms
  • the oxazoline oligosaccharide is selected from one or more of the following group: disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, Hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonasaccharide oxazoline, decasaccharide oxazoline and undecasaccharide oxazoline.
  • the oxazoline oligosaccharide has the following structure:
  • the first six-carbon sugar group or its derivative - (the second six-carbon sugar group or its derivative) f - ⁇ -D-glucopyranose oxazoline
  • the first six-carbon glycosyl or derivative thereof is selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idurosyl or derivatives thereof substance; and/or its 6-position carbon is in the form -C(O)-; and/or
  • the second six-carbon glycosyl group or derivative thereof, at each occurrence, is independently selected from the group consisting of: glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof; and/or the oligosaccharide
  • the individual monosaccharide moieties in the structure are connected by ⁇ -(1 ⁇ 4) glycosidic bonds; and/or
  • the derivatives are independently selected from the group consisting of uronic acids Or derivatives in which the hydroxyl group of a monosaccharide is replaced by an amido group.
  • the oxazoline oligosaccharide has the following structure:
  • the first six-carbon sugar group or its derivative - ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar group or its derivative is mannitol (mannosyl) or its derivative.
  • the oxazoline oligosaccharide has the following structure:
  • the first six-carbon sugar group or its derivative - ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar group or its derivative is galactosyl or its derivative.
  • the oxazoline oligosaccharide has the following structure:
  • the oxazoline oligosaccharide has the following structure: the first six-carbon sugar base or its derivative- ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar base or its derivative is glucose base or its derivatives; or
  • the oxazoline oligosaccharide has the following structure: the first six-carbon sugar group or its derivative - ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar group or its derivative is fructosyl or its derivatives; or
  • the oxazoline oligosaccharide has the following structure: the first six-carbon sugar base or its derivative- ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar base or its derivative is gulose base or derivatives thereof; or
  • the oxazoline oligosaccharide has the following structure: the first six-carbon sugar group or its derivative - ⁇ -D-glucopyranose oxazoline, the first six-carbon sugar group or its derivative is idose base or its derivatives.
  • the structure of the oxazoline oligosaccharide is:
  • the structure of the oxazoline oligosaccharide is:
  • the invention provides a method for preparing a conjugated drug.
  • the antibody conjugated drug is site-directed conjugated based on the N-glycosylation site of the Fc region.
  • the method includes the following steps:
  • the donor containing oxazoline oligosaccharide is the linker-loading compound of formula (I):
  • P is the load
  • the 6-position carbon of the first six-carbon sugar moiety or its derivative part is in the form of -C(O)-, and f is 0, 1, 2, 3, 4, 5 or 6;
  • L is a linker, and L is directly connected to the carbonyl group in D-C(O)- through -NH- in it.
  • L is a branchless linker, it is connected to 1 P, and t is 1, and when L is a branched linker, When branching joints, each branch can be connected to 1 P, and t is an integer greater than 1;
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Protein is a protein containing an Fc region.
  • -L-(P)t is represented by Formula I-1 or Formula I-2 below.
  • the first six-carbon glycosyl or derivative thereof is selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idurosyl or derivatives thereof; and /or
  • the second six-carbon glycosyl group or derivative thereof is independently selected at each occurrence from glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof; and/or
  • the individual monosaccharide moieties in the oligosaccharide structure are connected by ⁇ -(1 ⁇ 4) glycosidic bonds; and/or
  • the first six-carbon glycosyl derivative and the second six-carbon glycosyl derivative are independently selected from the group consisting of uronic acids or derivatives in which the hydroxyl group of a monosaccharide is replaced by an amido group.
  • the Fc region-containing protein is an antibody or Fc fusion protein.
  • the present invention provides a method for preparing an antibody-conjugated drug.
  • the antibody-conjugated drug is site-directed conjugated based on the N-glycosylation site of the antibody Fc region.
  • the method includes the following steps:
  • the donor containing oxazoline oligosaccharide is the linker-loading compound of formula (I):
  • the antibody-conjugated drug structure is shown in formula (II-1), (II-2), (II-3), (II-4) or (II-5):
  • P is the load
  • L is a linker, and L is directly connected to the carbonyl group in D-C(O)- through -NH- in it.
  • L is a branchless linker, it is connected to 1 P, and t is 1, and when L is a branched linker, When branching joints, each branch can be connected to 1 P, and t is an integer greater than 1;
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or an antigen-binding fragment thereof.
  • the invention provides a method for preparing an antibody-conjugated drug, wherein the antibody-conjugated drug is site-directed conjugated based on the N-glycosylation site of the antibody Fc region, and the method includes the following steps:
  • the donor containing oxazoline oligosaccharide is the linker-loading compound of formula (I):
  • P is the load
  • DC(O)- is a disaccharide structure
  • L is a linker, and L is directly connected to the carbonyl group in D-C(O)- through -NH- in it.
  • L is a branchless linker, it is connected to 1 P, and t is 1, and when L is a branched linker, When branching joints, each branch can be connected to 1 P, and t is an integer greater than 1;
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or an antigen-binding fragment thereof.
  • -L-(P) t in Formula (I) is -L 2 -L 1 -BP, when Formula (I) is:
  • B does not exist independently, or is the following 1), or is the following 2), or is a combination of the following 1) and 2): 1) self-cleaving spacer Sp1; 2) a divalent group, or two or more A combination of divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-;
  • L 1 independently does not exist; or is a non-cleavable sequence; or is a cleavable sequence containing an amino acid sequence that can be cleaved by an enzyme, and the amino acid sequence that can be cleaved by an enzyme includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;
  • L 2 does not exist independently; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • Amino acid residue sequence that is -*(AA) n **-, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-100, AA appears every time are independently amino acid residues, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C 2 H 4 -O) is optionally present between the amino group and the ⁇ -carbon of an amino acid m -(CH 2 ) p -, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end is with the disaccharide structure
  • the carbonyl group forms an amide bond;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 alkyl, C 4-10 cycloalkylene; or R 1 and R 2 and the carbon atoms to which they are connected together form a 3-6 membered cycloalkylene group, and/or R 3 and R 4 and the carbon atoms to which they are connected together form 3 -6-membered cycloalkylene;
  • the invention provides a method for preparing an antibody-conjugated drug, wherein the antibody-conjugated drug is site-directed conjugated based on the N-glycosylation site of the antibody Fc region, and the method includes the following steps:
  • linker-loader compounds of formula (I) are provided.
  • P is the load
  • D-C(O)- is the disaccharide oxazoline and has the following structure:
  • the first six-carbon sugar group or its derivative- ⁇ -D-glucopyranose oxazoline is galactosyl or its derivative
  • L is a linker, and L is directly connected to the carbonyl group in D-C(O)- through -NH- in it.
  • L is a branchless linker, it is connected to 1 P, and t is 1, and when L is a branched linker, When branching joints, each branch can be connected to 1 P, and t is an integer greater than 1;
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or an antigen-binding fragment thereof.
  • -L-(P) t in Formula (I) is -L 2 -L 1 -BP, when Formula (I) is:
  • B does not exist independently, or is the following 1), or is the following 2), or is a combination of the following 1) and 2): 1) self-cleaving spacer Sp1; 2) a divalent group, or two or more A combination of divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-;
  • L 1 independently does not exist; or is a non-cleavable sequence; or is a cleavable sequence containing an amino acid sequence that can be cleaved by an enzyme, and the amino acid sequence that can be cleaved by an enzyme includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;
  • L 2 does not exist independently; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • Amino acid residue sequence that is -*(AA) n **-, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-100, AA appears every time are independently amino acid residues, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C 2 H 4 -O) is optionally present between the amino group and the ⁇ -carbon of an amino acid m -(CH 2 ) p -, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end is with the disaccharide structure
  • the carbonyl group forms an amide bond;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 alkyl, C 4-10 cycloalkylene; or R 1 and R 2 and the carbon atoms to which they are connected together form a 3-6 membered cycloalkylene group, and/or R 3 and R 4 and the carbon atoms to which they are connected together form 3 -6-membered cycloalkylene;
  • P is the load connected to part B, or part L 1 , or part L 2 .
  • formula (I) is:
  • Ld2 and each Ld1 are independently bonds; or are selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-; or are each independently on the side chain Natural amino acids that are unsubstituted or substituted by -CO-(PEG) j -R 11 or oligomeric natural amino acids with a degree of polymerization of 2-10 (i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10) ; R 11 is C 1-10 alkyl;
  • d 0, 1, 2, 3, 4, 5 or 6;
  • -(PEG) i - and -(PEG) j - are each a PEG fragment containing a specified number of consecutive -(OC 2 H 4 )-structural units or consecutive -(C 2 H 4 -O)-structural units, optionally Attached to one end is a C 1-10 alkylene group; each i is independently an integer from 1 to 100, and each j is independently an integer from 1 to 100;
  • M is hydrogen or LKa-L 2 ⁇ L 1 ⁇ B ⁇ P;
  • Q is NH 2 or L 2 ⁇ L 1 ⁇ B ⁇ P
  • M is hydrogen and Q is NH 2 at the same time;
  • Each LKa is independently selected from
  • opSu is Or a mixture thereof; wherein, * represents the connecting part with L 2 ;
  • B does not exist independently, or is the following 1), or is the following 2), or is a combination of the following 1) and 2): 1) self-cleaving spacer Sp1; 2) a divalent group, or two or more A combination of divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-;
  • L 1 independently does not exist; or is a non-cleavable sequence; or is a cleavable sequence containing an amino acid sequence that can be cleaved by an enzyme, and the amino acid sequence that can be cleaved by an enzyme includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;
  • L 2 does not exist independently; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • Amino acid residue sequence that is -*(AA) n **-, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-100, AA appears every time are independently amino acid residues, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C 2 H 4 -O) is optionally present between the amino group and the ⁇ -carbon of an amino acid m -(CH 2 ) p -, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end is with the disaccharide structure
  • the carbonyl group forms an amide bond;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 alkyl, C 4-10 cycloalkylene; or R 1 and R 2 and the carbon atoms to which they are connected together form a 3-6 membered cycloalkylene group, and/or R 3 and R 4 and the carbon atoms to which they are connected together form 3 -6-membered cycloalkylene;
  • P is the load connected to part B, or part L 1 , or part L 2 .
  • L 2 is an amino acid residue sequence, namely -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, and * represents the corresponding amino acid The N - terminus of is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure.
  • AA independently on each occurrence, is Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr , Arg, His, or any combination thereof.
  • n is an integer from 1 to 100. In some embodiments, n is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 20, 22, 25, 28, 32, 34, 40, 50 , 52, 60, 70, 86, 90, 100, or any interval value (or endpoint value) between any two values. In some embodiments, n is approximately 1-50. In some embodiments, n is about 1-30. In some embodiments, n is about 1-20. In some embodiments, n is about 1-10.
  • L 1 is a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably -Gly-Gly-Phe-Gly-, - Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof.
  • L 1 is -Val-Cit-.
  • B is selected from: (-PABC-), -NH-CH 2 -U-, or -NH-CH 2 -U-(CH 2 ) g -(CO)-; where g is 1, 2, 3, 4, 5 or 6; U is absent or CH2 , O, S or NH, preferably O or S.
  • -L 1 -B- represents -Val-Cit-PABC-.
  • -L2 - L1- B- represents -Gly-Gly-Gly-Val-Cit-PABC-.
  • the load P is selected from small molecule compounds (for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine, etc.; small molecule targeted drugs, such as mesylate Imatinib, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores , chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptoids and proteins.
  • the payload P is a small molecule drugs with various mechanisms of
  • the cytotoxin is selected from the group consisting of taxanes, maytansinoids, aristatins, epothilones, combretastatin A-4 phosphate phosphate), combretastatin A-4 and its derivatives, indole-sulfonamides, vinblastines such as vinblastine, vincristine, and vindesine , vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10 and its analogs, halichondrin B, Eribulin, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), Discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone, mitoguanine hydrazone, nitrogen mustards, nitrosoureas, aziridines
  • Cytotoxicants are selected from the group consisting of vinblastines, colchicines, taxanes, oristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine and exatecan and their derivatives; and/or
  • the cytotoxin is selected from auristatin, especially MMAE, MMAF or MMAD; and/or
  • the cytotoxin is selected from exatecan and its derivatives, such as DX8951f; and/or
  • the cytotoxin is selected from DXd-(1) and DXd-(2); DXd-(1) is preferred.
  • the linker-loader compound is represented by Formula (I-2):
  • the antibody is selected from the group consisting of: anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30/TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 Antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117/KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227/MUC1 antibody, anti-CD352 antibody, Anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2/HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4/AGS-5 antibody, anti-CEACAM5 antibody, Anti-PSMA antibody, anti-TIM1 antibody, anti-LY
  • the endoglycosidase having glycosyltransfer activity is N-acetylglucosamine endohydrolase.
  • the N-acetylglucosamine endohydrolase includes Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola).
  • the endoglycosidase with glycosyl transfer activity is covalently linked to a Halo tag, and the endoglycosidase fusion protein is fixed on a support containing a haloalkyl linker through the Halo tag; the Halo tag is Dehalogenase or a variant thereof or a truncated functionally active portion thereof.
  • one end of the endoglycosidase is connected with a Halo tag, and the other end of the endoglycosidase is connected with a His tag (His tag is a histidine polypeptide, such as His 4 , His 5 , His 6 , His 8 , His 10 , His 12 , or His 14 ).
  • His tag is a histidine polypeptide, such as His 4 , His 5 , His 6 , His 8 , His 10 , His 12 , or His 14 ).
  • the amino terminus of the endoglycosidase is linked to a Halo tag
  • the carboxy terminus of the endoglycosidase is linked to a His tag
  • Halo-Endoglycosidase-His (His here refers to His tag is His 4 , His 5 , His 6 , His 8 , His 10 , His 12 , or His 14 ; the same below).
  • the amino terminus of the endoglycosidase is connected with a Halo tag
  • the carboxyl terminus of the endoglycosidase is connected with a His tag.
  • the endoglycosidase is Endo-S2, that is, Halo-Endo S2- His.
  • the support includes a chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support via covalent interaction between the chloroalkyl linker and the Halo tag. superior.
  • the chloroalkyl linker is generated from a chloroalkyl substrate having the structure of Formula (III):
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19.
  • the support has the structure of Formula (IV):
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19;
  • resin is agarose resin, silicone resin, polymethylmethacrylate resin or cellulose resin.
  • It is highly cross-linked agarose resin or polymethylmethacrylate resin.
  • the invention provides an endoglycosidase fusion protein comprising an endoglycosidase and a Halo tag covalently linked together; the Halo tag is a dehalogenase or a variant thereof or a truncated version thereof Functionally active part.
  • the endoglycosidase fusion protein consists of an endoglycosidase and Halo covalently linked together.
  • the fusion protein has a Halo tag covalently attached to one end of the endoglycosidase and a His tag covalently attached to the other end of the endoglycosidase.
  • the endoglycosidase has a Halo tag covalently attached to the amino terminus and a His tag is covalently attached to the carboxyl terminus of the endoglycosidase.
  • the endoglycosidase is selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3) , Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes) At least one of endoglycosidase-CC); or the endoglycosidase is selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo
  • the endoglycosidase is endo- ⁇ -N-acetylglucosaminidase. In one embodiment, the endoglycosidase is selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.
  • the His tag is a plurality of contiguous histidine residues. In some embodiments, the His tag is 3 histidines, 4 histidines, 5 histidines, 6 histidines, 7 histidines, 8 histidines, 9 histidines amino acids or 10 amino acids. In some embodiments, the His tag is His6 . In one embodiment, the His tag is His8 . In one embodiment, the His tag is His10 .
  • the endoglycosidase fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 1, or has at least 80% identity, at least 85% identity, as compared to SEQ ID NO: 1. At least 90% identical, or having 1 or more conservative amino acid substitutions compared to SEQ ID NO:1.
  • the endoglycosidase fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.
  • the endoglycosidase fusion protein comprises the amino acid sequence set forth in positions 1 to 1150 of SEQ ID NO: 1, or is identical to the amino acid sequence set forth in positions 1 to 1150 of SEQ ID NO: 1
  • the amino acids shown are at least 90% identical, or have one or more conservative amino acid substitutions compared to the amino acids shown at positions 1 to 1150 in SEQ ID NO:1.
  • the endoglycosidase fusion protein comprises or consists of the amino acid sequence set forth in positions 1 to 1150 of SEQ ID NO: 1.
  • the PI of the endoglycosidase fusion protein is about 4-7. In some embodiments, the endoglycosidase fusion protein has a PI of about 4, 4.1, 4.3, 4.5, 4.7, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, or the interval value between any two values (including endpoint values).
  • the invention provides a method for preparing the endoglycosidase fusion protein of the invention, which includes: (a) providing a nucleic acid sequence of the endoglycosidase; (b) one end of the nucleic acid sequence of the endoglycosidase The nucleic acid sequence connecting the Halo tag;
  • the obtained nucleic acid sequence is cloned into a suitable vector, and then the vector is transformed into a suitable host cell, and the endoglycosidase fusion protein of the present invention is expressed in the host cell.
  • a method for preparing an endoglycosidase fusion protein of the present invention includes: (a) providing a nucleic acid sequence of the endoglycosidase; (b) connecting one end of the nucleic acid sequence of the endoglycosidase to a Halo tag. Nucleic acid sequence; (c) The other end of the nucleic acid sequence of the endoglycosidase is connected to the nucleic acid sequence of the His tag.
  • nucleic acid sequence of the Halo tag is linked to the amino terminus of the sequence of the endoglycosidase. In some embodiments, the nucleic acid sequence of the Halo tag is linked to the amino terminus of the nucleic acid sequence of the endoglycosidase and the nucleic acid sequence of the His tag is linked to the carboxy terminus of the nucleic acid sequence of the endoglycosidase.
  • the invention provides an immobilized endoglycosidase fusion protein comprising an endoglycosidase fusion protein of the invention immobilized on a support.
  • the support includes a haloalkyl linker such that the endoglycosidase fusion protein is immobilized on the support through covalent interactions between the haloalkyl linker and the Halo tag.
  • the support includes a chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support via covalent interaction between the chloroalkyl linker and the Halo tag. superior.
  • the chloroalkyl linker is generated from a chloroalkyl substrate having the structure of Formula (III):
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19.
  • the support has the structure of Formula (IV):
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19;
  • resin is agarose resin, silicone resin, polymethylmethacrylate resin or cellulose resin.
  • It is highly cross-linked agarose resin or polymethylmethacrylate resin.
  • the present invention also provides a prepacked column filled with immobilized endoglycosidase fusion protein.
  • the present invention also provides the use of the immobilized endoglycosidase fusion protein and/or the prepacked column in the preparation and/or purification of conjugated drugs or antibody conjugates.
  • the immobilized endoglycosidase fusion protein and/or the prepacked column can be easily used for industrial scale-up production in the preparation (such as purification) of conjugated drugs or antibody conjugates.
  • Liquid-phase enzymatic catalysis requires the investment of a large amount of endoglycosidase, and a large number of enzyme-related impurities (such as host proteins and nucleic acids) are introduced into the reaction system, which requires high enzyme purity (complex process and high preparation cost).
  • enzyme-related impurities such as host proteins and nucleic acids
  • the downstream removal of impurities brings great challenges; enzyme immobilization does not require high purity of the starting endoglycosidase (the enzyme preparation process is simple and low-cost), and very few impurities are introduced into the reaction system, which greatly simplifies conjugation of drugs.
  • downstream impurity removal process; and enzyme immobilization are beneficial to separation and purification, making it easier to remove the tool enzyme from the product, reducing the possibility of product sugar chains and small molecules falling off due to tool enzyme residues, thus reducing potential product decomposition. toxicity;
  • the PI of the endoglycosidase fusion protein of the present invention is about 5.5, while the PI of the ADC conjugate is 8-9; using AEX and/or CEX facilitates the later removal of the endoglycosidase fusion protein from the ADC product;
  • the endoglycosidase fusion protein coupling column catalysis of the present invention can achieve excellent coupling efficiency, is easy to linearly amplify, and the residual enzyme is easy to remove, making it more suitable for the industrial production of ADC.
  • Figure 1 shows the SDS-PAGE electrophoresis diagram after purification of the endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention; in the figure, 1 indicates that the sample comes from the cell fluid, 2 indicates that the sample comes from the supernatant, and 3 indicates The sample comes from the flow-through fluid (the components of the equilibrium buffer used are: 50mM Tris, 150mM NaCl, 20mM imidazole, pH7.4), 4 means that the sample comes from the washing fluid (the components of the washing fluid are: 50mM Tris, 150mM NaCl, 80mM imidazole, pH7.4), 5 means that the sample comes from the eluent (the components of the eluent are: 50mM Tris, 150mM NaCl, 500mM imidazole, pH7.4), 6 means that the sample comes from the eluent (eluent The components are: 50mM Tris, 150mM Na
  • Figure 2 is an SDS-PAGE electrophoresis diagram showing the supernatant of the immobilized endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention at different incubation times;
  • Figure 3 shows the SDS-PAGE electrophoresis diagram of the product when the endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention is used in a suspension coupling reaction;
  • Figure 4 shows the HIC-HPLC spectrum of the product when the endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention is used in a suspension coupling reaction;
  • Figure 5A shows the chromatogram of ADC purified by Q Sepharose FF
  • Figure 5B shows the chromatogram of ADC and endoglycosidase fusion protein purified by Q Sepharose FF;
  • Figure 6A is a chromatogram showing the purification after ADC hanging column Capto S impact
  • Figure 6B is a chromatogram showing the purification after ADC and endoglycosidase fusion protein hanging column Capto S impact;
  • Figure 7 shows the affinity detection of ADC-1 to ErbB2/Her2 on the cell surface (EC50, nM);
  • Figure 8 shows the inhibitory effects of different drugs on the proliferation of tumor cells BT474 (IC50, nM);
  • Figure 9 shows the inhibitory effects of different drugs on the proliferation of tumor cells NCI-N87 (IC50, nM);
  • Figure 10 shows the inhibitory effects (IC50, nM) of different drugs on the proliferation of tumor cells HepG2.
  • Figure 11 shows the inhibitory effect of ADC-1 on the NCI-N87CDX mouse model.
  • Figure 12 shows the HIC-HPLC pattern of ADC-2.
  • Figure 13 shows the inhibitory effect (IC50, nM) of ADC-2 and different drugs on the proliferation of tumor cells SK-BR-3.
  • Figure 14 shows the inhibitory effect (IC50, nM) of ADC-2 and different drugs on the proliferation of tumor cell HCC1954.
  • Figure 15 shows the inhibitory effect (IC50, nM) of ADC-2 and different drugs on the proliferation of tumor cells MDA-MB-468.
  • Figure 16 shows the high-resolution mass spectrum deconvolution plot of ADC-3.
  • Figure 17 shows the inhibitory effect (IC50, nM) of ADC-3 and different drugs on the proliferation of tumor cells BxPC-3.
  • Figure 18 shows the inhibitory effect (IC50, nM) of ADC-3 and different drugs on tumor cell FaDu proliferation.
  • Figure 19 shows the inhibitory effect (IC50, nM) of ADC-3 and different drugs on the proliferation of tumor cells HepG2.
  • Figure 20 shows the tumor inhibitory effect of ADC-3 on NCI-N87CDX mice.
  • Figure 21 shows the effect of ADC-3 on body weight of NCI-N87CDX mice.
  • Figure 22 is a HIC-HPLC detection analysis chart of ADC-4.
  • Figure 23 is a HIC-HPLC detection analysis chart of ADC-5.
  • Figure 24 shows the in vitro inhibitory activity of ADC-4 and ADC-5 on BxPC-3.
  • Figure 25 shows the in vitro inhibitory activity of ADC-4 and ADC-5 on FaDu.
  • Figure 26 shows the in vitro inhibitory activity of ADC-4 and ADC-5 on HepG2-3.
  • Figure 27 shows HIC-HPLC detection analysis of ADC-6.
  • Figure 28 shows the in vitro inhibitory activity of ADC-6 against SK-BR-3.
  • Figure 29 shows the in vitro inhibitory activity of ADC-6 against NCI-N87.
  • the expression “at least one” or “one or more” or “one” or “a plurality” means one, two, three, four, five, six, seven, eight, nine, one hundred, two One hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred or more, and so on.
  • “a” and “an” shall be understood to mean “at least one” or “at least one” unless expressly stated to the contrary.
  • biomolecule encompasses proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids, and their derivatives.
  • nucleic acid or “polynucleotide” refers to a polymer formed by at least two nucleotides or nucleotide derivatives linked together by a phosphodiester bond, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a “vector” is a vehicle used to transfer exogenous nucleic acid into a host cell in which it is amplified or expressed.
  • the definition of “vector” encompasses plasmids (eg, linearized plasmids), viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like.
  • a vector that is expressible and/or replicable within a host cell means that the vector is capable of expressing an RNA polynucleotide or polypeptide and/or producing multiple copies of the vector in the host cell.
  • a vector may contain a nucleic acid sequence or element operably linked to a promoter or replicon.
  • operably linked in relation to nucleic acid sequences or elements means that these nucleic acid sequences are functionally associated with each other.
  • a promoter can be operably linked to a nucleic acid sequence encoding a polypeptide, whereby the promoter regulates or mediates transcription of the nucleic acid.
  • Fc fusion protein refers to a recombinant protein obtained by fusion expression of a biologically active protein or polypeptide with the hinge region and Fc fragment of IgG; among which biologically active proteins or polypeptides such as ligands, cytokines, receptors, and antigens , cyclic polypeptides that bind to cell surface antigens.
  • Fc fusion proteins endow the fusion protein with more antibody properties, including extending its plasma half-life and exerting the unique functional effects of the Fc fragment.
  • the GlcNAc motif refers to a sugar chain with two conserved N-glycosylation sites on aspartic acid (Asn) at position 297 in the Fc region of the antibody, and the sugar chain contains N-acetyl covalently linked to Asn. Glucose- ⁇ -(1,4)-N-acetylglucose. Glycosylation of the Fc region is a complex post-translational modification process. With the participation of various enzymes, a variety of sugar chains with different lengths, compositions and structures are formed. Different sugar chains have an impact on protein biological activity, conformation and stability. , solubility, pharmacokinetics, etc. have different effects.
  • the Fc glycosylated sugar molecule has a complex dual-antenna core structure composed of two pentose sugar molecules, mannose and N-acetylglucosamine, and different sugar forms additionally contain Different numbers of sugar molecules, such as fucose, mannose, N-acetylglucosamine, galactose, bisection N-acetylglucosamine and sialic acid, due to galactosylation and sialic acid groups of terminal sugars ation produces heterogeneity. According to the number of terminal galactose, it is divided into three different subtypes (G0, G1 and G2).
  • Each subtype contains different forms with or without core fucose and two halves of N-acetylglucosamine, that is, shared 16 neutral composite structures.
  • peptide “polypeptide,” or “protein” refers to two or more amino acids covalently linked. These terms are interchangeable unless otherwise specifically stated.
  • Amino acid refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code.”
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine Acid (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I ), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • sequence identity has an art-recognized meaning, and the percent sequence identity between two polypeptides can be calculated by aligning the two sequences using publicly available algorithms, such as local alignment-based algorithm search tools (BLAST) and Fast Adaptive Shrinking/Thresholding Algorithm (FASTA) (see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds. Humana Press, New Jersey, 1994).
  • BLAST local alignment-based algorithm search tools
  • FASTA Fast Adaptive Micronking/Thresholding Algorithm
  • identity is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • the term "variant" refers to a protein (or nucleic acid) that has one or more substitutions, deletions, or insertions of residues (or nucleotides) compared to a reference protein (or nucleic acid).
  • the reference protein may be a naturally occurring protein that can be isolated from natural sources (i.e., a wild-type protein), or an engineered protein.
  • the function or activity of a variant such as an EndoS variant, EndoS2 variant or Halo tag variant, is substantially similar or equivalent or greater than the function or activity of the reference EndoS, EndoS2 or Halo tag, respectively.
  • the term “endoglycosidase” refers to an enzyme that catalyzes the hydrolysis of the internal glycosidic bonds of oligosaccharide chains and polysaccharides, and can be used to cleave polysaccharides from glycoproteins; in some embodiments, the term “endoglycosidase” herein “Dicer” can be used to hydrolyze the ⁇ -(1 ⁇ 4) glycosidic bond between two N-acetylglucose at the N-glycosylation site in the Fc region of the antibody.
  • Glycoside transfer activity refers to the connection of catalytically activated sugars to different acceptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules; in this application, donors with oxazoline-containing oligosaccharides can be connected to the antibody Fc region GlcNAc.
  • Halo tag refers to a haloalkane dehalogenase, or a variant thereof, that generates a haloalkane dehalogenase from a haloalkyl substrate (e.g., a reagent containing a haloalkyl moiety -( CH2 ) 2-30 -X, where X is halogen Such as F, Cl, Br, I, especially Cl or Br) to remove the halogen and form a covalent bond with the remaining part of the substrate.
  • a haloalkyl substrate e.g., a reagent containing a haloalkyl moiety -( CH2 ) 2-30 -X, where X is halogen
  • F, Cl, Br, I, especially Cl or Br e.g., a reagent containing a haloalkyl moiety -( CH2 ) 2-30 -X, where X is halogen
  • Mutated haloalkane dehalogenases have been described, for example, in WO2006/093529 and WO2008/054821, the relevant contents of which are incorporated herein by reference.
  • Mutated haloalkane dehalogenases useful in the present invention may include, but are not limited to, Xanthobacter dehalogenases (such as Xanthobacter autotrophicus dehalogenase (DhIA)) or Rhodococcus dehalogenases.
  • Mutants of halogenases e.g., Rhodococcus rhodochrous dehalogenase (DhaA)
  • DhaA Rhodococcus rhodochrous dehalogenase
  • containing one or more substitutions at the catalytic triad residues e.g., substitutions with Phe/Ala/Gly/Gln/Asn His272 or Asp106 replaced with Cys or other substitutions as described in WO2008/054821.
  • the premise is that the mutated haloalkane dehalogenase is able to form covalent bonds with haloalkyl substrates.
  • His tag is a polypeptide composed of histidine, such as His-His(His 2 ), His-His-His(His 3 ), His-His-His-His(His 4 ), His-His-His- His-His(His 5 ), His-His-His-His-His(His 6 ), His-His-His-His-His-His-His-His(His 10 ).
  • fusion protein refers to the co-expressed protein product of two or more genes obtained by DNA recombinant technology. For example, co-expression of the two genes can be achieved by deleting the stop codon of the gene encoding the first protein and then connecting the second protein gene with the stop codon.
  • the endoglycosidase fusion protein may further comprise one or more additional elements, such as additional polypeptides or tags, such as Halo tags and/or His tags. In some embodiments, the endoglycosidase fusion protein substantially retains desired properties.
  • resin refers to an organic polymer that has a softening or melting range after being heated, has a tendency to flow under the action of external force when softened, and is solid, semi-solid or liquid at room temperature.
  • Halogenated resin refers to a new compound formed by replacing at least one functional group in the resin with a halogen group.
  • the term “coupling” refers to the covalent attachment of at least two moieties (eg, at least two molecules or at least two termini of the same molecule).
  • a “conjugate” can be prepared by covalent attachment of at least two moieties (eg, at least two molecules or at least two termini/side chains of the same molecule).
  • antibody-drug conjugate refers to a conjugate that includes an antibody or antibody fragment covalently coupled to a cargo that is a small molecule compound, an agonist, a nucleic acid, a nucleic acid analog, or a fluorescent molecule. .
  • drug conjugate refers to a conjugate comprising an Fc region-containing protein covalently coupled to a payload, which is a small molecule compound, an agonist, a nucleic acid, a nucleic acid analog, or a fluorescent molecule. .
  • antibodies includes a wide variety of biochemically distinguishable polypeptides. Those skilled in the art will understand that classes of heavy chains include gamma, mu, alpha, delta or epsilon (gamma, mu, alpha, delta, epsilon), of which there are also subclasses (eg ⁇ 1- ⁇ 4). The nature of this chain determines the "class" of the antibody: IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been They are characterized and the functional specificity conferred is also known.
  • the immunoglobulin molecule is of the IgG class.
  • IgG typically contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000.
  • the four chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be combined with a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequence extends from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the immunoglobulin lambda light chain is V ⁇ .
  • Both light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used according to function.
  • the variable regions of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antibody's antigen-binding site, or amino terminus.
  • the N-terminal part is the variable region and the C-terminal part is the constant region; the CH3 and CL domains contain the carboxyl termini of the heavy chain and light chain respectively.
  • each antigen-binding domain is short, short, “complementarity-determining regions” or “CDRs” that form the antigen-binding domain, assuming the antibody assumes its three-dimensional configuration in an aqueous environment.
  • the remaining other amino acids in the antigen-binding domain known as the "framework” region, show less inter-molecular variability.
  • Most of the framework region adopts a ⁇ -sheet conformation, and the CDRs form a ring structure connected to it, or in some cases form part of the ⁇ -sheet structure.
  • the framework region forms a scaffold that positions the CDR in the correct orientation through non-covalent interactions between chains.
  • the antigen-binding domain with CDRs in specific positions forms a surface complementary to the epitope on the antigen that promotes non-covalent binding of the antibody to its epitope.
  • those of ordinary skill in the art can identify the amino acids containing the CDR and framework regions by known methods (see Kabat, E. et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • CDRs defined according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbering comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDR contains based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • Kabat numbering refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983).
  • Antibodies can also use the EU numbering system.
  • a “light chain constant region” includes a portion of the amino acid sequence from an antibody light chain.
  • the light chain constant region (CL) includes at least one of a constant kappa domain or a constant lambda domain.
  • a “light chain-heavy chain pair” is Refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • the "Fc region” is the tail region of an antibody, which interacts with cell surface receptors and some proteins of the complement system. Should Properties allow antibodies to activate the immune system.
  • the Fc region consists of two identical protein fragments, derived from the second and third constant regions of the two heavy chains of the antibody; in the IgM and IgE antibody isotypes, The Fc region contains three heavy chain constant domains (CH2-4); the Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is necessary for Fc receptor-mediated activity, and different glycoforms have different effects on the pharmaceutical properties of therapeutic antibodies.
  • Antibodies can be produced using conventional recombinant DNA techniques. Antibody-producing cell lines can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, technical references suitable for use in the present invention are described below, such as Current Protocols in Immunology, Coligan et al., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017; the entire content of which, including supplementary content, is incorporated by reference into the full text.
  • DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein according to conventional methods, placed into an expression vector, and then transfected into host cells, and the transfected host cells are cultured in culture medium to produce Monoclonal antibodies.
  • an expression antibody vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus. Promoters from other cells such as muscle can also be used. Kinesin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain a selection marker.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection. Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired protein.
  • isoelectric point (pi) is the pH (hydrogen concentration index) value of an aqueous solution of a molecule (such as a protein) without a net surface charge and is expressed in pH units.
  • the pI of a protein can be measured experimentally using methods well known in the art, such as imaging capillary isoelectric focusing (iCIEF) and capillary isoelectric focusing (CIEF).
  • iCIEF imaging capillary isoelectric focusing
  • CIEF capillary isoelectric focusing
  • Different biomolecules (proteins, nucleic acids, polysaccharides, etc.) with different pIs may carry different charges at a given pH value, allowing their separation by methods such as ion exchange chromatography or isoelectric focusing.
  • a molecule with a "basic pi” means that the molecule has a pi greater than 7.0.
  • a molecule with an “acidic pi” means that the molecule has a pi below 7.0.
  • ion exchange chromatography refers to a technique that separates biomolecules based on differences in their net surface charge and their affinity for ion exchangers (also called media, resins, or stationary phases).
  • IEX ion exchange chromatography
  • a protein with a pI lower than the buffer pH will have a negative net surface charge and bind to a positively charged anion exchanger; however, another protein with a pI higher than the buffer pH will have positive net surface charge and no Binds to a positively charged anion exchanger and will therefore pass through the medium with the buffer.
  • support refers to a water-insoluble material such as a surface, gel, polymer, matrix, particle, resin, bead or membrane that can be separated from a reaction mixture in solid or semi-solid form.
  • Spacer refers to a structure located between different structural modules that can spatially separate structural modules. The definition of a spacer does not limit whether it has a certain function or whether it can be cut off or degraded in the body. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, where the non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. "Spacer sequence” refers to an amino acid sequence as a spacer. Examples thereof include but are not limited to a single amino acid, a sequence containing multiple amino acids, such as a sequence containing two amino acids, such as GA, etc., or such as GGGGS, GGGSGGGGS , GGGGSGGGGSGGGGS, etc.
  • Self-cleaving spacers are covalent components that allow two chemical bonds to be sequentially cleaved upon activation of a protective moiety in the precursor: the protective moiety (e.g., cleavable sequence) is removed upon activation, triggering A cascade of decomposition reactions results in the sequential release of smaller molecules.
  • the protective moiety e.g., cleavable sequence
  • self-cleavable spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms connected to the rest of the molecule by a single bond.
  • the alkyl group may have 1-20 carbon atoms and refers to "C 1 -C 20 alkyl", such as C 1 -C 4 alkyl, C 1 -C 3 alkyl, C 1 -C 2 alkyl, C 3 alkyl base, C 4 alkyl, C 3 -C 6 alkyl.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2- Methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or their isomers body.
  • a divalent free radical refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of the corresponding monovalent free radical.
  • a divalent radical has two attachment sites to the rest of the molecule.
  • alkylene or “alkylene” refers to a saturated linear or branched divalent hydrocarbon group.
  • alkylene examples include, but are not limited to, methylene (-CH 2 -), ethylene (-C 2 H 4 -), propylene (-C 3 H 6 -), butylene (- C 4 H 8 -), pentylene (-C 5 H 10 -), hexylene (-C 6 H 12 -), 1-methylethylene (-CH(CH 3 )CH 2 -), 2 -Methylethylene (-CH 2 CH(CH 3 )-), methylpropylene or ethylpropylene, etc.
  • cycloalkyl refers to a cyclic saturated aliphatic group composed of carbon and hydrogen atoms that is attached to the rest of the molecule by a single bond.
  • the cycloalkyl group can have 3-10 carbon atoms, that is, "C 3 -C 10 cycloalkyl", such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl base or cyclodecyl.
  • Cycloalkylene refers to a divalent cycloalkyl group.
  • heterocyclyl means that one or more carbon atoms in the above-mentioned cycloalkyl group are replaced by heteroatoms selected from nitrogen, oxygen and sulfur, such as aza, oxa or thiiryl, aza, oxa hetero or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl.
  • Heterocyclylene refers to a divalent cycloalkyl group.
  • the relevant substituent is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl , carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl (monoalkyl)amino or alkyl Carbonyl (monoalkyl)amino.
  • the linkage between the groups may be linear or branched, provided that a chemically stable structure is formed.
  • the structure formed by such a combination can be connected to other parts of the molecule through any suitable atoms in the structure, preferably through designated chemical bonds.
  • Divalent groups join together to form a combination, and two or more divalent groups can form a linear connection with each other, for example -CR 1 R 2 -C 1-10 alkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-C 1-10 alkylene-(CO)-, -CR 1 R 2 - CR 1 R 2' -(CO)-, -CR 1 R 2 -CR 1 'R 2 '-CR 1” R 2” -(CO)-, etc.
  • the resulting bivalent structure can be further linked to other parts of the molecule.
  • the invention provides linker-loader compounds having formula (I):
  • D-C(O)- is a disaccharide structure or
  • L is a linker (for example, it can be chemically (for example, hydrolytically) or biologically (for example, enzymatically) disconnected from P to release P), and L is connected through -NH- and D-C(O)- therein
  • the carbonyl group is directly connected, where when L is a branchless joint, it is connected to 1 P, and t is 1, and when L is a branched joint, each branch can be connected to 1 P, and t is greater than 1 an integer (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10).
  • -L-(P) t is
  • formula (I) is:
  • Ld2 and each Ld1 are independently bonds; or are selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-; or are each independently on the side chain Natural amino acids that are unsubstituted or substituted by -CO-(PEG) j- R 11 or oligomeric natural amino acids with a degree of polymerization of 2-10 (i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10) ;
  • -(PEG) i - and -(PEG) j - are each a PEG fragment containing a specified number of consecutive -(OC 2 H 4 )-structural units or consecutive -(C 2 H 4 -O)-structural units, optionally Attach a C 1-10 alkylene group to one end;
  • M is hydrogen or LKa-L 2 ⁇ L 1 ⁇ B ⁇ P;
  • Q is NH 2 or L 2 ⁇ L 1 ⁇ B ⁇ P
  • M is hydrogen and Q is NH 2 at the same time;
  • Each LKa is independently selected from
  • opSu is Or a mixture thereof; wherein, * represents the connecting part with L 2 ;
  • Each L 2 does not exist independently; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • Amino acid residue sequence namely -*(AA)n**-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
  • AA is an amino acid residue independently each time base
  • * represents the N-terminus of the corresponding amino acid
  • ** represents the C-terminus of the corresponding amino acid
  • p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure ;
  • L 1 independently does not exist; or is a non-cleavable sequence; or is a cleavable sequence containing an amino acid sequence that can be cleaved by an enzyme, and the amino acid sequence that can be cleaved by an enzyme includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;
  • Each B does not exist independently, or is the following 1), or is the following 2), or a combination of the following 1) and 2): 1) self-cleaving spacer Sp1; 2) a divalent group, or two or A combination of more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heteroylene ring group and -(CO)-;
  • P is the load connected to part B, or part L 1 , or part L 2 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 alkyl, C 4-10 cycloalkylene; or R 1 and R 2 and the carbon atoms to which they are connected together form a 3-6 membered cycloalkylene group, and/or R 3 and R 4 and the carbon atoms to which they are connected together form 3 -6-membered cycloalkylene;
  • R 11 is C 1-10 alkyl
  • d 0, 1, 2, 3, 4, 5 or 6;
  • Each i is independently an integer from 1 to 100; in some embodiments, i is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 30, 34, 40 , 50, 60, 70, 90, 100, or the interval value between any two values (including endpoint values);
  • Each j is independently an integer from 1 to 100; in some embodiments, j is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 30, 34, 40 , 50, 60, 70, 90, 100, or an interval value between any two values (including endpoint values); in some embodiments, j is about 1-20; in some embodiments, j is about 8 -12; in some embodiments, j is about 8, 9, 12, or 13.
  • At least one of B, L 1 and L 2 is not “absent”.
  • L 2 is selected from: -NH-(CH2) a -(CH2) 2 (CO)-, a is an integer of 0, 1, 2, 3, 4 or 5; b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the carbonyl group in the above L2 structure is attached to L1 .
  • a is 0, 1, 2 or 3, preferably 3.
  • L 2 is an amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, AA is in each times are independently amino acid residues, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and there is optionally -(C 2 H 4 - between the amino group and the ⁇ -carbon of an amino acid O) m -(CH 2 ) p -, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end is with a disaccharide
  • the carbonyl group in the structure forms an amide bond.
  • AA is, independently each time, Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Any one of Arg, His or any combination thereof;
  • n is an integer of 2-100, preferably an integer of 2-50, preferably an integer of 2-30, preferably 2-20 The integer is preferably an integer from 2 to 10, preferably 2, 3, 4, 5, 6, 7, 8, or 9.
  • L1 comprises a cleavable sequence of an enzyme-cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the enzymatically cleavable amino acid sequence is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu- Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; the preferred enzymatically cleavable amino acid sequence is -Gly-Gly-Phe-Gly-.
  • L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably -Gly-Gly-Phe-Gly-, - Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof.
  • L 1 represents -Val-Cit-.
  • Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and combinations thereof; preferably, Sp1 is an acetal, heteroacetal or PABC; further preferably, heteroacetal Selected from N,O-heteroacetals; more preferably, Sp1 is -O-CH 2 -U- or -NH-CH 2 -U-, where -O- or -NH- is associated with an amino acid sequence that can be cleaved by an enzyme Linked, U is absent or CH 2 , O, S or NH, preferably O or S.
  • PABC p-aminobenzyloxycarbonyl
  • B is absent, or is -NH-CH 2 -U-, or -NH-CH 2 -U-(CH 2 ) g - (CO)-, where g is 1, 2, 3, 4, 5 or 6 and U is absent or CH2 , O, S or NH, preferably O or S.
  • B is absent.
  • B is the following 1), 2) or a combination of 1) and 2): 1) self-cleaving spacer Sp1; 2) one divalent group, or two or more divalent groups A combination of groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene and -(CO)-.
  • B is -NH-CH 2 -U- or -NH-CH 2 -U-(CH 2 ) g -(CO)-, U is absent, or CH 2 , O, S or NH, preferably O or S. In one embodiment, B is linked to the load via an amide bond or an ester bond or an ether bond. In one embodiment, B is selected from: (-PABC-), -NH-CH 2 -U-, or -NH-CH 2 -U-(CH 2 ) g -(CO)-; where g is 1, 2, 3, 4, 5 or 6; U is absent or CH 2 , O, S or NH, preferably O or S.
  • -L 1 -B- represents -Val-Cit-PABC-.
  • -L2 - L1- B- represents -Gly-Gly-Gly-Val-Cit-PABC-.
  • Ld2 and each Ld1 are independently selected from bonds, or
  • Each i, j and k is independently selected as an integer from 1-100.
  • each i, j, and k is independently selected from an integer of 1-20. In one embodiment, each i, j, and k is independently selected from an integer from 1 to 12.
  • each i is independently selected from an integer from 2 to 8; in one embodiment, i is about 4.
  • each j is independently selected from an integer from 8 to 12; in one embodiment, j is about 8 or 12.
  • each k is independently selected from an integer from 1 to 7; in one embodiment, k is about 1 or 3 or 5.
  • Ld2 and each Ld1 are independently selected from a bond; or a C 1-20 alkylene group with amino and carbonyl groups at both ends, or a PEG fragment of a certain length with amino and carbonyl groups at both ends ( Denoted as -(PEG) i -), or one or more natural amino acids whose side chains are each independently unsubstituted or substituted by a PEG fragment of a certain length (denoted as -CO-(PEG) j -) .
  • -(PEG) i - comprises -(OC 2 H 4 ) i - or -(C 2 H 4 -O) i -, and optionally a C 1-10 alkylene group is appended to one end ;
  • -(PEG) j - includes -(OC 2 H 4 ) j - or -(C 2 H 4 -O) j -, and optionally has a C 1-10 alkylene group attached to one end.
  • -(PEG) i - includes -C 2 H 4 -(OC 2 H 4 ) i - or -(C 2 H 4 -O) i -C 2 H 4 -.
  • the support may be in solid or semi-solid form made of any material.
  • supports may include, but are not limited to, resins (e.g., agarose resin, silicone resin, polymethylmethacrylate resin, cyclic oxy resin or cellulose resin), gels (e.g., alginate hydrogels), beads/microspheres/particles (e.g., polystyrene beads, magnetic particles), plates, wells, tubes, films, membranes, matrices, and Glass (e.g., slide).
  • resins e.g., agarose resin, silicone resin, polymethylmethacrylate resin, cyclic oxy resin or cellulose resin
  • gels e.g., alginate hydrogels
  • beads/microspheres/particles e.g., polystyrene beads, magnetic particles
  • plates wells, tubes, films, membranes, matrices, and Glass (e.g., slide).
  • the support is a resin. In some embodiments, the support is selected from the group consisting of agarose resin, silicone resin, polymethyl methacrylate resin, and cellulose resin. In some embodiments, the support is a highly cross-linked agarose resin.
  • Enzyme immobilization methods include adsorption, covalent or non-covalent binding, encapsulation, encapsulation and cross-linking. It is desirable to retain maximum enzymatic activity of the endoglycosidase after immobilization and to have a minimum amount of free endoglycosidase present in the conjugate product after the coupling reaction.
  • the support surface is modified to include one or more functional groups such that the endoglycosidase fusion protein can be covalently immobilized on the support.
  • the support contains one or more chemically reactive functional groups that are reactive with groups in the endoglycosidase fusion protein (such as amines, thiols, and carboxylates) or with groups in the haloalkyl substrate A covalent bond is formed, or the support contains one or more binding partners corresponding to the binding tag/affinity tag contained in the endoglycosidase fusion protein.
  • groups in the endoglycosidase fusion protein such as amines, thiols, and carboxylates
  • binding partners corresponding to the binding tag/affinity tag contained in the endoglycosidase fusion protein.
  • the support contains chemically reactive functional groups that can form covalent bonds with reactive groups on the endoglycosidase fusion protein (such as amines, thiols, and carboxylates) or with reactive groups in the haloalkyl substrate. key.
  • the support includes functional groups selected from the group consisting of: cyanate, isothiocyanate, isocyanate, carbodiimide, N-hydroxysuccinimide (NHS) ester, amine, carbonic acid Esters, epoxides, maleimides, haloacetyl, aziridines, ethyl chloroformate and aliphatic aldehydes.
  • the support is an epoxy activated resin, CNBr (cyanobromide)-activated resin, or NHS-activated resin.
  • the support is an epoxy activated resin.
  • the support is epoxy-activated agarose resin.
  • the support is an epoxy-activated highly cross-linked agarose resin.
  • the epoxy activated resin is pretreated to introduce amino groups prior to reaction with the haloalkyl substrate.
  • pretreatment of the epoxy activated resin is performed using ammonia.
  • pretreatment of the epoxy activated resin results in the introduction of amino groups on the ethylene oxide ring and ring opening of the ethylene oxide ring provides a hydroxyl group.
  • hydroxyl groups are optionally capped in subsequent steps to prepare the support.
  • pretreatment of the epoxy activated resin results in the introduction of amino groups on the ethylene oxide ring and ring opening of the ethylene oxide ring provides hydroxyl groups, which are optionally esterified in subsequent steps to prepare the support Esterification with a reagent (eg, an acetylating reagent such as Ac2O ).
  • a reagent eg, an acetylating reagent such as Ac2O
  • Epoxy activated resins thus pretreated are within the scope of "epoxy activated resins" as defined above.
  • the resin is an agarose resin (eg, highly cross-linked agarose resin) or polymethylmethacrylate resin.
  • the support contains one or more binding partners of a corresponding binding tag/affinity tag contained in an endoglycosidase fusion protein, such as an tag or affinity tag. mark.
  • binding partners of a corresponding binding tag/affinity tag contained in an endoglycosidase fusion protein such as an tag or affinity tag. mark.
  • binding tags/affinity tags and corresponding binding partners may include, but are not limited to, His tag and Ni 2+ , biotin/SPB tag/Strep tag/Strep tag II, and Streptavidin/Avidin/Medium Sex avidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca 2+ , MBP and amylose, S tag and ribonuclease S-protein, SNAP tag and benzylbird Purine (BG) derivatives, as well as CLIP tags and benzylcytosine (BC) derivatives.
  • the support is functionalized by containing a haloalkyl linker to form a covalent interaction with the Halo tag.
  • the haloalkyl linker can be introduced into the support by covalently linking one or more functional groups contained in the support to one or more reactive groups in the haloalkyl substrate.
  • the support thus obtained is also called a haloalkyl linker modification. of support.
  • Haloalkyl linker modified supports are within the scope of "supports" as defined above. Examples of haloalkyl substrates include, but are not limited to, those described in US20060024808A1 and WO2006093529. Haloalkyl substrates and methods for preparing such supports are described, for example, in U.S. Patent Nos. 7,429,472, 7,888,086, and 8,202,700, and Japanese Patent No. 4748685, the relevant contents of which are incorporated herein by reference.
  • the haloalkyl substrate may contain a haloalkyl moiety, which contains primary or secondary halo groups.
  • the haloalkyl substrate contains a primary halo group.
  • the halo group in the haloalkyl moiety is selected from the group consisting of F, Cl, Br and I.
  • the halo group in the haloalkyl moiety is selected from Cl and Br.
  • the haloalkyl substrate has the structure of Formula (V): (F1 o ⁇ H1 e ) r ⁇ Lh ⁇ (F2 e ⁇ H2 o ) s (V)
  • F1 and F2 are independently moieties containing reactive groups that can form covalent bonds with chemically active functional groups contained in the support;
  • H1 and H2 are independently selected from halogenated C 2-30 alkyl
  • Lh is a chemical bond or a C 3-200 alkylene group, and one or more (-CH 2 -) structures in the alkylene group are optionally replaced by -O-, -NH-, -(CO)-, -NH(CO)- and -(CO)NH- instead;
  • Lh is optionally substituted with 1, 2 or 3 substituents selected from -OC 1-10 alkyl, -NH-C 1-10 alkyl, -(CO)-C 1-10 alkyl , -NH(CO)-C 1-10 alkyl and -(CO)NH-C 1-10 alkyl;
  • o is 0 or 1
  • e is 0 or 1, provided that o and e are different;
  • r is an integer from 1 to 100;
  • s is an integer from 1 to 100.
  • r is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, s is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the reactive group in F1 or F2 is selected from the group consisting of amino, amine, thiol, and active ester.
  • the active ester contains one or more carboxylic acid radicals (as in a suitable alcohol or carbonic acid monoester of a phenol, e.g., an electron-deficient phenol such as 4-nitrophenol; or as in an NHS ester or sulfo radical -NHS ester) or one or more sulfonic acid radicals (as in methanesulfonic acid active esters, for example, MsO-).
  • F1 or F2 is
  • H1 and H2 are independently selected from haloC 2-20 alkyl. In some embodiments, H1 and H2 are independently selected from haloC 2-10 alkyl, especially haloC 6 alkyl. In some specific embodiments, the alkyl group in H1 or H2 is a straight chain alkyl group. In some embodiments, H1 or H2 is ( CH2 ) 2-30 -X. In some embodiments, H1 or H2 is ( CH2 ) 2-20 -X. In some embodiments, H1 or H2 is ( CH2 ) 2-10 -X, particularly ( CH2 ) 6 -X, where X is a halogen selected from F, Cl, Br and I.
  • the support is HaloLink TM resin (Promega).
  • the support is a resin that can include a haloalkyl linker comprising the structure -( CH2 ) 2-30 -X, where X is selected from the group consisting of F, Cl, Br, and I of halogen.
  • the support is a haloaryl linker modified resin.
  • the support is agarose resin or polymethylmethacrylate resin.
  • the support is highly cross-linked agarose resin.
  • o is 1, e is 0, r is 1, s is 1, and F1 is Lh is H2 is (CH 2 ) 2-20 -Cl, and the haloalkyl substrate is a chloroalkyl substrate, which has the structure of formula (III):
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19.
  • u is 3, v is 2, and w is 5, and the chloroalkyl substrate has the structure of the following formula (III-1):
  • the support is a chloroalkyl linker modified support and has the structure of Formula (IV):
  • u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; means a support which is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide or surface.
  • the support is a resin.
  • the support is an agarose resin, a silicone resin, a polymethylmethacrylate resin, or a cellulose resin.
  • the support is highly cross-linked agarose resin. Note that for the sake of clarity, only a single chloroalkyl-linker moiety is described attached to the support, but it is understood that there can be many such chloroalkyl-linker moieties attached to the support.
  • a chloroalkyl linker-modified support as shown in Formula (IV) is represented by Preparation of resins, beads, membranes, gels, matrices, films, plates, wells, tubes, slides or surfaces and a chloroalkyl substrate of formula (III).
  • a chloroalkyl linker-modified support as shown in formula (IV) is prepared from a pretreated epoxy-activated resin by adding ethylene oxide to the epoxy-activated resin. It is prepared by introducing an amino group into the alkane ring. During the pretreatment process, the ring opening of the ethylene oxide ring provides a hydroxyl group, which is optionally esterified with Ac 2 O in the subsequent step of preparing the support.
  • the support represented by formula (IV) It has the structure of formula (IV-1):
  • substructure represents pretreated epoxy activated resin, where The moiety represents the ethylene oxide ring, which reacts with the amino group and opens the ring to give the hydroxyl group, which is subsequently esterified to form AcO-, Parts represent other parts of the pretreated epoxy activated resin.
  • the immobilized endoglycosidase fusion protein has the following structure:
  • Support is a support (eg a solid support), for example selected from resins, beads, membranes, gels, matrices, films, plates, wells, tubes, slides or surfaces.
  • the support is a resin.
  • the support is an agarose resin, a silicone resin, a polymethylmethacrylate resin, or a cellulose resin.
  • the support is highly cross-linked agarose resin or polymethylmethacrylate resin.
  • the support is selected from the group consisting of NHS activated resin, CNBr activated resin, and epoxy activated resin;
  • a linker is a linker moiety covalently bound to a support, e.g., a chain containing 10-60 carbon atoms, optionally containing one or more ether, ester, urethane, and/or amide linkages; e.g., formula The linker part of (VI) or (VI-1)
  • u is an integer from 1 to 20
  • v is an integer from 0 to 20
  • w is an integer from 1 to 19;
  • HaloTag is Halo tag (haloalkane dehalogenase), covalently bound to the linker;
  • EndoS/EndoS2 are endoglycosidases
  • HisTag is a histidine tag
  • the coupling equipment disclosed in the patent application WO2022170676A or CN114480115A can be used for fixed-site coupling of antibodies.
  • the body Fc region N-glycosylation site and the load are specifically used in the method of preparing conjugates or antibody-conjugated drugs of the present invention.
  • the conjugation equipment includes a flow reactor and a fluid delivery unit, and the flow The reactor is filled with immobilized endoglycosidase, the fluid delivery unit is in fluid communication with the inlet of the flow reactor, and delivers a donor containing oxazoline oligosaccharides and an antibody containing a GlcNAc motif to the flow reactor ( or Fc region-containing proteins).
  • the coupling device includes: at least one flow reactor having an inlet and an outlet, the flow reactor is filled with a medium (such as a matrix such as chromatography beads, fibers, or films), and the endoglycosidase is immobilized onto the medium; a fluid delivery unit that is in fluid communication with the inlet of the flow reactor and is configured to continuously provide at least one reaction fluid to the flow reactor according to different stages of the coupling process, the at least one reaction fluid including a donor containing an oxazoline oligosaccharide and an antibody (or Fc region-containing protein) containing a GlcNAc motif; and a fluid collection unit in fluid communication with an outlet of the flow reactor and configured to perform a coupling process according to Different stages control the collection of fluid flowing out of the outlet of the flow reactor.
  • a medium such as a matrix such as chromatography beads, fibers, or films
  • the endoglycosidase is immobilized onto the medium
  • a fluid delivery unit that is in fluid communication
  • an oxazoline oligosaccharide-containing donor and a GlcNAc motif-containing antibody occur under the catalytic action of an endoglycosidase
  • the coupling reaction produces antibody conjugates or conjugated drugs.
  • the endoglycosidase is directionally immobilized on the medium and filled in the flow reactor, so that when the reaction fluid flows through the flow reactor, the reaction fluid contains a concentration of the conjugate to be generated.
  • the two reaction components are continuously and stably coupled.
  • the coupling device of the present invention greatly reduces process steps, significantly reduces process complexity, and is very suitable for saving expensive manufacturing costs. Moreover, by using flow reactors, linear scale-up and continuous flow production of the coupling process can be achieved to meet industrial demands for higher throughput, shorten unit coupling time and reduce the space occupied in the manufacturing area.
  • the production of bioconjugates through this coupling device can achieve site-specific coupling between the payload-linker and the antibody (or protein containing the Fc region), improving homogeneity and thus broadening the therapeutic window.
  • the conjugation process can be integrated with the production process of biomolecules such as monoclonal antibodies. For example, coupling can be completed during the production stages of monoclonal antibody intermediates and monoclonal antigen solutions. Therefore, the process is highly flexible and consistent.
  • At least one reaction fluid comprises a first reaction fluid comprising an oxazoline oligosaccharide-containing donor or a GlcNAc motif-containing antibody (or Fc-containing region), the second reaction fluid includes an antibody containing a GlcNAc motif (or a protein containing an Fc region) or a donor containing an oxazoline oligosaccharide.
  • the coupling process sequentially includes the following stages: pre-reaction equilibrium, coupling reaction, post-reaction recovery and post-recovery flushing, and the fluid delivery unit is further configured to: pre-reaction equilibrium, post-reaction recovery and recovery.
  • buffer is continuously provided to the flow reactor; and during the coupling reaction, the donor containing oxazoline oligosaccharide and the antibody containing the GlcNAc motif (or Fc region-containing protein).
  • the buffer, first reaction fluid, and second reaction fluid are stored in a first container, a second container, and a third container, respectively.
  • the fluid delivery unit includes a first delivery pump and a second delivery pump.
  • the first container and the second container are respectively connected to the first delivery pump via the first container outlet line and the second container outlet line.
  • the third container is connected to the second delivery pump via the third container outlet line.
  • the first delivery pump and the second delivery pump are respectively connected to the inlet main pipe via the first inlet branch pipe and the second inlet branch pipe, and the inlet main pipe is connected to the inlet of the flow reactor.
  • the first delivery pump pumps the buffer solution in the first container.
  • the first delivery pump pumps the first reaction fluid in the second container to the inlet main pipe
  • the second delivery pump pumps the second reaction fluid in the third container to the inlet main pipe. Import supervisor.
  • the fluid delivery unit further includes a first valve, a second valve, a third valve, and a fourth valve.
  • the first valve, the second valve and the third valve are respectively provided on the first container outlet pipeline, the second container outlet pipeline and the third container outlet pipeline, and are respectively used to control the first container outlet pipeline and the second container outlet.
  • the fluid flows in the pipeline and the outlet pipeline of the third container, and a fourth valve is disposed on the first inlet branch pipe for controlling the fluid flow in the first inlet branch pipe.
  • the first valve and the fourth valve are open, the second valve and the third valve are closed, and during the coupling reaction, the first valve is closed, The second valve, the third valve and the fourth valve are opened.
  • the first container outlet line, the second container outlet line, the third container outlet line, the first inlet branch pipe, the second inlet branch pipe, and the inlet main pipe are disposable or non-disposable, and Each is made of one of stainless steel, titanium, and silicone.
  • the first container, the second container and the third container are each selected from one of the following: disposable liquid storage bags, disposable liquid storage bottles, stainless steel containers, and disposable and non-disposable glass or plastic containers.
  • the fluid collection unit is further configured to: allow the fluid flowing out from the outlet of the flow reactor to be collected into the fourth container during the pre-reaction equilibrium and post-recovery flushing; and during the coupling reaction and post-reaction During recovery, fluid flowing out of the outlet of the flow reactor is collected into a fifth container.
  • the fourth container and the fifth container are respectively connected to the outlet main pipe via the fourth container inlet pipe and the fifth container inlet pipe, the outlet main pipe is connected to the outlet of the flow reactor, and the fluid collection unit includes a third
  • the fifth valve and the sixth valve are respectively arranged on the fourth container inlet pipeline and the fifth container inlet pipeline, and are respectively used to control the fourth container inlet pipeline and the fifth container inlet pipeline. of fluid flow.
  • the fifth valve is open and the sixth valve is closed, and during the coupling reaction and post-reaction recovery, the fifth valve is closed and the sixth valve is open.
  • the fourth container inlet line and the fifth container inlet line are disposable or non-disposable, and are respectively made of one of stainless steel, titanium, and silicone.
  • the fourth container and the fifth container are each selected from one of the following: disposable liquid storage bags, disposable liquid storage bottles, stainless steel containers, and disposable and non-disposable glass or plastic containers.
  • the coupling device further includes: a temperature control unit configured to: during the coupling process, control the temperatures of the fluid flowing into the inlet of the flow reactor and the fluid flowing out of the outlet of the flow reactor.
  • the temperature control unit includes: a heating module, which is disposed at the inlet of the flow reactor, for heating the fluid flowing into the inlet; and a cooling module, which is disposed at the outlet of the flow reactor, for Cool the fluid flowing out of the outlet.
  • the coupling device further includes: a sampling detection unit, which is in fluid communication with the outlet of the flow reactor, and is configured to: collect sample fluid from the fluid flowing out of the outlet of the flow reactor according to a preset sampling time ; and detecting the conjugate in the sample fluid to obtain a test result, and the test result indicates whether the conjugate reaches a preset standard.
  • a sampling detection unit which is in fluid communication with the outlet of the flow reactor, and is configured to: collect sample fluid from the fluid flowing out of the outlet of the flow reactor according to a preset sampling time ; and detecting the conjugate in the sample fluid to obtain a test result, and the test result indicates whether the conjugate reaches a preset standard.
  • the sampling and detection unit includes a sampling pump, a first switching valve, an elution pump, at least one analytical column and a detector.
  • the sampling pump is connected to the outlet of the flow reactor through the sampling pipeline, and the first switching valve is provided with a sample product ring, and the first switching valve switches between the first state and the second state according to the preset sampling time.
  • the sampling pump When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, and collects the sample fluid from the fluid flowing out of the outlet of the flow reactor via the sampling pipeline and pumps it into the sample loop, and during the first switching
  • the valve When the valve is in the second state, the elution pump, the sample loop, at least one analytical column and the detector are fluidly connected through the detection pipeline, and the elution pump pumps the eluent into the detection pipeline to allow the eluent to flow through the sample loop, thereby driving the sample fluid in the sample loop to flow through one of the at least one analytical columns and then enter the detector.
  • the sampling and detection unit further includes a second switching valve and a cleaning pump, and the second switching valve switches between two states.
  • the second switching valve When the second switching valve is in any state, the sample loop and the detector are in fluid communication with one of the two analytical columns, and the eluent drives the sample fluid in the sample loop to flow into the one analytical column, and the cleaning pump In fluid communication with the other of the two analytical columns, buffer is pumped to the other analytical column to equilibrate it.
  • the first switching valve is a six-way valve
  • the second switching valve is a ten-way valve
  • the elution pump is a quaternary pump.
  • the coupling device further includes: a recycling unit disposed between the inlet and the outlet of the flow reactor.
  • the fluid collection unit is configured to stop collecting the fluid flowing out of the outlet of the flow reactor
  • the recycling unit is configured to control the fluid flowing out of the outlet of the flow reactor to be re- into the inlet to carry out the coupling reaction again in the flow reactor.
  • the recycling recovery unit includes a seventh valve disposed on the recovery pipeline.
  • the recovery pipeline is connected between the inlet and outlet of the flow reactor, and a recovery container is provided on the recovery pipeline.
  • the seventh valve is opened, and the fluid flowing out of the outlet of the flow reactor flows through the recovery pipeline and the recovery container and then flows into the inlet.
  • the flow reactor is a coupled column (i.e., a prepacked column).
  • the coupling device further includes at least one selected from the group consisting of: a pressure sensing module, a flow detection module, a pH metering module, a conductivity metering module, and a UV detection module, a pressure sensing module, a flow detection module , pH measurement module, conductivity measurement module and UV detection module.
  • the modules may be located at the inlet and/or outlet respectively.
  • Ni Sepharose 6 FF Metal chelate affinity chromatography medium, also known as fixed metal ion affinity chromatography.
  • the principle is to use some amino acids on the protein surface, such as histidine, tryptophan, cysteine, etc., to bind to metals.
  • the principle of special interaction between ions Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , Fe 3+ ) can be used to analyze proteins. Leave. These functions include valence bonding, electrostatic adsorption, and covalent bonding. Among them, valence bonding is the main one, and the 6-histidine tag (His-Tag) is the most widely used.
  • Q Sepharose FF/Capto S impact The separation of molecules by ion exchange is based on the difference in surface electrostatic charges. Proteins are composed of many different amino acids containing weak acid and weak base groups. The surface electrostatic charge will gradually change as the pH of the surrounding environment changes. That is, the protein molecules are amphoteric molecules. In the ion exchange chromatography separation process, the binding and elution of specific molecules are achieved by controlling the reversible interaction between charged molecules and oppositely charged ion exchange packing, thereby achieving the separation effect. Protein surfaces at the same pH value as their isoelectric point have zero electrostatic charge and will not interact with charged fillers.
  • the protein When the pH value of the environment is higher than its isoelectric point, the protein will bind to the positively charged filler, that is, the anion exchanger; when the pH value of the environment is lower than its isoelectric point, the protein will bind to the negatively charged filler. , that is, combined with a cation exchanger.
  • SDS-PGAE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS is an anionic detergent. As a denaturant and co-solvent, it can break intra- and intermolecular hydrogen bonds and remove molecules. Internal hydrophobicity causes molecules to unfold and destroy the secondary and tertiary structures of protein molecules. SDS binds to the denatured protein to make it negatively charged. The amount of bound SDS is positive with the molecular weight. When reaching saturation, each gram of protein can be combined with 1.4g SDS, and the negative charge greatly exceeds the charge of the original protein.
  • Halo-EndoS2-His (Halo is underlined, His Tag is in italics, GGGGSGGGGS is the connecting sequence)
  • LB Lia-Bertani
  • Ni column affinity purification the Ni chromatography column is connected to the protein purification system, and is first fully washed with purified water and equilibrium buffer; the low flow rate supernatant is loaded. Wash with equilibration buffer and 80mM imidazole buffer respectively; elute with 500mM imidazole buffer. SDS-PAGE detection and analysis.
  • Halo-Endo S2-His is hung on Ni magnetic beads. There is no target sample in the flow-through, and Halo-Endo S2-His is eluted under the condition of 500mM imidazole. This result shows that Halo-Endo S2-His can be cultured in a scale-up reactor and linear production scale-up can be achieved through a Ni chromatography column.
  • the resin was filtered with isopropyl alcohol, and the filter cake was washed once with DMF (N,N-dimethylformamide) and then sucked dry. Transfer the filter cake to the flask with DMF and stir. Next, ethylenediamine was added to the mixture and stirred for 10 to 15 hours. Filter and wash the filter cake with DMF. Then drain the liquid;
  • DMF N,N-dimethylformamide
  • the resin was filtered with isopropanol, and the filter cake was washed once with H 2 O and then sucked dry. Transfer the filter cake to the flask and stir. Then, add 25% to 28% concentrated ammonia water to the mixture, slowly heat the system to 40-50°C, and react with stirring at 40-50°C. Lower the system temperature to 20-30°C and filter the mixture. Wash the filter cake with H2O until the pH of the filtrate reaches about 7-8. Next, wash the filter cake with DMF and drain the liquid;
  • step (2) Transfer the filter cake from step (1) to the flask and stir. Next, DMF and triethylamine containing the chlorinated alkyl substrate of formula (III-1) were added to the system in sequence. Stir the reaction. Next, filter the system and wash the filter cake with DMF, and finally drain the liquid;
  • step (3) Transfer the filter cake from step (2) to the flask. Turn on stirring. Next, Ac 2 O and triethylamine were added sequentially to the mixture. Stir the reaction. Then, the mixture was filtered and the filter cake was washed with DMF. The filter cake was then washed with H2O and the liquid drained. Finally, transfer the mixture to a container with 20% ethanol for storage.
  • Halo-Endo S2-His and chlorine resin Chloro resin
  • 20mM Tris-HCl, 150mM NaCl, pH 6.0-10.0 buffer for washing steps, repeat 3 times.
  • the immobilized endoglycosidase fusion protein (Halo-Endo S2-His is covalently linked to chlorine resin) is tested for activity. After passing the test, it is washed with 20mM Tris-HCl and 150mM NaCl, and finally stored at 2-8°C.
  • Compound 1 was prepared using the following steps and its structure is as follows:
  • Step B Synthesis of compound 2-1c
  • Step D Synthesis of compound 2-1e
  • Step E Add 2-1e (1 equivalent), iodobenzene acetate (3 equivalents), TEMPO (0.5 equivalents) and tert-butanol/dichloromethane/water (volume ratio 4:4:1) to the single-neck bottle.
  • saturated sodium thiosulfate was added to the system to quench the reaction. Extract and separate the layers with dichloromethane. The combined organic phases are washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated.
  • the resulting carboxylic acid intermediate is directly used in the next reaction without further purification.
  • Step G Synthesis of compound 2-1g
  • Step A Synthesis of compound 2-2a
  • Step B Synthesis of compound 2-2c
  • Step D Synthesis of compound 2-2e
  • the structure of the connector payload 1 (LP-1) is as follows:
  • the structure of the connector payload 2 (LP-2) is as follows:
  • the preparation process is as follows:
  • Step A Synthesis of intermediate LP-6-1b
  • Step B Synthesis of intermediate LP-6-1d
  • Step C Synthesis of intermediate LP-6-1e
  • Step D Synthesis of intermediate LP-6-1g
  • Step E Synthesis of intermediate LP-6-1h
  • Step F Synthesis of intermediate LP-6-1j
  • reaction solution of LP-6-1h in the previous step was cooled to 0 ⁇ 5°C, added PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 3i (0.85 equivalent), and reacted at 0 ⁇ 10°C 3 ⁇ 4h, LCMS monitors the reaction progress. After the reaction is complete, add the reaction solution to ice water, add 2-methyltetrahydrofuran to extract once, and then extract the aqueous phase twice with 2-methyltetrahydrofuran.
  • Step G Synthesis of compound LP-6-1
  • LP-6-2 is synthesized using the polypeptide solid-phase synthesis method. The steps are as follows,
  • Step 1 Preparation of NH2-Asp(OtBu)-Rink amide resin
  • Step 2 Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin
  • Step 3 Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin
  • Step 4 Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin
  • Step 5 Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin
  • Step 6 Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin
  • Step 7 Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin
  • Step A Synthesis of intermediate LP-6-3a
  • Step B Synthesis of intermediate LP-6-3b
  • LP-7 The structure of LP-7 is as follows:
  • Compound LP-7a was synthesized by solid-phase polypeptide synthesis, with reference to the similar method of LP-6-2.
  • Step B Synthesis of intermediate LP-7c
  • Step D Synthesis of compound LP-7e
  • ADC drugs are prepared based on the coupling reaction of Halo-Endo S2-His catalyzed antibody and linker-payload.
  • endonuclease buffer mix the antibody and linker-loader in an appropriate molar ratio (1:1 to 1:100) mix thoroughly, add Halo-Endo S2-His medium and mix well.
  • the coupling reaction in a mixed state is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, centrifuge and take out the solid-phase coupling reaction mixture, which is purified, ultrafiltrated or dialyzed to remove unreacted drug intermediates.
  • Purified ADC1 is stored in 1 ⁇ PBS pH7.4 at 4°C or -80°C.
  • ADC-1 After the coupling reaction is completed, the purity and coupling efficiency of ADC-1 can be detected by SDS-PAGE.
  • the SDS-PAGE detection results of ADC1 are shown in Figure 3.
  • the coupling reaction is targeted at the heavy chain of the antibody (there is an obvious molecular weight jump compared to the heavy chain coupled to the cytotoxin compared with the heavy chain uncoupled to the cytotoxin). ; No uncoupled cytotoxin heavy chain was detected in the coupling product, and the coupling efficiency was as high as over 95%.
  • test results are shown in Figure 4 and Table 2.
  • the antibodies that are not coupled to compound LP-1 are less than 1%; the conjugated products are mainly DAR2, and the DAR value of the ADC-1 drug is 1.93.
  • the concentration of the antibody reaction solution is 1-100mg/ml, and the concentration of the small molecule reaction solution is 0.1-50mg/ml.
  • the treated immobilized enzyme (Halo-Endo S2-His) medium is packed into the column, heated to 10-40°C by air or water bath conduction, and preheated for >30 minutes in advance.
  • the antibody reaction solution and the small molecule reaction solution are mixed evenly according to a fixed ratio, and reacted through the coupling column.
  • the effluent is the ADC drug, and the retention time on the coupling column is 5 minutes to 24 hours.
  • Coupling reaction solution samples were tested by SDS-PAGE, HIC-HPLC and RP-HPLC.
  • ADC-1 the purity and coupling efficiency of ADC-1 can be detected by SDS-PAGE.
  • the SDS-PAGE test results of ADC-1 showed that the coupling reaction occurred on the heavy chain of the antibody at the specific site, and no uncoupled cytotoxin heavy chain was detected in the coupling product, and the coupling efficiency was as high as over 95%.
  • the specific operation process of column coupling is as follows: install the appropriate pipeline (such as peristaltic pump silicone tube) to the appropriate pump head (such as peristaltic pump head (Chongqing Jieheng, BT-600CA/DG4(10))), Perform line balancing. Then connect the outlet end of the pipeline to the inlet end of the coupling column, and connect another section of pipeline to the lower end of the coupling column. Place the connected coupling column in a temperature control device (such as a hybridization oven (UVP/HB-1000Hybridizer)) set to a temperature of 10-40°C. The outlet end of the pipeline at the lower end of the coupling column is placed outside the temperature control device.
  • a temperature control device such as a hybridization oven (UVP/HB-1000Hybridizer)
  • the tube Place the inlet end of the coupling reaction solution in the coupling reaction solution, turn on the pump speed to perform the coupling reaction, and collect the coupling reaction solution.
  • the coupling reaction solution After loading the coupling reaction solution, rinse the coupling column with coupling equilibrium buffer and use the same collection volume. Collect; the effluent is the ADC drug.
  • the collection use a UV spectrophotometer to detect the concentration of the collected solution, and remove unreacted drug intermediates through purification, ultrafiltration or dialysis.
  • the purified ADC is stored in 1 ⁇ PBS pH7. 4. Store at 4°C or -80°C. Samples of the coupling reaction solution are tested by SDS-PAGE, HIC-HPLC or RP-HPLC.
  • Solid-phase coupling column reactions can achieve linear amplification. While ensuring the same key process parameters (such as retention time), the corresponding process (such as pump flow rate) is adjusted according to the size of the coupling column to achieve linear amplification of the coupling column to hundreds of milliliters. (500mL), several liters (3L) or larger coupling columns.
  • Glycosidase coupling column catalysis and offline DAR detection were realized on a laboratory scale; a larger coupling column was used to achieve linear amplification of conjugated drugs through higher flow rates and online DAR value detection.
  • the coupling device in patent application WO2022170676A is suitable for the glycosylation process of the present invention. This example verifies the feasibility of applying coupling equipment of different scales to the glycosylation coupling platform.
  • the protein When the pH value of the environment is higher than its isoelectric point, the protein will bind to the positively charged filler, that is, the anion exchanger; when the pH value of the environment is lower than its isoelectric point, the protein will bind to the negatively charged filler. , that is, combined with a cation exchanger.
  • the isoelectric point of Halo-Endo S2-His is 5.5. Under pH6.0-pH8.0 conditions, the surface of the Halo-Endo S2-His protein is negatively charged; the Halo-Endo S2-His protein is also combined with the negatively charged filler. This is the cation exchanger flow-through mode; the Halo-Endo S2-His protein combines with the positively charged packing, which is the anion exchanger flow.
  • the isoelectric point of ADC is generally 8-9. Under pH6.0-pH8.0 conditions, the ADC protein surface is positively charged; ADC will be combined with negatively charged fillers, that is, cation exchangers; ADC will be combined with positively charged Packing, that is, anion exchanger flow-through mode.
  • the isoelectric point of Halo-Endo S2-His and ADC has a significant difference of more than 2. Select pH6.0-pH8.0/buffer to effectively separate ADC and Halo-Endo S2-His through ion exchange chromatography. That is, Halo-Endo S2-His is used to catalyze ADC on site. Even if a small amount of the Halo-Endo S2-His protein immobilized on the column is shed in the ADC product, subsequent anion and cation chromatography can effectively remove Halo-Endo S2-His.
  • Halo-Endo S2-His sample was passed through Q FF chromatography column hanging column mode at pH 7.5, 20mM Tris-HCl, 1M NaCl pH 7.5 to elute the sample; as shown in Figure 5B, ADC-1 and Halo-Endo S2-His mixed loading sample can achieve effective separation after passing through the Q FF chromatography column, ADC-1 flows through, and Halo-Endo S2-His is hung on the column.
  • the Halo-Endo S2-His sample passes through the Capto S impact cation chromatography column in flow-through mode under pH 7.5 conditions; as shown in Figure 6B: ADC-1 and Halo-Endo S2-His are mixed to load the sample, and the sample passes through the Capto S impact After the cation chromatography column, effective separation can be achieved, ADC-1 is hung on the column, and Halo-Endo S2-His flows through.
  • ErbB2/Her2-positive human breast cancer cell BT-474, ErbB2/Her2-positive human gastric cancer cell NCI-N87, and ErbB2/Her2-negative human liver cancer cell HepG2 were inoculated into 96-well cells at 100 ⁇ l per well (containing 1,000 to 10,000 cells). The plate was cultured overnight in a cell culture incubator at 37°C, 5% CO 2 , and 100% humidity.
  • Example 10 In vivo activity test of ADC-1 (NCI-N87CDX mouse model)
  • V 0.5a x b 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor).
  • V 0.5a x b 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor).
  • the animals were randomly divided into vehicle control group and ADC-1 3 mg/kg group, with 5 animals in each group.
  • the animals were administered via tail vein injection, and the control group was administered an equal volume of vehicle (Vehicle).
  • the day of group administration was defined as Day 0.
  • the tumor volume of animals in each group was measured twice a week within 35 days after administration.
  • T/C% TRTV /C RTV ⁇ 100% ( TRTV : RTV of treatment group; CRTV : RTV of vehicle control group).
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • TGI (%) [1-(Average tumor volume at the end of treatment in a certain treatment group - Average tumor volume at the beginning of administration in this treatment group)/(Average tumor volume at the end of treatment in the vehicle control group - Vehicle The average tumor volume in the control group at the beginning of treatment)] ⁇ 100%.
  • Table 4 and Figure 11 show that ADC-1 can significantly inhibit tumor growth in the NCI-N87CDX mouse model compared with the vehicle control group.
  • ADC-2 was prepared with reference to the above preparation method. The difference from ADC-1 is that the linker-payload is LP-6.
  • the characterization data of the antibody conjugated drug ADC-2 is as follows.
  • SDS-PAGE detection and analysis of antibody conjugated drug ADC-2 After the coupling reaction is completed, the purity and coupling efficiency of ADC-2 are detected by SDS-PAGE.
  • the SDS-PAGE test results of ADC-2 show that the coupling reaction occurs at the specific site on the heavy chain of the antibody, and the ADC-2 heavy chain coupled to the linker-payload is significantly better than the heavy chain of the monoclonal antibody with the sugar chain cut off.
  • the molecular weight jump of the linker-payload proves that the linker-payload is successfully coupled to the monoclonal antibody heavy chain molecule at a specific site; almost no uncoupled linker-payload antibody is observed in the coupling product, the coupling efficiency is as high as 95%, and the purity of the coupling product is In line with expectations.
  • ADC-2 Take mixed healthy human plasma (5 males and 5 females mixed in equal volumes), add ADC-2 to a specific final concentration, divide into 4 portions, 450 ⁇ L each, and incubate in a 37°C incubator.
  • the sample collection times are 0h, 24h, 48h and 96h, and then stored in a refrigerator at -60 ⁇ -90°C after collection to detect the free load shedding rate and DAR change rate.
  • Double Blank For each sample (except Double Blank), take 40 ⁇ L and add a certain amount of internal standard precipitant (1ng/mL DXd), and shake for at least 10 minutes; for Double Blank, take 40 ⁇ L of blank matrix and add 120 ⁇ L of no internal standard precipitant, shake for at least 10 minutes, 3600g , 4°C, centrifuge for 15 minutes, pipet 60 ⁇ L of the supernatant into the injection bottle, add a certain amount of 0.1% FA ultrapure water, shake for at least 3 minutes, and detect by HPLC.
  • internal standard precipitant 1ng/mL DXd
  • Double Blank take 40 ⁇ L of blank matrix and add 120 ⁇ L of no internal standard precipitant, shake for at least 10 minutes, 3600g , 4°C, centrifuge for 15 minutes, pipet 60 ⁇ L of the supernatant into the injection bottle, add a certain amount of 0.1% FA ultrapure water, shake for at least 3 minutes, and detect by HPLC.
  • HER2 ECD Take an appropriate amount of HER2 ECD, replace it with a 30kD ultrafiltration tube (Buffer is 0.1M NaHCO 3 ), then mix the replaced HER2 ECD with the filler, place it in a rotating mixer, and incubate at 25°C and 10RPM for 2h ⁇ 10min for coupling. Then add 5 mL of 0.1M NaHCO 3 buffer, centrifuge and wash to remove uncoupled proteins, centrifuge for a certain period of time, and wash 3 times. Add a certain amount of glycine (pH 8.0) to the filler and let it stand for 16 hours at 2 to 8°C to block unreacted chemical groups on the filler.
  • Buffer is 0.1M NaHCO 3
  • Example 13 In vitro activity test of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468)
  • the cancer cell proliferation effect of the antibody conjugate drug ADC-2 at various ErbB2/HER2 expression levels was tested.
  • ErbB2/HER2-positive human tumor cells such as SK-BR-3 and HCC1954, and MDA-MB-468 ErbB2/HER2-negative human tumor cells were selected.
  • the results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 5 and Figure 13- As shown in 15, ADC-2 and small molecule toxins have significant inhibitory effects on the proliferation of ErbB2/HER2-positive cells.
  • the antibody mAb-1 monoclonal antibody has a certain inhibitory effect on the proliferation of ErbB2/HER2-positive cells, and ADC-2 is obviously superior. to mAb-1.
  • ADC-2 and mAb-1 monoclonal antibodies have no inhibitory effect on ErbB2/HER2-negative cells, showing good targeting properties.
  • the antibody used is the anti-TROP2 antibody
  • mAb-2 the linker-payload used is LP-6
  • the antibody conjugated drug ADC-1 Characterization data for 3 are described below.
  • SEC-HPLC of ADC-3 shows that high molecular weight aggregates are ⁇ 5%, ADC samples mainly exist in the form of monomers, and the damage to the antibody caused by the coupling reaction is negligible.
  • the molecular weight of ADC-3 was analyzed by a high-resolution mass spectrometer.
  • the deconvoluted mass spectrum is shown in Figure 16. Based on the measured molecular weight information and comparison with the theoretical molecular weight, each major molecular weight variant can be assigned and The DAR value analysis was performed using the mass spectrum abundance of each major molecular weight variant, and the average DAR value was calculated to be 3.93.
  • Example 15 In vitro activity test of ADC-3 (BxPC-3, FaDu, HepG2)
  • Example 16 In vivo activity test of ADC-3 (NCI-N87CDX mouse model)
  • the antibody conjugated drug ADC-4 was prepared with reference to the above preparation method. The difference between it and ADC-1 is that the antibody used is an anti-TROP2 antibody, that is, mAb-2.
  • the characterization data of the antibody conjugated drug ADC-4 is as follows.
  • ADC-5 Refer to the above preparation method to prepare the antibody conjugated drug ADC-5.
  • the difference from ADC-1 is that the antibody is anti-TROP2 antibody mAb-2, the linker-payload uses LP-2, and the characterization of the antibody conjugated drug ADC-5 The data are described below.
  • the cancer cell proliferation effects of the antibody conjugate drugs ADC-4 and ADC-5 at various TROP2 expression levels were tested.
  • TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells) and FaDu (human throat cancer cells), and TROP2-negative tumor cells HepG2 (human liver cancer cells) were selected.
  • the results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 7 and Figures 24-26.
  • ADC-4, ADC-5 and MMAE small molecule toxins all have obvious inhibitory effects on positive cells, and ADC-4 and There was no significant difference in ADC-5 activity, and the monoclonal antibody had no significant inhibitory effect on the proliferation of TROP2-positive cells.
  • ADC-4, ADC-5 and monoclonal antibodies have no inhibitory effect on antigen-negative cells, showing good targeting properties.
  • ADC-6 antibody-conjugated drugs.
  • the antibody used is mAb-3 (Trastuzumab)
  • the linker-payload is LP-6
  • the characterization data of the antibody-conjugated drug ADC-6 As described below.
  • SEC-HPLC detection of ADC-6 shows that high molecular weight polymers in ADC drugs are less than 5%, and ADC samples mainly exist in the form of monomers.
  • Example 21 In vitro activity test of ADC-6 (SK-BR-3, NCI-N87)
  • Example 9 the proliferation effect of the antibody-conjugated drug ADC-6 on cancer cells at various HER2 expression levels was tested.
  • the results showed that there is no Srt A enzyme Specific recognition of short peptide sequences (Sortase recognition sequence), the target ADC has no difference in the cell killing activity of the above two cell lines (see Figure 28-29).
  • the present invention illustrates the detailed methods of the present invention through the above embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must rely on the above detailed methods to be implemented.
  • Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

一种制备抗体偶联药物的方法，抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括：(1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；(2)通过糖苷内切酶的催化作用将活化的含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体；实现一步偶联。还涉及一种糖苷内切酶融合蛋白，其包含共价连接的糖苷内切酶、Halo标签和/或His标签。还涉及一种糖苷内切酶融合蛋白固定在支持物上而得到的固定化的糖苷内切酶融合蛋白和填充有固定化的融合蛋白的预装柱，以及固定化的糖苷内切酶融合蛋白用于抗体偶联物的纯化方法。

Description

固定化的内切糖苷酶融合蛋白及其应用 技术领域
本发明涉及生物技术领域，具体涉及一种使用固定化的、具有糖苷转移活性的糖苷内切酶融合蛋白制备药物偶联物或抗体偶联药物的方法，其中所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行一步法定点偶联。本发明还涉及一种包含共价连接的糖苷内切酶、Halo标签和/或His标签的糖苷内切酶融合蛋白、将所述糖苷内切酶融合蛋白固定在支持物上而得到的固定化的糖苷内切酶融合蛋白。
背景技术
对于高质量偶联物的需求，特别是如用于生物科学研究、诊断或治疗目的生物偶联物的需求正在迅速增加。然而，生物偶联物的高质量稳定生产远不能令人满意，部分原因是生物分子的复杂性和放大困难性，使得难以达到生物偶联物的高质量标准。
抗体偶联药物(antibody-drug conjugate，ADC)是一类纳入生物偶联物范畴的新型靶向药物，其通过化学链接方式将高活性小分子药物与单克隆抗体有机连接，兼具小分子药物的高活性和抗体药物的靶向性，能较好地克服小分子毒素对人体大的毒副作用，同时克服抗体疗法对实体瘤疗效有限的局限性，而成为一类有较大发展前景的抗肿瘤药物。
伴随抗体偶联药物的开发与逐渐成熟，抗体偶联技术经历了几次迭代升级，大致经历了随机偶联、基于抗体基因工程的定点偶联和不依赖抗体基因工程的定点偶联技术等三代技术发展历程(Walsh SJ,Bargh JD,Dannheim FM等,Site-selective modification strategies in antibody-drug conjugates,Chem.Soc.Rev.2021,50,1305-1353)。第一代偶联技术主要采用基于赖氨酸或半胱氨酸残基的随机偶联，一般得到偶联位点随机、药抗比(drug/antibody ratio,DAR)不均一的高度异质混合物，存在工艺稳定性、质量控制、药物稳定性、代谢一致性、安全性等方面的问题。第二代偶联技术通常需要通过工程化突变向抗体中引入特定氨基酸或插入特定序列的多肽片段，在此基础上经位点专一性的化学偶联或酶促偶联实现抗体的定点偶联。第三代偶联技术则无需抗体的工程化改造，聚焦于选择特定的氨基酸位点、利用特定的条件或临近效应进行定点偶联，具体有链间二硫键的改造、化学选择性的修饰、基于临近效应的修饰以及糖基修饰。综合对比，糖基修饰由于具有无需抗体工程化改造、不涉及抗体核心骨架结构的改变、不影响抗体的抗原结合区的结合能力、无需精细调节化学反应条件、偶联重复性及均一性好等优势成为抗体偶联技术的研究热点。抗体Fc区域的297位天冬酰胺存在高度保守糖基化(N-297Glycan)，通过该位点的糖链重塑可以实现不同分子在抗体上的定点连接(Wang LX,Tong X,Li C.Glycoengineering of Antibodies for Modulating Functions,Annu Rev Biochem,2019,88,433-459)。基于抗体Fc糖基修饰的定点偶联技术主要有：
1)基于糖基的化学修饰，利用高碘酸钠氧化核心岩藻糖(Zuberbuhler K,Casi G,Bernardes GJ等,Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting  monoclonal antibody in IgG format,Chem Commun,2012,48,7100-7102)或糖基末端唾液酸中邻二醇(Zhou Q,Stefano JE,Manning C等,Site-specific antibody–drug conjugation through glycoengineering,Bioconjugate Chem,2014,25,510-520)，得到相应的醛，醛羰基可用于与小分子毒素片段的偶联制备ADC，此类方法由于N-297位糖基结构多样性，并非所有单抗糖基上都含有核心岩藻糖或唾液酸反应位点，具有一定的底物局限性；
2)基于酶催化的修饰，利用糖苷内切酶和糖苷转移酶等工具酶的去糖基-转糖基(deglycosylation-transglycosylation)接力催化，实现糖链重塑引入生物正交反应基团，再通过后续化学反应，实现ADC的定点偶联制备(Wang LX,Tong X,Li C,Glycoengineering of antibodies for modulating functions,Annu Rev Biochem,2019,88,433-459；和Zeng Y,Tang F,Shi W等,Recent advances in synthetic glycoengineering for biological applications,Current Opinion in Biotechnol,2022,74,247-255)。
2012年王莱曦等报道了基于糖苷内切酶Endo S及其突变体接力催化的抗体糖链重塑定点偶联技术，合成了糖型结构单一的、含有叠氮基修饰的糖链重塑抗体(Huang W,Giddens J,Fan SQ等,Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions,J.Am.Chem.Soc.,2012,134,12308)。在此工作基础上，王莱曦和黄蔚等开发了一大类基于糖苷内切酶Endo S或Endo S2及其突变体等工具酶接力催化的去糖基-转糖基-点击化学反应三步法的ADC定点偶联合成技术(Zeng Y,Tang F,Shi W等,Recent advances in synthetic glycoengineering for biological applications,Current Opinion in Biotechnol,2022,1074,247-255)。2021年王莱曦等报道了一锅法的Endo S2催化的抗体糖链重塑定点偶联，向抗体上引入生物正交的叠氮基官能团，再经一步点击化学反应得到ADC分子(Zhang X,Ou C,Liu H等,General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies:facile synthesis of homogeneous antibody-drug conjugates,ACS Chem.Biol.,2021,16,11,2502-2514)。
糖链重塑定点偶联技术中另一类常用的工具酶是β-1,4-半乳糖基转移酶(β-1,4-Gal-T1)及其突变体(β-1,4-Gal-T1Y289L)，该酶以尿苷二磷酸半乳糖(Gal-UDP)为供体，将半乳糖Gal转移到糖蛋白的乙酰氨基葡萄糖(Glc-NAc)非还原性末端。基于此策略，2009年Qasba等(Boeggeman E,Ramakrishnan B,Pasek M等,Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases:application for cell surface antigen detection,Bioconjugate Chem.,2009,20,6,1228-1236)首次报道了以β-1,4-Gal-T1和β-1,4-Gal-T1Y289L为工具酶、C2-keto-Gal-UDP或N-azidoacetylgalactosamine-UDP(GalNAz-UDP)为供体的抗体定点偶联技术，制备了酮羰基或叠氮基修饰的抗体。随后，他们在2014年报道了首例基于β-1,4-Gal-T1及其突变体催化的去糖基-转糖基-生物正交反应三步法的ADC分子合成方法(Zhu Z,Ramakrishnan B,Li J等,Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar,mAbs,2014,6,1190-1200)。在上述工作基础上，Synaffix公司组合利用糖苷内切酶Endo S和β-1,4-Gal-T1Y289L工具酶接力催化，开发了去糖基-转糖基-生物正交反应的ADC三步合成法(Van Geel R,Wijdeven MA,Heesbeen R等,Chemoenzymatic conjugation of toxic payloads to the globally conserved N- glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates,Bioconjugate Chem.,2015,26,2233-2242)。此外文献中还报道了去糖基-转糖基-转糖基-生物正交反应的ADC四步合成法(Li X,Fang T,Boons GJ,Preparation of well-defined antibody–drug conjugates through glycan remodeling and strain-promoted azide–alkyne cycloadditions,Angew.Chem.Int.Ed.,2014,53,7179-7182)，该方法共使用了三种工具酶，即β-1,4-Gal-T1、β-1,4-Gal-T1Y289L及唾液酸转移酶。
上述糖链重塑偶联技术均利用液相酶催化的多步反应，存在一定的局限性，如液相酶催化需要大比例糖苷内切酶催化当量，对酶纯度要求高(工艺复杂，制备成本高)，易在反应体系引进大量酶相关杂质(如宿主蛋白和核酸)，对于后续偶联药物下游去除杂质带来较大挑战。
目前仅有少数几例公开报道的、基于固定酶催化的糖链偶联反应模式，例如2018年王莱曦等报道了基于共价固定化的Endo S2酶和共价固定化的Endo S2D184M酶的串联柱式偶联，实现了去糖基-转糖基的连续流接力催化得到糖基修饰的糖链均一性抗体(Li T,Li C,Quan DN等,Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies,Carbohydr.Res.2018,458-459,77-84)。2021年吴宗益等则报道了液相混悬式的固定化酶Endo S2及Endo S2T138Q的接力催化得到糖基修饰的糖链均一性抗体(Chuang H,Huang C,Hung T等，Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies,Bioorg.Chem.2021,112,104863)。目前尚未有基于固定化酶催化的糖链重塑一步制备ADC药物的报道。总之，目前现有糖链重塑反应避免了其他定点偶联技术需对抗体进行工程化改造的问题，但仍存在非常大的局限性，如偶联步骤冗长、操作繁琐，最少需要两步酶促反应外加一步化学反应，即至少需三步反应、三次完全纯化才能得到ADC。另一方面，现有糖链重塑绝大多数均基于液相酶催化反应，存在放大困难、糖苷酶难以分离除去、产业化挑战大等问题，据文献报道ADC产品中即使残留痕量的工具酶都有可能造成ADC产物去糖基分解而使毒素分子脱落(Li T,Li C,Quan DN等,Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies,Carbohydrate Research 2018,458-459,77-84)，可能造成严重毒性反应，给药物研发和生产带来重大挑战，也给ADC药物安全性带来重大隐患。本发明旨在解决这些问题。
发明内容
抗体偶联药物可基于糖链重塑的定点偶联技术进行偶联，抗体Fc区的297位天冬酰胺存在高度保守糖基化，通过该位点的糖链重塑可以实现不同分子在抗体上的定点偶联。含有Fc区的蛋白(如Fc融合蛋白)也能基于抗体Fc区297位天冬酰胺实现定点偶联不同分子；其中含有Fc区的蛋白中第297位天冬酰胺是以抗体氨基酸顺序来进行定位。
本申请通过固定化的、具有糖基转移酶活性的糖苷内切酶重塑抗体的Fc端糖链，经混悬式液相催化或柱式连续流催化工艺将连接子-负载物高效、特异性地偶联到含Fc 的蛋白或抗体上，一步得到均一性好的抗体偶联药物。
在一个方面，本发明提供了一种制备偶联药物的方法，所述偶联药物是基于Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
(1)提供含有噁唑啉低聚糖的供体、含有Fc区的蛋白以及固定化的具有糖苷转移活性的糖苷内切酶，所述Fc含有GlcNAc基序；
(2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有Fc基序的蛋白。
在一些实施方案中，所述含有Fc区的蛋白为抗体或Fc融合蛋白。在一个方面，本发明提供了一种制备抗体偶联药物的方法，其中所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：(1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；(2)通过所述糖苷内切酶的催化作用将活化的所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体。
在一些实施方案中，所述含有噁唑啉低聚糖的供体还含有负载物。在一些实施方案中，所述负载物选自下组：小分子化合物、激动剂、核酸、核酸类似物、荧光分子、放射性核素和免疫调节蛋白(如白介素)。在一些实施方案中，所述负载物选自由小分子化合物(例如，各种作用机制的小分子药物，包括各种传统小分子药物、光声动力疗法药物、光热疗法药物等，例如化疗药、小分子靶向药、免疫激动剂等，例如传统细胞毒性药物，如顺铂、紫杉醇、5-氟尿嘧啶、环磷酰胺和苯达莫司汀等；小分子靶向药，如甲磺酸伊马替尼、吉非替尼和安罗替尼等；免疫激动剂，如STING激动剂、TLR激动剂等)、核酸及核酸类似物、示踪分子(包括荧光分子、生物素、荧光团、发色团、自旋共振探针及放射性标记等)、短肽、多肽、拟肽和蛋白质组成的组。
在一些实施方案中，所述噁唑啉低聚糖选自以下组中的一种或多种：二糖噁唑啉、三糖噁唑啉、四糖噁唑啉、五糖噁唑啉、六糖噁唑啉、七糖噁唑啉、八糖噁唑啉、九糖噁唑啉、十糖噁唑啉和十一糖噁唑啉。在一些实施方案中，所述噁唑啉低聚糖具有如下结构：
第一六碳糖基或其衍生物-(第二六碳糖基或其衍生物)f-β-D-吡喃葡萄糖噁唑啉，
其中f为0、1、2、3、4、5或6；β-D-吡喃葡萄糖噁唑啉的结构为：
在一些实施方案中，所述第一六碳糖基或其衍生物选自下组：葡萄糖基、甘露糖基、半乳糖基、果糖基、古洛糖基、艾杜糖基或它们的衍生物；和/或其6位碳呈-C(O)-形式；和/或
所述第二六碳糖基或其衍生物在每次出现时独立地选自下组：葡萄糖基、甘露糖基、半乳糖基、果糖基或它们的衍生物；和/或所述寡糖结构中的各个单糖部分通过β-(1→4)糖苷键相连；和/或
所述衍生物(即第一六碳糖基衍生物和第二六碳糖基衍生物)独立地选自糖醛酸 或单糖的羟基被酰氨基替换的衍生物。
在一些实施方案中，所述噁唑啉低聚糖具有如下结构：
第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为甘露醇(甘露糖基)或其衍生物。
在一些实施方案中，所述噁唑啉低聚糖具有如下结构：
第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为半乳糖基或其衍生物。
在一些实施方案中，所述噁唑啉低聚糖具有如下结构：
所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为葡萄糖基或其衍生物；或
所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为果糖基或其衍生物；或
所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为古洛糖基或其衍生物；或
所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为艾杜糖基或其衍生物。
在一些实施方案中，所述噁唑啉低聚糖的结构为：
在一些实施方案中，所述噁唑啉低聚糖的结构为：
在一个方面，本发明提供了一种制备偶联药物的方法，所述抗体偶联药物是基于Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
(1)提供含有噁唑啉低聚糖的供体、含有Fc区的蛋白以及固定化的具有糖苷转移活性的糖苷内切酶，所述Fc区包含GlcNAc基序；
其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
(2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有Fc区的蛋白；
其中，药物偶联药物结构如式(II)所示：
其中，P为负载物；
D-C(O)-L-为连接子；
第一六碳糖基或其衍生物部分的6位碳呈-C(O)-形式，f为0、1、2、3、4、5或6；
L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
R为氢或α-L-岩藻糖基；
q为1或2；
Protein为含有Fc区的蛋白。
在一些实施方案中，-L-(P)t为下述式I-1或式I-2所示。
在一些实施方案中，所述第一六碳糖基或其衍生物选自葡萄糖基、甘露糖基、半乳糖基、果糖基、古洛糖基、艾杜糖基或它们的衍生物；和/或
所述第二六碳糖基或其衍生物在每次出现时独立地选自葡萄糖基、甘露糖基、半乳糖基、果糖基或它们的衍生物；和/或
低聚糖结构中的各个单糖部分通过β-(1→4)糖苷键相连；和/或
第一六碳糖基衍生物和第二六碳糖基衍生物独立地选自糖醛酸或单糖的羟基被酰氨基替换的衍生物。在一些实施方案中，所述含有Fc区的蛋白为抗体或Fc融合蛋白。
在一个方面，本发明提供一种制备抗体偶联药物的方法，所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
(1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；
其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
(2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体；
其中，抗体偶联药物结构如式(II-1)，(II-2)，(II-3)，(II-4)或(II-5)所示：

其中，P为负载物；
D-C(O)-L-为连接子；
D-C(O)-为二糖结构；
L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
R为氢或α-L-岩藻糖基；
q为1或2；
Ab为抗体或其抗原结合片段。
在一个方面，本发明提供了一种制备抗体偶联药物的方法，其中所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
(1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；
其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
(2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体；
其中，抗体偶联药物结构如式(II)所示：
其中，P为负载物；
D-C(O)-L-为连接子；
D-C(O)-为二糖结构
L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
R为氢或α-L-岩藻糖基；
q为1或2；
Ab为抗体或其抗原结合片段。
在一些实施方案中，式(I)中的-L-(P)t为-L2-L1-B-P，此时式(I)为：
其中
B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、- C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
P是与B部分，或L1部分，或L2部分连接的负载物。在一个方面，本发明提供了一种制备抗体偶联药物的方法，其中所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
提供式(I)的连接子-负载物化合物、抗体以及固定化的具有糖苷转移活性的糖苷内切酶：
通过所述糖苷内切酶的催化作用形成抗体偶联药物；
其中，P为负载物；
D-C(O)-L-为连接子；
D-C(O)-为二糖噁唑啉，具有如下结构：
第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为半乳糖基或其衍生物；
L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
R为氢或α-L-岩藻糖基；
q为1或2；
Ab为抗体或其抗原结合片段。
在一些实施方案中，式(I)中的-L-(P)t为-L2-L1-B-P，此时式(I)为：
其中
B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、-C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
P是与B部分，或L1部分，或L2部分连接的负载物。
在一些实施方案中，其中-L-(P)t为
即式(I)为：
其中，
Ld2和每个Ld1独立地是键；或选自-NH-C1-20亚烷基-(CO)-、-NH-(PEG)i-(CO)-；或为侧链上各自独立地未取代或被-CO-(PEG)j-R11取代的天然氨基酸或聚合度为2-10(即2、3、4、5、6、7、8、9或10)的寡聚天然氨基酸；R11是C1-10烷基；
d是0、1、2、3、4、5或6；
-(PEG)i-和-(PEG)j-各自为PEG片段，包含指定数量的连续-(O-C2H4)-结构单元或连续-(C2H4-O)-结构单元，任选地在一个末端附加C1-10亚烷基；每个i独立地为1-100的整数，每个j独立地为1-100的整数；
M是氢或LKa-L2―L1―B―P；
Q是NH2或L2―L1―B―P；
条件是不包括以下情况：M是氢且同时Q是NH2；
每个LKa独立选自
opSu为或其混合物；其中，*表示和L2的连接部分；
B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、-C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
P是与B部分，或L1部分，或L2部分连接的负载物。
在一些实施方案中，L2为氨基酸残基序列，即-*(AA)n**-，n为1-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键。在一些实施方案中，AA在每次出现时独立地为Phe、Lys、Gly、Ala、Leu、Asn、Val、Ile、Pro、Trp、Ser、Tyr、Cys、Met、Asp、Gln、Glu、Thr、Arg、His中的任意一种或其任意地组合。
在一些实施方案中，n为1-100的整数。在一些实施方案中，n约为1、2、3、4、5、6、7、8、9、10、11、12、14、20、22、25、28、32、34、40、50、52、60、70、86、90、100，或任何两个值之间的区间值(或端点值)。在一些实施方案中，n约为 1-50。在一些实施方案中，n约为1-30。在一些实施方案中，n约为1-20。在一些实施方案中，n约为1-10。
在一些实施方案中，L1是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸。在一些实施方案中，L1为Val、Cit、Phe、Lys、Gly、Ala、Leu、Asn中的任意一种或其任意地组合，优选地，为-Gly-Gly-Phe-Gly-、-Phe-Lys-、-Val-Cit-、-Val-Lys-、-Gly-Phe-Leu-Gly-、-Ala-Leu-Ala-Leu-、-Ala-Ala-Ala-及其组合。在一些实施方案中，L1为-Val-Cit-。
在一些实施方案中，其中B选自：(-PABC-)，-NH-CH2-U-，或-NH-CH2-U-(CH2)g-(CO)-；其中g为1、2、3、4、5或6；U不存在，或是CH2、O、S或NH，优选O或S。
在一些实施方案中，-L1-B-表示-Val-Cit-PABC-。在一些实施方案中，-L2-L1-B-表示-Gly-Gly-Gly-Val-Cit-PABC-。
在一些实施方案中，所述负载物P选自由小分子化合物(例如，各种作用机制的小分子药物，包括各种传统小分子药物、光声动力疗法药物、光热疗法药物等，例如化疗药、小分子靶向药、免疫激动剂等，例如传统细胞毒性药物，如顺铂、紫杉醇、5-氟尿嘧啶、环磷酰胺和苯达莫司汀等；小分子靶向药，如甲磺酸伊马替尼、吉非替尼和安罗替尼等；免疫激动剂，如STING激动剂、TLR激动剂等)、核酸及核酸类似物、示踪分子(包括荧光分子、生物素、荧光团、发色团、自旋共振探针及放射性标记等)、短肽、多肽、拟肽和蛋白质组成的组。在一些实施方案中，所述负载物P是细胞毒素或其片段，具有任选的衍生化以连接到式(I)化合物中的L部分。
在一些实施方案中，细胞毒素选自由紫杉烷类、美登木素类、奥利斯他汀类、埃博霉素类(epothilones)、康普瑞丁A-4磷酸盐(combretastatin A-4 phosphate)、康普瑞丁A-4(combretastatin A-4)及其衍生物、吲哚-磺胺类、长春碱类如长春碱(vinblastine)、长春新碱(vincristine)、长春地辛(vindesine)、长春瑞滨(vinorelbine)、长春氟宁(vinflunine)、长春甘酯(vinglycinate)、脱水长春碱(anhy-drovinblastine)、尾海兔素10(dolastatin 10)及其类似物、软海绵素B、艾瑞布林(eribulin)、吲哚-3-氧乙酰酰胺类、鬼臼毒素类、7-二乙氨基-3-(2'-苯并噁唑基)-香豆素(DBC)、圆皮海绵内酯(discodermolide)、laulimalide、喜树碱类及其衍生物、米托蒽醌、米托胍腙、氮芥类、亚硝基脲类、氮杂环丙烷类、苯并多巴、卡波醌、美妥替哌、乌瑞替哌、达内霉素(dynemicin)、埃斯培拉霉素(esperamicin)、新制癌菌素、阿克拉霉素、放线菌素、安曲霉素、博来霉素、放线菌素C、卡拉比星、洋红霉素、抗癌霉素、洋红霉素、放线菌素D、柔红霉素、地托比星、阿霉素、表柔比星、依索比星、依达比星、麻西罗霉素、丝裂霉素类、诺拉霉素、橄榄霉素、培洛霉素、泊非霉素、嘌罗霉素、铁阿霉素、罗多比星、链黑霉素、链佐星、净司他丁、佐柔比星、单端孢霉烯类、T-2毒素、verracurin A、杆孢菌素A、安归啶(anguidine)，乌苯美司、重氮丝氨酸、6-重氮基-5-氧代-L-正亮氨酸，二甲叶酸、甲氨蝶呤、蝶罗呤、三甲曲沙、依达曲沙、氟达拉滨、6-巯基嘌呤、硫咪嘌呤、硫鸟嘌呤、安西他滨、吉西他滨、依诺他滨、阿扎胞苷、6-氮尿苷、卡莫氟、阿糖胞苷、二脱氧 尿苷、去氧氟尿苷、氟尿苷、卡普睾酮、丙酸屈他雄酮、环硫雄醇、美雄烷、睾内酯、氨鲁米特、米托坦、曲洛司坦、氟他胺、尼鲁米特、比卡鲁胺、醋酸亮丙瑞林、蛋白激酶抑制剂和蛋白酶体抑制剂组成的组；和/或
细胞毒素选自长春碱类、秋水仙碱类、紫杉烷类、奥利斯他汀类、美登木素类、calicheamicin、doxonubicin、duocarmucin、SN-38、念珠藻素类似物(cryptophycin analog)、deruxtecan、duocarmazine、calicheamicin、centanamycin、dolastansine、pyrrolobenzodiazepine和exatecan及其衍生物；和/或
细胞毒素选自奥利斯他汀(auristatin)，尤其是MMAE、MMAF或MMAD；和/或
细胞毒素选自exatecan及其衍生物，例如DX8951f；和/或
细胞毒素选自DXd-(1)和DXd-(2)；优选DXd-(1)。
在一些实施方案中，所述连接子-负载物化合物如式(I-2)所示：

在一些实施方案中，所述抗体选自下组：抗CD19抗体、抗CD20抗体、抗CD22抗体、抗CD25抗体、抗CD30/TNFRSF8抗体、抗CD33抗体、抗CD37抗体、抗CD44v6抗体、抗CD56抗体、抗CD70抗体、抗CD71抗体、抗CD74抗体、抗CD79b抗体、抗CD117/KITk抗体、抗CD123抗体、抗CD138抗体、抗CD142抗体、抗CD174抗体、抗CD227/MUC1抗体、抗CD352抗体、抗CLDN18.2抗体、抗DLL3抗体、抗ErbB2/HER2抗体、抗CN33抗体、抗GPNMB抗体、抗ENPP3抗体、抗Nectin-4抗体、抗EGFRvⅢ抗体、抗SLC44A4/AGS-5抗体、抗CEACAM5抗体、抗PSMA抗体、抗TIM1抗体、抗LY6E抗体、抗LIV1抗体、抗Nectin4抗体、抗SLITRK6抗体、抗HGFR/cMet抗体、抗SLAMF7/CS1抗体、抗EGFR抗体、抗BCMA抗体、抗AXL抗体、抗NaPi2B抗体、抗GCC抗体、抗STEAP1抗体、抗MUC16抗体、抗间皮素(Mesothelin)抗体、抗ETBR抗体、抗EphA2抗体、抗5T4抗体、抗FOLR1抗体、抗LAMP1抗体、抗Cadherin 6抗体、抗FGFR2抗体、抗FGFR3抗体、抗CA6抗体、抗CanAg抗体、抗整合素αV抗体、抗TDGF1抗体、抗肝配蛋白(Ephrin)A4抗体、抗TROP2抗体、抗PTK7抗体、抗NOTCH3抗体、抗C4.4A抗体、抗FLT3抗体、抗B7H3/4抗体、抗TF(组织因子(Tissue Factor))抗体、抗ROR1/2/抗体；优选为抗CD19抗体、抗ErbB2/HER2抗体、抗CLDN18.2抗体、抗Nectin-4抗体、抗FGFR3抗体、抗Trop2抗体。
在一些实施方案中，所述具有糖苷转移活性的糖苷内切酶为N-乙酰葡萄糖胺内切水解酶。在一些实施方案中，所述N-乙酰葡萄糖胺内切水解酶包括选自Endo S(酿脓链球菌(Streptococcus pyogenes)内切糖苷酶-S)、Endo F3(米尔伊丽莎白菌(Elizabethkingia miricola)内切糖苷酶-F3)、Endo S2(Endoglycosidase-S2，酿脓链球菌内切糖苷酶-S2)、Endo Sd(Endoglycosidase-Sd，酿脓链球菌内切糖苷酶-Sd)和Endo CC(Endoglycosidase-CC，酿脓链球菌内切糖苷酶-CC)中的至少一种；或所述N-乙酰葡萄糖胺内切水解酶包括选自Endo H、Endo D、Endo F2、Endo F3、Endo M、Endo CC1、Endo CC2、Endo Om、Endo S和Endo S2中的至少一种。
在一些实施方案中，所述具有糖苷转移活性的糖苷内切酶共价连接有Halo标签，糖苷内切酶融合蛋白通过Halo标签固定在含卤代烷基连接子的支持物上；所述Halo标签为脱卤素酶或其变体或其截短的功能活性部分。在一些实施方案中，所述糖苷内切酶的一端连接有Halo标签，所述糖苷内切酶的另一端连接有His标签(His标签为组氨酸多肽，如His4，His5，His6，His8，His10，His12，或His14)。在一些实施方案中，所述糖苷内切酶的氨基端连接有Halo标签，所述糖苷内切酶的羧基端连接有His标签，即Halo-糖苷内切酶-His(此处His指的是His标签，为His4，His5，His6，His8，His10，His12，或His14；下同)。在一些实施方案中，所述糖苷内切酶的氨基端连接有Halo标签，所述糖苷内切酶的羧基端连接有His标签，所糖苷内切酶为Endo-S2，即Halo-Endo S2-His。
在一些实施方案中，所述支持物包含氯代烷基连接子，使得所述糖苷内切酶融合蛋白通过氯代烷基连接子和Halo标签之间的共价相互作用固定在所述支持物上。在一些实施方案中，所述氯代烷基连接子由具有式(III)结构的氯代烷基底物产生：
其中，u为1-20的整数，v为0-20的整数，w为1-19的整数。
在一些实施方案中，所述支持物具有式(IV)的结构：
其中u为1-20的整数，v为0-20的整数，w为1-19的整数；
为树脂、珠子、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面。在一些实施方案中，为树脂。在一些实施方案中，为琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂。在一些实施方案中，为高度交联的琼脂糖树脂或聚甲基丙烯酸甲酯树脂。
在一个方面，本发明提供了一种糖苷内切酶融合蛋白，其中包含共价连接在一起的糖苷内切酶和Halo标签；所述Halo标签为脱卤素酶或其变体或其截短的功能活性部分。在一些实施方案中，所述糖苷内切酶融合蛋白由共价连接在一起的糖苷内切酶和Halo组成。
在一些实施方案中，所述融合蛋白在所述糖苷内切酶的一端共价连接有Halo标签，且在所述糖苷内切酶的另一端共价连接有His标签。在一些实施方案中，所述糖苷内切酶的氨基端端共价连接有Halo标签，所述糖苷内切酶的羧基端共价连接有His标签。
在一些实施方案中，所述糖苷内切酶选自Endo S(酿脓链球菌(Streptococcus pyogenes)内切糖苷酶-S)、Endo F3(米尔伊丽莎白菌(Elizabethkingia miricola)内切糖苷酶-F3)、Endo S2(Endoglycosidase-S2，酿脓链球菌内切糖苷酶-S2)、Endo Sd(Endoglycosidase-Sd，酿脓链球菌内切糖苷酶-Sd)和Endo CC(Endoglycosidase-CC，酿脓链球菌内切糖苷酶-CC)中的至少一种；或所述糖苷内切酶选自Endo H、Endo D、Endo F2、Endo F3、Endo M、Endo CC1、Endo CC2、Endo Om、Endo S和Endo S2中的至少一种。
在一个实施方案中，所述糖苷内切酶为内切-β-N-乙酰氨基葡糖苷酶。在一个实施方案中，所述糖苷内切酶选自下组：Endo H、Endo D、Endo F2、Endo F3、Endo M、Endo CC1、Endo CC2、Endo Om、Endo S和Endo S2。
在一些实施方案中，所述His标签为多个连续的组氨酸残基。在一些实施方案中，所述His标签为3个组氨酸、4个组氨酸、5个组氨酸、6个组氨酸、7个组氨酸、8个组氨酸、9个组氨酸或10个氨基酸。在一些实施方案中，所述His标签为His6。在一个实施方案中，所述His标签为His8。在一个实施方案中，所述His标签为His10。
在一个实施方案中，所述糖苷内切酶融合蛋白包含如SEQ ID NO:1所示的氨基酸序列，或与SEQ ID NO:1相比具有至少80％同一性、至少85％同一性性、至少90％同一性，或与SEQ ID NO:1相比具有1个或多个保守氨基酸取代。
在一些实施方案中，所述糖苷内切酶融合蛋白包含如SEQ ID NO:1所示的氨基酸序列或由其组成。
在一些实施方案中，所述糖苷内切酶融合蛋白包含如SEQ ID NO:1中第1位至第1150位所示的氨基酸序列，或与SEQ ID NO:1中第1位至第1150位所示的氨基酸相比具有至少90％同一性，或与SEQ ID NO:1中第1位至第1150位所示的氨基酸相比具有1个或多个保守氨基酸取代。在一些实施方案中，所述糖苷内切酶融合蛋白包含如SEQ ID NO:1中第1位至第1150位所示的氨基酸序列或由其组成。
在一些实施方案中，所述的糖苷内切酶融合蛋白的PI约为4-7。在一些实施方案中，糖苷内切酶融合蛋白的PI约为4、4.1、4.3、4.5、4.7、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7，或任何两个值之间的区间值(包括端点值)。
在一个方面，本发明提供了一种制备本发明的糖苷内切酶融合蛋白的方法，其包括：(a)提供糖苷内切酶的核酸序列；(b)糖苷内切酶的核酸序列的一端连接Halo标签的核酸序列；
将所得到的核酸序列克隆到合适的载体中，再将所述载体转化在合适的宿主细胞中，并在宿主细胞中表达本发明的糖苷内切酶融合蛋白。
在一些实施方案中，制备本发明的糖苷内切酶融合蛋白的方法，其包括：(a)提供糖苷内切酶的核酸序列；(b)糖苷内切酶的核酸序列的一端连接Halo标签的核酸序列；(c)糖苷内切酶的核酸序列的另一端连接His标签的核酸序列。
在一些实施方案中，将Halo标签的核酸序列连接在糖苷内切酶的序列的氨基端。在一些实施方案中，将Halo标签的核酸序列连接在糖苷内切酶的核酸序列的氨基端，并将His标签的核酸序列连接在糖苷内切酶的核酸序列的羧基端。
在一个方面，本发明提供一种固定化的糖苷内切酶融合蛋白，其包含固定在支持物上的本发明的糖苷内切酶融合蛋白。
在一些实施方案中，所述支持物包含卤代烷基连接子，使得所述糖苷内切酶融合蛋白通过卤代烷基连接子和Halo标签之间的共价相互作用固定在所述支持物上。
在一些实施方案中，所述支持物包含氯代烷基连接子，使得所述糖苷内切酶融合蛋白通过氯代烷基连接子和Halo标签之间的共价相互作用固定在所述支持物上。在一些实施方案中，所述氯代烷基连接子由具有式(III)结构的氯代烷基底物产生：
其中，u为1-20的整数，v为0-20的整数，w为1-19的整数。
在一些实施方案中，所述支持物具有式(IV)的结构：
其中u为1-20的整数，v为0-20的整数，w为1-19的整数；
为树脂、珠子、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面。在一些实施方案中，为树脂。在一些实施方案中，为琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂。在一些实施方案中，为高度交联的琼脂糖树脂或聚甲基丙烯酸甲酯树脂。
另一方面，本发明还提供一种预装柱，所述预装柱填充有固定化的糖苷内切酶融合蛋白。
另一方面，本发明还提供所述的固定化的糖苷内切酶融合蛋白，和/或所述的预装柱在偶联药物或抗体偶联物的制备和/或纯化中的应用。在一些实施方案中，所述的固定化的糖苷内切酶融合蛋白，和/或所述的预装柱在偶联药物或或抗体偶联物的制备(如纯化)中易于工业化放大生产。
本申请固定化的糖苷内切酶催化的一步法偶联体系的优点：
1)液相酶催化需要投入大量糖苷内切酶，反应体系中同时引进大量酶相关杂质(如宿主蛋白和核酸)，对酶纯度要求高(工艺复杂，制备成本高)，对于后续偶联药物下游去除杂质带来挑战很大；酶固定化对于起始糖苷内切酶纯度要求不高(酶制备工艺简单，成本低)，引进到反应体系中的杂质非常少，极大简化了偶联药物下游杂质去除工艺；以及酶固定化有利于分离纯化，使得工具酶更容易从产物中除去，减少因工具酶残留造成的产物糖链及小分子脱落的可能，因而可以降低潜在的产物分解造成的毒性；
2)酶固定化有利于酶的重复使用；
3)连续流智能化偶联技术，大大简化了反应和纯化的流程，大幅提高了药物发现的效率，保障药物生产过程中工艺稳健性；
4)利用柱上固定的糖苷内切酶，实现抗体和连接子-负载物的一步法偶联；
5)本发明的糖苷内切酶融合蛋白的PI为5.5左右，而ADC偶联物的PI为8-9；采用AEX和/或CEX便于后期从ADC产物中去除糖苷内切酶融合蛋白；
6)本发明的糖苷内切酶融合蛋白偶联柱催化能够实现优异的偶联效率，易线性放大，残留酶容易去除，从而更适合ADC的工业化生产。
附图说明
图1为显示本发明的糖苷内切酶融合蛋白(Halo-Endo S2-His)纯化后的SDS-PAGE电泳图；图中，1表示样品来自细胞液，2表示样品来自上清液，3表示样品来自流穿液(采用的平衡缓冲液的组分为：50mM Tris，150mM NaCl，20mM咪唑，pH7.4)，4表示样品来自洗涤液(洗涤液的组分为：50mM Tris，150mM NaCl，80mM咪唑，pH7.4)，5表示样品来自洗脱液(洗脱液的组分为：50mM Tris，150mM NaCl，500mM咪唑，pH7.4)，6表示样品来自于洗脱液(洗脱液的组分为：50mM Tris，150mM NaCl，500mM咪唑，pH7.4)，7表示样品来自磁珠；糖苷内切酶融合蛋白(Halo-Endo S2-His)的 分子量约为130kDa；
图2为显示本发明固定化的糖苷内切酶融合蛋白(Halo-Endo S2-His)在不同温育时间上清液的SDS-PAGE电泳图；
图3为显示将本发明的糖苷内切酶融合蛋白(Halo-Endo S2-His)用于混悬式偶联反应，产物的SDS-PAGE电泳图；
图4为显示将本发明的糖苷内切酶融合蛋白(Halo-Endo S2-His)用于混悬式偶联反应，产物的HIC-HPLC谱图；
图5A为显示ADC通过Q Sepharose FF纯化的图谱，图5B显示ADC和糖苷内切酶融合蛋白通过Q Sepharose FF纯化的图谱；
图6A为显示ADC挂柱Capto S impact后纯化的图谱，图6B为显示ADC和糖苷内切酶融合蛋白挂柱Capto S impact后纯化的图谱；
图7显示ADC-1对细胞表面ErbB2/Her2的亲和力检测(EC50,nM)；
图8显示不同药物对肿瘤细胞BT474增殖的抑制作用(IC50,nM)；
图9显示不同药物对肿瘤细胞NCI-N87增殖的抑制作用(IC50,nM)；
图10显示不同药物对肿瘤细胞HepG2增殖的抑制作用(IC50,nM)。
图11显示ADC-1对NCI-N87CDX小鼠模型的抑制效果。
图12显示ADC-2的HIC-HPLC图谱。图13显示ADC-2和不同药物对肿瘤细胞SK-BR-3增殖的抑制作用(IC50,nM)。
图14显示ADC-2和不同药物对肿瘤细胞HCC1954增殖的抑制作用(IC50,nM)。
图15显示ADC-2和不同药物对肿瘤细胞MDA-MB-468增殖的抑制作用(IC50,nM)。
图16显示ADC-3的高分辨质谱去卷积图。
图17显示ADC-3和不同药物对肿瘤细胞BxPC-3增殖的抑制作用(IC50,nM)。
图18显示ADC-3和不同药物对肿瘤细胞FaDu增殖的抑制作用(IC50,nM)。
图19显示ADC-3和不同药物对肿瘤细胞HepG2增殖的抑制作用(IC50,nM)。
图20显示ADC-3对NCI-N87CDX小鼠的抑瘤效果。
图21显示ADC-3对NCI-N87CDX小鼠体重影响。
图22为ADC-4的HIC-HPLC检测分析图。
图23为ADC-5的HIC-HPLC检测分析图。
图24显示ADC-4、ADC-5对BxPC-3的体外抑制活性。
图25显示ADC-4、ADC-5对FaDu的体外抑制活性。
图26显示ADC-4、ADC-5对HepG2-3的体外抑制活性。
图27显示ADC-6的HIC-HPLC检测分析。
图28显示ADC-6对SK-BR-3的体外抑制活性。
图29显示ADC-6对NCI-N87的体外抑制活性。
1.定义
除非另有定义，否则本发明使用的所有技术和科学术语具有与本领域技术人员通常理解的相同含义。此外，与蛋白和核酸化学、分子生物学、细胞和组织培养、微生物学 和免疫学有关的术语和实验方法是本领域广泛使用的术语和常用方法。当本文中出现商品名时，意在指代其相应的商品或其活性成分。本文所引用的所有专利、公开的专利申请和出版物均援引加入本文。同时，为了更好地理解本发明，相关术语的定义和解释提供如下。
如本文所用，表述“至少一个”或“一个或多个”或“一种”或“多种”是指一、二、三、四、五、六、七、八、九、一百、两百、三百、四百、五百、六百、七百、八百、九百或更多，等等。如本文所用，除非明确指出相反，“一个”和“一种”应理解为“至少一个”或“至少一种”。
当以范围、优选范围、或者优选数值上限或优选数值下限的形式表述特定量、浓度或其他值或参数的时候，应当理解为相当于具体揭示了通过将任意数值上限或优选数值与任意数值下限或优选数值组合起来的任何范围，不管所述范围是否明确列举。除非另有说明，本文所列出的数值范围旨在包括范围的端点、该范围内的所有整数和分数(小数)，以及任意两个数值的区间值。例如，表述“u”为1-20的整数”应理解为u为1到20的任一整数，例如，u可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。其他类似的表述也应以类似的方式理解。
术语“约”和“大约”，当与数值变量结合使用时，如浓度、等电点(pI)、pH、温度或特定范围，通常是指变量的数值和变量的所有数值都在实验误差范围内(例如，平均值的95％置信区间内)或具体值±10％以内，或更宽的范围。
术语“任选”或“任选地”是指其后描述的事件可能发生但不是一定发生，该表述说明包括所述事件或情况发生或不发生的情况。
术语“包含”、“包括”、“含有”和“具有”等是开放性的，不排除额外的未列举的元素、步骤或成分。术语“由…组成”排除未指明的任何元素、步骤或成分。术语“基本上由…组成”指范围限制在指定的元素、步骤或成分，加上任选存在的元素、步骤或成分，其不会实质上影响所要求保护主题的关键和新的特征。应当理解，术语“包含”涵盖术语“基本上由…组成”和“由…组成”。
如本文所用，“生物分子”的定义涵盖蛋白、核酸、脂质、碳水化合物、小核苷酸、氨基酸及其衍生物。
如本文所用，“核酸”或“多核苷酸”是指至少两个核苷酸或核苷酸衍生物通过磷酸二酯键连接在一起形成的聚合物，包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。
如本文所用，“载体”是一种用于将外源核酸转移到宿主细胞中的媒介物，外源核酸在宿主细胞中扩增或表达。如本文所用，“载体”的定义涵盖质粒(如线性化质粒)、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。如本文所用，载体可在宿主细胞内表达和/或复制，是指载体能够在宿主细胞中表达RNA多核苷酸或多肽和/或产生载体的多个拷贝。为了“可表达”或“可复制”，载体可以包含与启动子或复制子可操作连接的核酸序列或元件。如本文所用，与核酸序列或元件相关的“可操作连接”是指这些核酸序列在功能上相互关联。例如，启动子可以与编码多肽的核酸序列可操作连接，由此启动子调节或介导核酸的转录。本领域技术人员可根据特定目的选择和使用合适的载体。
Fc融合蛋白(Fc fusion protein)是指将生物活性蛋白或多肽与IgG的铰链区、Fc片段进行融合表达所获得的重组蛋白；其中生物活性蛋白或多肽如配体、细胞因子、受体、抗原、与细胞表面抗原结合的环状多肽。Fc融合蛋白赋予了所融合蛋白更多抗体的特性，包括延长了其血浆半衰期、发挥Fc片段特有的功能效应等。
GlcNAc基序指的是抗体Fc区第297位天冬氨酸(Asn)上有两个保守的N-糖基化位点的糖链，且该糖链包含与Asn共价连接的N-乙酰基葡萄糖-β-(1,4)-N-乙酰基葡萄糖。Fc区糖基化是一种复杂的翻译后修饰过程，在各种不同酶的参与下形成多种长度、组成和结构各不相同的糖链，不同糖链对蛋白生物活性、构象、稳定性、溶解度、药物动力学等的影响各不相同。在一些实施方案中，Fc糖基化糖分子具有复杂的双天线型核心结构，该结构由甘露糖和N-乙酰葡糖胺两种戊糖分子组成，不同糖型除核心结构外还另外含有不同数目的糖分子，如岩藻糖、甘露糖、N-乙酰葡糖胺、半乳糖、二等分N-乙酰葡糖胺和唾液酸，多因末端糖的半乳糖基化和唾液酸基化产生异质性。根据末端半乳糖数量，分为三个不同的亚型(G0,G1和G2)，每种亚型中包含有无核心岩藻糖及二等分N-乙酰葡糖胺的不同形式，即共有16种中性复合型结构。如本文所用，“肽”、“多肽”或“蛋白”是指共价连接的两个或多个氨基酸。除非另有具体说明，这些术语可以互换。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：Ala，一字母代码：A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
如本文所用，“序列同一性”具有本领域公认的含义，并且两个多肽之间的序列同一性百分比可通过使用公开可用的算法比对两个序列来计算，如基于局部比对算法搜索工具(BLAST)和快速自适应收缩/阈值算法(FASTA)(参见，例如：Computational Molecular Biology,Lesk,A.M.编,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.编,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.和Griffin,H.G.编,Humana Press,New Jersey,1994)。虽然有多种方法来测量两个多肽之间的同一性，但术语“同一性”是本领域技术人员所熟知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
如本文所用，术语“变体”是指与参考蛋白(或核酸)相比，具有一个或多个残基(或核苷酸)的取代、缺失、插入的蛋白(或核酸)。参考蛋白可以是可从自然资源中分离的天然存在的蛋白(即野生型蛋白)，或者工程化蛋白。如本文所用，变体的功能或活性，如EndoS变体、EndoS2变体或Halo标签变体，分别与参考EndoS、EndoS2或Halo标签的功能或活性基本相似或相当或更高。
如本文所用，术语“糖苷内切酶”是指催化水解寡糖链及多糖的内部糖苷键的酶，可用于从糖蛋白上切割多糖的酶；在一些实施方案中，本申请的“糖苷内切酶”可用于水解抗体Fc区N-糖基化位点的两个N-乙酰基葡萄糖之间的β-(1→4)糖苷键。“糖苷转移活性”是指催化活化的糖连接到不同的受体分子，如蛋白、核酸、寡糖、脂和小分子上；本申请中可将具有含有噁唑啉低聚糖的供体连接到抗体Fc区GlcNAc上。
如本文所用，术语“Halo标签”是指卤代烷烃脱卤酶或其变体，其从卤代烷基底物(例如，包含卤代烷基部分-(CH2)2-30-X的试剂，其中X是卤素如F、Cl、Br、I，特别是Cl或Br)中除去卤素，并与底物的剩余部分形成共价键。突变的卤代烷烃脱卤酶已在，例如，WO2006/093529和WO2008/054821中描述，其相关内容援引加入本文。可用于本发明的突变卤代烷烃脱卤酶可包括但不限于黄杆菌属(Xanthobacter)脱卤酶(如自养黄杆菌(Xanthobacter autotrophicus)脱卤酶(DhIA))或红球菌属(Rhodococcus)脱卤酶(如玫瑰红红球菌(Rhodococcus rhodochrous)脱卤酶(DhaA))的突变体，如在催化三联体残基处包含一个或多个取代，如用Phe/Ala/Gly/Gln/Asn取代His272或用Cys取代Asp106或其他取代，如WO2008/054821中所述。前提是突变的卤代烷烃脱卤酶能够与卤代烷基底物形成共价键。
“His标签”是由组氨酸组成的多肽，如His-His(His2)、His-His-His(His3)、His-His-His-His(His4)、His-His-His-His-His(His5)、His-His-His-His-His-His(His6)、His-His-His-His-His-His-His-His-His-His(His10)。
如本文所用，术语“融合蛋白”是指通过DNA重组技术得到的两个或多个基因的共同表达的蛋白产物。例如，将第一个蛋白的编码基因的终止密码子删除，再接上带有终止密码子的第二个蛋白基因，可以实现两个基因的共同表达。糖苷内切酶融合蛋白可以进一步包含一个或多个附加元件，如附加多肽或标签，例如Halo标签和/或His标签。在一些实施方案中，糖苷内切酶融合蛋白基本上保留期望的特性。
如本文所用，术语“树脂”是指受热后有软化或熔融范围，软化时在外力作用下有流动倾向，常温下是固态、半固态或液态的有机聚合物。“卤代树脂”是指树脂中的至少一个官能团被卤基所取代而形成的新化合物。
如本文所用，术语“偶联”是指至少两部分(例如，至少两个分子或同一分子的至少两个末端)的共价连接。
如本文所用，“偶联物(conjugate)”可通过至少两部分(例如，至少两个分子或同一分子的至少两个末端/侧链)的共价连接制备。
如本文所用，术语“抗体偶联药物”是指偶联物，其包含共价偶联至负载物的抗体或抗体片段，负载物为小分子化合物、激动剂、核酸、核酸类似物或荧光分子。
如本文所用，术语“偶联药物”是指偶联物，其包含共价偶联至负载物的含有Fc区的蛋白，负载物为小分子化合物、激动剂、核酸、核酸类似物或荧光分子。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充 分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。IgG通常包含分子量约23,000道尔顿的两条相同的轻链多肽和分子量约为53,000-70,000的两条相同的重链多肽。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为Vλ。
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链(VL)和重链(VH)链部分的可变区决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域分别包含重链和轻链的羧基端。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.等,U.S.Department of Health and Human Services,Sequences of Proteins of Immunological Interest,(1983)及Chothia和Lesk,J.Mol.Biol.,196:901-917(1987))。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat等,U.S.Dept.of Health and Human Services在“Sequence of Proteins of Immunological Interest”(1983)提出的编号系统。抗体还可以采用EU编号系统。
轻链恒定区“包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区(CL)包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。
“Fc区”是抗体的尾区，其与细胞表面受体和补体系统的一些蛋白质相互作用。该 特性允许抗体激活免疫系统。在IgG、IgA和IgD抗体同种型中，Fc区由两个相同的蛋白质片段组成，衍生自抗体的两条重链的第二和第三恒定区；在IgM和IgE抗体同种型中，Fc区含有三个重链恒定结构域(CH2-4)；IgG的Fc区具有高度保守的N-糖基化位点。Fc片段的糖基化对于Fc受体介导的活性是必需的，且不同的糖型对治疗性抗体的药学性质有不同的影响。
抗体可以通过使用常规重组DNA技术制备。使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的细胞系。这些技术在各种实验室手册和主要出版物中均有描述。在这方面，下文描述的适合本发明使用的技术参考文献如Current Protocols in Immunology,Coligan等编,Green Publishing Associates and Wiley-Interscience,John Wiley and Sons,New York(1991)，Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells,D.L.Hacker,F.M.Wurm,in Reference Module in Life Sciences,2017；其全部内容包括补充内容通过引用并入全文。
在一些实施方案中，可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生单克隆抗体。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
如本文所用，术语“等电点(pI)”是分子(如蛋白)在不带表面净电荷时的水溶液的pH(氢浓度指数)值并表达为pH单位。蛋白的pI可以采用本领域所熟知的方法实验测量，如，成像毛细管等电聚焦(iCIEF)和毛细管等电聚焦(CIEF)。具有不同pI的不同生物分子(蛋白、核酸、多糖等)在给定的pH值下可能带有不同的电荷，从而允许通过离子交换层析或等电聚焦等方法将它们分离。
如本文所用，具有“碱性pI”的分子是指该分子的pI高于7.0。如本文所用，具有“酸性pI”的分子是指该分子的pI低于7.0。
如本文所用，“离子交换层析(IEX)”是指基于生物分子的净表面电荷差异及其对离子交换剂(也称为介质、树脂或固定相)的亲和力差异来分离生物分子的技术。例如，在阴离子交换层析中，pI低于缓冲液pH的蛋白将具有负的净表面电荷并与带正电荷的阴离子交换剂结合；然而，另一种pI高于缓冲液pH的蛋白将具有正的净表面电荷并且不 与带正电的阴离子交换剂结合，因此将与缓冲液一起通过介质。
如本文所用，术语“支持物”是指可以以固体或半固体形式从反应混合物中分离的水不溶性物质，如表面、凝胶、聚合物、基质、颗粒、树脂、珠子或膜。
“间隔子(spacer)”是指位于不同结构模块之间，可以从空间上将结构模块间隔开的结构。间隔子的定义并不限定是否具有一定的功能，也不限定是否能在体内被切断或降解。间隔子的实例包括但不限于氨基酸和非氨基酸结构，其中非氨基酸结构可以但不限于是氨基酸衍生物或类似物。“间隔序列(Spacer sequence)”是指作为间隔子的氨基酸序列，其实例包括但不限于单个氨基酸、含有多个氨基酸的序列，例如含有两个氨基酸的序列，如GA等，或者例如GGGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS等。自切除间隔子(例如自切除间隔子Sp1)是共价组件，其使得前体中的保护性部分激活后两个化学键相继裂解：保护性部分(例如可切割序列)在激活后被去除，引发级联的分解反应，导致按先后顺序释放较小分子。自切除间隔子的实例包括但不限于PABC(对氨基苄氧基羰基)、缩醛、杂缩醛及其组合。
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和的脂肪烃基团，其通过单键与分子的其余部分连接。烷基可以具有1-20个碳原子，指“C1-C20烷基”，例如C1-C4烷基、C1-C3烷基、C1-C2烷基、C3烷基、C4烷基、C3-C6烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1,2-二甲基丙基、新戊基、1,1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、2,3-二甲基丁基、1,3-二甲基丁基或1,2-二甲基丁基，或者它们的异构体。二价自由基是指由相应的一价自由基的具有自由价电子的碳原子去除一个氢原子从而获得的基团。二价自由基具有两个与分子其余部分相连的连接位点。例如“亚烷基”或“烷基亚基”指饱和的直链或支链的二价烃基。“亚烷基”的实例包括但不限于如亚甲基(-CH2-)、亚乙基(-C2H4-)、亚丙基(-C3H6-)、亚丁基(-C4H8-)、亚戊基(-C5H10-)、亚己基(-C6H12-)、1-甲基亚乙基(-CH(CH3)CH2-)、2-甲基亚乙基(-CH2CH(CH3)-)、甲基亚丙基或乙基亚丙基等。
术语“环烷基”是指由碳原子和氢原子组成的环状饱和脂肪族基团，其通过单键与分子的其余部分连接。环烷基可以具有3-10个碳原子，即“C3-C10环烷基”，例如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基或环癸基。“亚环烷基”是指二价的环烷基。
术语“杂环基”是指上述环烷基中的一个或多个碳原子被选自氮、氧和硫的杂原子替换，例如氮杂、氧杂或硫杂环丙基，氮杂、氧杂或硫杂环丁基，吡咯烷基，吡唑烷基，咪唑烷基，四氢呋喃基，四氢噻吩基，哌啶基，哌嗪基，四氢吡喃基，或四氢噻喃基。“亚杂环基”是指二价的环烷基。
当在本文中提及“取代”时，除非另有说明，相关取代基选自烷基、卤素、氨基、单烷基氨基、二烷基氨基、硝基、氰基、甲酰基、烷基羰基、羧基、烷基氧基羰基、烷基羰基氧基、氨基羰基、单烷基氨基羰基、二烷基氨基羰基、甲酰基氨基、烷基羰基氨基、甲酰基(单烷基)氨基或烷基羰基(单烷基)氨基。
如本文所用，当基团与另一基团进行组合时，基团之间的连接可以是线性或支化的，前提是形成化学上稳定的结构。通过这样的组合形成的结构可以通过该结构中的任意合适的原子与分子的其他部分连接，优选通过指定的化学键连接。例如，当两个或更多个选自-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-的二价基团结合在一起形成组合，两个或更多个二价基团可以相互形成线性连接，例如-CR1R2-C1-10亚烷基-(CO)-、-CR1R2-C4-10亚环烷基-(CO)-、-CR1R2-C4-10亚环烷基-C1-10亚烷基-(CO)-、-CR1R2-CR1R2’-(CO)-、-CR1R2-CR1'R2'-CR1”R2”-(CO)-等。得到的二价结构可以进一步连接到分子的其他部分。
2.连接子-负载物化合物
在一个方面，本发明提供具有式(I)的连接子-负载物化合物：
其中
P为负载物；
D-C(O)-L-为连接子；
D-C(O)-为二糖结构或；
L为接头(例如，其能够以化学方式(例如水解方式)或生物方式(例如酶催化方式)与P断开连接而释放P)，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数(例如2、3、4、5、6、7、8、9或10)。
在一些实施方案中，-L-(P)t为
即式(I)为：
其中，
Ld2和每个Ld1独立地是键；或选自-NH-C1-20亚烷基-(CO)-、-NH-(PEG)i-(CO)-；或为侧链上各自独立地未取代或被-CO-(PEG)j-R11取代的天然氨基酸或聚合度为2-10(即2、3、4、5、6、7、8、9或10)的寡聚天然氨基酸；
-(PEG)i-和-(PEG)j-各自为PEG片段，包含指定数量的连续-(O-C2H4)-结构单元或连续-(C2H4-O)-结构单元，任选地在一个末端附加C1-10亚烷基；
M是氢或LKa-L2―L1―B―P；
Q是NH2或L2―L1―B―P；
条件是不包括以下情况：M是氢且同时Q是NH2；
每个LKa独立选自
opSu为或其混合物；其中，*表示和L2的连接部分；
每个L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、-C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10，AA在每次时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
每个B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
P是与B部分，或L1部分，或L2部分连接的负载物；
R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
R11是C1-10烷基；
d是0、1、2、3、4、5或6；
每个i独立地为1-100的整数；在一些实施方案中，i约为1、2、3、4、5、6、7、8、9、10、14、20、30、34、40、50、60、70、90、100，或任何两个值之间的区间值(包括端点值)；
每个j独立地为1-100的整数；在一些实施方案中，j约为1、2、3、4、5、6、7、8、9、10、14、20、30、34、40、50、60、70、90、100，或任何两个值之间的区间值(包括端点值)；在一些实施方案中，j约为1-20；在一些实施方案中，j约为8-12；在一些实施方案中，j约为8、9、12或13。
在一个实施方案中，B、L1和L2中的至少一个不为“不存在”。
在一个实施方案中，L2选自：-NH-(CH2)a-(CH2)2(CO)-，a是0、1、2、3、4或5的整数； b是1、2、3、4、5、6、7、8、9或10。在一个实施方案中，上述L2结构中的羰基与L1相连。在一个实施方案中，a是0、1、2或3，优选为3。
在一个实施方案中，L2为氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10，AA在每次时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键。在一个实施方案中，AA在每次时独立地为Phe、Lys、Gly、Ala、Leu、Asn、Val、Ile、Pro、Trp、Ser、Tyr、Cys、Met、Asp、Gln、Glu、Thr、Arg、His中的任意一种或其任意地组合；在一个实施方案中，n为2-100的整数，优选为2-50的整数，优选为2-30的整数，优选为2-20的整数，优选为2-10的整数，优选为2、3、4、5、6、7、8、9。
在一个实施方案中，L1包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸。在一个实施方案中，可被酶裂解的氨基酸序列选自-Gly-Gly-Phe-Gly-、-Phe-Lys-、-Val-Cit-、-Val-Lys-、-Gly-Phe-Leu-Gly-、-Ala-Leu-Ala-Leu-、-Ala-Ala-Ala-及其组合；优选的可被酶裂解的氨基酸序列是-Gly-Gly-Phe-Gly-。在一个实施方案中，L1为Val、Cit、Phe、Lys、Gly、Ala、Leu、Asn中的任意一种或其任意地组合，优选地，为-Gly-Gly-Phe-Gly-、-Phe-Lys-、-Val-Cit-、-Val-Lys-、-Gly-Phe-Leu-Gly-、-Ala-Leu-Ala-Leu-、-Ala-Ala-Ala-及其组合。在一个实施方案中，L1表示-Val-Cit-。
在一个实施方案中，Sp1选自PABC(对氨基苄氧羰基)、乙缩醛、杂缩醛及其组合；优选地，Sp1为缩醛、杂缩醛或PABC；进一步优选地，杂缩醛选自N，O-杂缩醛；更优选地，Sp1为-O-CH2-U-或-NH-CH2-U-，其中-O-或-NH-与可被酶裂解的氨基酸序列相连，U不存在，或是CH2、O、S或NH，优选O或S。
在一个实施方案中，B不存在，或者是-NH-CH2-U-，或者是-NH-CH2-U-(CH2)g- (CO)-，其中g为1、2、3、4、5或6，U不存在，或是CH2、O、S或NH，优选O或S。在一个实施方案中，B不存在。在一个实施方案中，B为以下1)、2)或1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个的二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基和-(CO)-。在一个实施方案中，B是-NH-CH2-U-或者是-NH-CH2-U-(CH2)g-(CO)-，U不存在，或是CH2、O、S或NH，优选O或S。在一个实施方案中，B通过酰胺键或酯键或醚键连接至负载物。在一个实施方案中，B选自：(-PABC-)，-NH-CH2-U-，或-NH-CH2-U-(CH2)g-(CO)-；其中g为1、2、3、4、5或6；U不存在，或是CH2、O、S或NH，优选O或S。
在一个实施方案中，-L1-B-表示-Val-Cit-PABC-。
在一个实施方案中，-L2-L1-B-表示-Gly-Gly-Gly-Val-Cit-PABC-。
在一个实施方案中，Ld2和每个Ld1独立选自键，或
每个i、j和k独立选自1-100的整数。
在一个实施方案中，每个i、j和k独立选自1-20的整数。在一个实施方案中，每个i、j和k独立选自1-12的整数。
在一个实施方案中，每个i独立选自2-8的整数；在一个实施方案中，i约为4。
在一个实施方案中，每个j独立选自8-12的整数；在一个实施方案中，j约为8或12。
在一个实施方案中，每个k独立选自1-7的整数；在一个实施方案中，k约为1或3或5。
在一个实施方案中，Ld2和每个Ld1独立选自键；或两端分别带有氨基和羰基的C1- 20亚烷基，或两端分别带有氨基和羰基的一定长度的PEG片段(表示为-(PEG)i-)，或一个或多个天然氨基酸，所述天然氨基酸侧链上各自独立地未取代或被一定长度的PEG片段(表示为-CO-(PEG)j-)取代。
在一个实施方案中，-(PEG)i-包含-(O-C2H4)i-或-(C2H4-O)i-，并且任选地在一个末端附加C1-10亚烷基；-(PEG)j-包含-(O-C2H4)j-或-(C2H4-O)j-，并且任选地在一个末端附加C1- 10亚烷基。在一个实施方案中，-(PEG)i-中包含-C2H4-(O-C2H4)i-或-(C2H4-O)i-C2H4-。
3.糖苷内切酶的固定化
支持物可以是由任何材料制成的固体形式或半固体形式。支持物的非限制性实例可包括但不限于，树脂(例如，琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂、环 氧树脂或纤维素树脂)、凝胶(如藻酸盐水凝胶)、珠/微球/颗粒(例如，聚苯乙烯珠、磁性颗粒)、板、孔、管、薄膜、膜、基质和玻璃(例如，载玻片)。
在一些实施方案中，支持物是树脂。在一些实施方案中，支持物选自由以下组成的组：琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂和纤维素树脂。在一些实施方案中，支持物是高度交联的琼脂糖树脂。
酶固定化的方法有吸附、共价或非共价结合、包埋、包封和交联。期望固定后能保留糖苷内切酶的最大酶活性并且在偶联反应后偶联物产物中存在最小量的游离糖苷内切酶。在一些实施方案中，支持物表面修饰为包含一个或多个官能团，使得糖苷内切酶融合蛋白可以共价固定在支持物上。
在一些实施方案中，支持物包含一个或多个化学活性官能团，其可与糖苷内切酶融合蛋白的反应基团(如胺、巯基和羧酸盐)或与卤代烷基底物中的反应基团形成共价键，或者支持物包含相应结合标签/亲和标记的一个或多个结合配偶体，所述结合标签/亲和标记包含在糖苷内切酶融合蛋白中。化学活性官能团和反应基团之间的对应关系，或结合标签/亲和标记和结合配偶体之间的对应关系是本领域已知的。
在一些实施方案中，支持物包含化学活性官能团，其可与糖苷内切酶融合蛋白上的反应基团(如胺、巯基和羧酸盐)或与卤代烷基底物中的反应基团形成共价键。在一些实施方案中，支持物包含的官能团选自由以下组成的组：氰酸酯、异硫氰酸酯、异氰酸酯、碳二亚胺、N-羟基琥珀酰亚胺(NHS)酯、胺、碳酸酯、环氧化物、马来酰亚胺、卤代乙酰基、氮丙啶、氯甲酸乙酯和脂肪醛。
在一些实施方案中，支持物是环氧活化树脂、CNBr(溴化氰)-活化树脂或NHS-活化树脂。在一些实施方案中，支持物是环氧活化树脂。在一些实施方案中，支持物是环氧活化的琼脂糖树脂。在一些实施方案中，支持物是环氧活化的高度交联琼脂糖树脂。在一些实施方案中，与卤代烷基底物反应之前，将环氧活化树脂预处理以引入氨基。在一些实施方案中，环氧活化树脂的预处理使用氨进行。在一些实施方案中，环氧活化树脂的预处理导致在环氧乙烷环上引入氨基并且环氧乙烷环的开环提供羟基。这样的羟基在制备支持物的后续工序中任选地被封端。在一些实施方案中，环氧活化树脂的预处理导致在环氧乙烷环上引入氨基并且环氧乙烷环的开环提供羟基，其在制备支持物的后续工序中任选地被酯化试剂(例如，乙酰化试剂，如Ac2O)酯化。这样预处理的环氧活化树脂在如上定义的“环氧活化树脂”的范围内。在一些实施方案中，所述树脂是琼脂糖树脂(如高度交联琼脂糖树脂)或聚甲基丙烯酸甲酯树脂。
在一些其他实施方案中，支持物包含相应结合标签/亲和标记的一个或多个结合配偶体，所述结合标签/亲和标记包含在糖苷内切酶融合蛋白中，如附加标签或亲和标记。反应基团之间或结合标签/亲和标记与结合配偶体之间的对应关系是本领域已知的。结合标签/亲和标签和对应的结合配偶体的实例可以包括但不限于His标签和Ni2+、生物素/SPB标签/Strep标签/Strep标签Ⅱ和链霉亲和素/亲和素/中性亲和素、GST标签和谷胱甘肽、Fc标签和蛋白A、钙调素标签和Ca2+、MBP和直链淀粉、S标签和核糖核酸酶S-蛋白、SNAP标签和苄基鸟嘌呤(BG)衍生物，以及CLIP标签和苄基胞嘧啶(BC)衍生物。
在一些实施方案中，支持物通过包含卤代烷基连接子进行官能化以与Halo标签形成共价相互作用。卤代烷基连接子可通过支持物包含的一个或多个官能团与卤代烷基底物中的一个或多个反应基团共价连接引入到支持物，由此获得的支持物也称为卤代烷基连接子修饰的支持物。卤代烷基连接子修饰的支持物在如上定义的“支持物”的范围内。卤代烷基底物的实例包括但不限于例如US20060024808A1和WO2006093529中所述。卤代烷基底物和制备这样的支持物的方法参见，例如，美国专利号7,429,472、7,888,086和8,202,700，日本专利号4748685中的描述，其相关内容援引加入本文。
卤代烷基底物可包含卤代烷基部分，其包含伯或仲卤基。在一些实施方案中，卤代烷基底物包含伯卤基。卤代烷基部分中的卤基选自F、Cl、Br和I。在一些实施方案中，卤代烷基部分中的卤基选自Cl和Br。在一些实施方案中，卤代烷基底物具有下式(V)的结构：
(F1o―H1e)r―Lh―(F2e―H2o)s      (V)
其中，
F1和F2独立地为包含反应基团的部分，其可以与支持物所包含的化学活性官能团形成共价键；
H1和H2独立地选自卤代C2-30烷基；
Lh为化学键或为C3-200亚烷基，并且其中所述亚烷基中的一个或多个(-CH2-)结构任选被-O-、-NH-、-(CO)-、-NH(CO)-和-(CO)NH-代替；
Lh任选地被1、2或3个取代基取代，所述取代基选自-O-C1-10烷基、-NH-C1-10烷基、-(CO)-C1-10烷基、-NH(CO)-C1-10烷基和-(CO)NH-C1-10烷基；
o为0或1，e为0或1，条件是o和e不同；
r为1-100的整数；
s为1-100的整数。
在一些实施方案中，r为1-10的整数，例如1、2、3、4、5、6、7、8、9或10。在一些实施方案中，s为1-10的整数，例如，1、2、3、4、5、6、7、8、9或10。在一些实施方案中，F1或F2中的反应基团选自氨基、胺、巯基和活性酯。在一些实施方案中，活性酯含有一个或多个羧酸自由基(如在合适的醇或苯酚的碳酸单酯中，例如缺电子苯酚如4-硝基苯酚；或如在NHS酯或磺基-NHS酯中)或一个或多个磺酸自由基(如在甲磺酸活性酯中，例如，MsO-)。在一具体实施方案中，F1或F2为
在一些实施方案中，H1和H2独立地选自卤代C2-20烷基。在一些实施方案中，H1和H2独立地选自卤代C2-10烷基，特别是卤代C6烷基。在一些具体实施方案中，H1或H2中的烷基为直链烷基。在一些实施方案中，H1或H2为(CH2)2-30-X。在一些实施方案中，H1或H2为(CH2)2-20-X。在一些实施方案中，H1或H2为(CH2)2-10-X，特别是(CH2)6-X，其中X为选自F、Cl、Br和I的卤素。
在一些实施方案中，支持物是HaloLinkTM树脂(Promega)。
在一些实施方案中，支持物是可包含卤代烷基连接子的树脂，所述卤代烷基连接子包含-(CH2)2-30-X的结构，其中X为选自F、Cl、Br和I的卤素。在一具体实施方案中，支持物是卤代芳基连接子修饰的树脂。在一些实施方案中，支持物是琼脂糖树脂或聚甲基丙烯酸甲酯树脂。在一些实施方案中，支持物是高度交联琼脂糖树脂。
在一些实施方案中，o为1，e为0，r为1，s为1，F1为Lh为H2为(CH2)2-20-Cl，并且所述卤代烷基底物为氯代烷基底物，其具有式(III)的结构：
其中，u为1-20的整数，v为0-20的整数，并且w为1-19的整数。
在一具体实施方案中，u为3，v为2，并且w为5，并且所述氯代烷基底物具有下式(III-1)的结构：
在一些实施方案中，支持物是氯代烷基连接子修饰的支持物并且具有式(IV)的结构:
其中u为1-20的整数，v为0-20的整数，并且w为1-19的整数；表示支持物，其为树脂、珠、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面。在一些实施方案中，支持物是树脂。在一些实施方案中，支持物是琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂。在一些实施方案中，支持物是高度交联琼脂糖树脂。注意，为清楚起见，仅对单个氯代烷基-连接子部分连接到支持物上进行描述，但应理解，会有许多这样的氯代烷基-连接子部分连接到支持物上。
在一些实施方案中，如式(IV)所示的氯代烷基连接子修饰的支持物使用表示为的树脂、珠、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面和式(III)的氯代烷基底物制备。
在一些实施方案中，如式(IV)所示的氯代烷基连接子修饰的支持物由预处理的环氧活化树脂制备，所述环氧活化树脂通过在环氧活化树脂的环氧乙烷环上引入氨基而制备，预处理过程中环氧乙烷环的开环提供羟基，其在制备支持物的后续工序中任选地用Ac2O酯化，式(IV)所示支持物具有式(IV-1)的结构：
其中，
亚结构代表预处理的环氧活化树脂，其中部分代表环氧乙烷环，其与氨基反应并开环得到羟基，随后酯化形成AcO-，部分代表预处理的环氧活化树脂的其他部分。
在一些实施方案中，固定化的糖苷内切酶融合蛋白具有以下结构：
Support----Linker----Halo Tag----EndoS/EndoS2----HisTag
其中，
Support是支持物(如固体支持物)，例如选自树脂、珠、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面。在一些实施方案中，支持物是树脂。在一些实施方案中，支持物是琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂。在一些实施方案中，支持物是高度交联琼脂糖树脂或聚甲基丙烯酸甲酯树脂。在一些实施方案中，支持物选自下组：NHS活化树脂、CNBr活化树脂和环氧活化树脂；
Linker是与支持物共价结合的连接子部分，例如，包含10-60个碳原子的链，任选地包含一个或多个醚、酯、氨基甲酸酯和/或酰胺键；例如，式(VI)或(VI-1)的连接子部分
其中u为1-20的整数，v为0-20的整数，并且w为1-19的整数；
HaloTag是Halo标签(卤代烷烃脱卤酶)，与连接子共价结合；
EndoS/EndoS2是糖苷内切酶；
HisTag是组氨酸标签；
其中一个或多个“----Linker----HaloTag----EndoS----HisTag”部分结合到相同的支持物上。
4.偶联设备
专利申请WO2022170676A或CN114480115A中公开的偶联设备可用于定点偶联抗 体Fc区N-糖基化位点和负载物，具体地用于本发明的制备偶联物或抗体偶联药物的方法，所述偶联设备包括流动反应器和流体输送单元，所述流动反应器填充有固定化的糖苷内切酶，所述流体输送单元与流动反应器的进口流体连通，并且向流动反应器输送含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体(或含Fc区的蛋白)。
在一些实施例中，所述偶联设备包括：至少一个流动反应器，其具有进口和出口，流动反应器中填充有介质(例如色谱珠、纤维或薄膜等基质)，并且糖苷内切酶固定到所述介质上；流体输送单元，其与流动反应器的进口流体连通，并且被配置为根据偶联过程的不同阶段，向流动反应器连续提供至少一种反应流体，该至少一种反应流体包括含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体(或含Fc区的蛋白)；以及流体收集单元，其与流动反应器的出口流体连通，并且被配置为根据偶联过程的不同阶段，控制对流出流动反应器的出口的流体的收集。在至少一种反应流体连续地流过流动反应器期间，含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体(或含Fc区的蛋白)在糖苷内切酶的催化作用下发生偶联反应而生成抗体偶联物或偶联药物。
在一些实施例中，将糖苷内切酶定向固定到所述介质上，并填装在流动反应器中，使得当反应流体流经流动反应器时，反应流体中包含的待生成偶联物的两个反应成分连续稳定地偶联。
与化学偶联相比，本发明的偶联设备极大地减少了工艺步骤，显著降低了工艺复杂性，并非常适于节约昂贵的制造成本。而且，通过使用流动反应器，能实现偶联工艺的线性放大和连续流生产，以满足对更高产量的工业需求，缩短单位偶联时间并减小制造区域中的占据空间。通过该偶联设备生产生物偶联物，能实现负载物-连接子与抗体(或含Fc区的蛋白)之间的定点特异性偶联，改善了同质性，因此拓宽了治疗窗。此外，偶联工艺能与诸如单抗之类的生物分子的生产工艺流程进行集成。例如，在单抗中间体和单抗原液的生产阶段都可以完成偶联。因此，该工艺灵活性高，连贯性好。
在一些实施例中，至少一种反应流体包含第一反应流体和第二反应流体，所述第一反应流体包含含有噁唑啉低聚糖的供体或含有GlcNAc基序的抗体(或含Fc区的蛋白)，所述第二反应流体包含含有GlcNAc基序的抗体(或含Fc区的蛋白)或含有噁唑啉低聚糖的供体。
在一些实施例中，偶联过程依次包括以下阶段：反应前平衡、偶联反应、反应后回收和回收后冲洗，并且，流体输送单元被进一步配置为：在反应前平衡、反应后回收和回收后冲洗期间，向流动反应器连续提供缓冲液；以及在偶联反应期间，向流动反应器连续并同时提供含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体(或含Fc区的蛋白)。
在一些实施例中，缓冲液、第一反应流体和第二反应流体分别被储存在第一容器、第二容器和第三容器中。流体输送单元包括第一输送泵和第二输送泵。第一容器和第二容器分别经由第一容器出口管路和第二容器出口管路连接至第一输送泵，第三容器经由第三容器出口管路连接至第二输送泵，第一输送泵和第二输送泵分别经由第一进口支管和第二进口支管连接至进口主管，进口主管连接至流动反应器的进口。并且，在反应前平衡、反应后回收和回收后冲洗期间，第一输送泵将第一容器中的缓冲液泵 送到进口主管中，在偶联反应期间，第一输送泵将第二容器中的第一反应流体泵送到进口主管中，第二输送泵将第三容器中的第二反应流体泵送到进口主管中。
在一些实施例中，流体输送单元进一步包括第一阀、第二阀、第三阀和第四阀。第一阀、第二阀和第三阀分别设置在第一容器出口管路、第二容器出口管路和第三容器出口管路上，分别用于控制第一容器出口管路、第二容器出口管路和第三容器出口管路中的流体流动，并且，第四阀设置在第一进口支管上，用于控制第一进口支管中的流体流动。
在一些实施例中，在反应前平衡、反应后回收和回收后冲洗期间，第一阀和第四阀开启，第二阀和第三阀关闭，并且在偶联反应期间，第一阀关闭，第二阀、第三阀和第四阀开启。
在一些实施例中，第一容器出口管路、第二容器出口管路、第三容器出口管路、第一进口支管、第二进口支管和进口主管为一次性的或非一次性的，并分别由不锈钢、钛和硅胶中的一个制成。第一容器、第二容器和第三容器分别选自以下各项中的一项：一次性储液袋、一次性储液瓶、不锈钢容器、以及一次性和非一次性玻璃或塑料容器。
在一些实施例中，流体收集单元被进一步配置为：在反应前平衡和回收后冲洗期间，使从流动反应器的出口流出的流体被收集到第四容器中；以及在偶联反应和反应后回收期间，使从流动反应器的出口流出的流体被收集到第五容器中。
在一些实施例中，第四容器和第五容器分别经由第四容器进口管路和第五容器进口管路连接至出口主管，出口主管连接至流动反应器的出口，并且，流体收集单元包括第五阀和第六阀，第五阀和第六阀分别设置在第四容器进口管路和第五容器进口管路上，并分别用于控制第四容器进口管路和第五容器进口管路中的流体流动。
在一些实施例中，在反应前平衡和回收后冲洗期间，第五阀开启，第六阀关闭，并且在偶联反应和反应后回收期间，第五阀关闭，第六阀开启。
在一些实施例中，第四容器进口管路和第五容器进口管路为一次性的或非一次性的，并分别由不锈钢、钛和硅胶中的一个制成。第四容器和第五容器分别选自以下各项中的一项：一次性储液袋、一次性储液瓶、不锈钢容器、以及一次性和非一次性玻璃或塑料容器。
在一些实施例中，偶联设备进一步包括：控温单元，其被配置为：在偶联过程中，控制流入流动反应器的进口的流体和流出流动反应器的出口的流体的温度。
在一些实施例中，控温单元包括：加热模块，其设置在流动反应器的进口处，用于对流入进口的流体进行加热；以及冷却模块，其设置在流动反应器的出口处，用于对流出出口的流体进行冷却。
在一些实施例中，偶联设备进一步包括：取样检测单元，其与流动反应器的出口流体连通，并且被配置为：根据预设的取样时间从流出流动反应器的出口的流体中采集样品流体；以及对样品流体中的偶联物进行检测，以获得检测结果，检测结果指示偶联物是否达到预设标准。
在一些实施例中，取样检测单元包括取样泵、第一切换阀、洗脱泵、至少一个分析柱和检测器。取样泵经由取样管路连接至流动反应器的出口，第一切换阀上设有样 品环，并且，第一切换阀根据预设的取样时间在第一状态与第二状态之间切换。在第一切换阀处于第一状态时，取样泵与样品环流体连通，并经由取样管路从流出流动反应器的出口的流体中采集样品流体并泵送至样品环中，并且在第一切换阀处于第二状态时，洗脱泵、样品环、至少一个分析柱和检测器经由检测管路流体连通，洗脱泵将洗脱液泵送至检测管路中，使洗脱液流经样品环，从而带动样品环中的样品流体流过至少一个分析柱中的一个分析柱后进入检测器。
在一些实施例中，分析柱为两个，并且取样检测单元进一步包括第二切换阀和清洗泵，第二切换阀在两个状态之间切换。在第二切换阀处于任一状态下，样品环和检测器与两个分析柱中的一个分析柱流体连通，洗脱液带动样品环中的样品流体流入该一个分析柱中，并且，清洗泵与两个分析柱中的另一个分析柱流体连通，并将缓冲液泵送至该另一个分析柱中以对其进行平衡。
在一些实施例中，第一切换阀为六通阀，第二切换阀为十通阀，并且洗脱泵为四元泵。
在一些实施例中，偶联设备进一步包括：循环回收单元，其设置在流动反应器的进口与出口之间。当检测结果指示偶联物未达到预设标准时，流体收集单元被配置为停止对流出流动反应器的出口的流体的收集，并且，循环回收单元被配置为控制流出流动反应器的出口的流体重新进入进口中，以在流动反应器中再次进行偶联反应。
在一些实施例中，循环回收单元包括第七阀，其设置在回收管路上。回收管路连接在流动反应器的进口与出口之间，并且，在回收管路上设有回收容器。当检测结果指示偶联物未达到预设标准时，第七阀开启，流出流动反应器的出口的流体流经回收管路和回收容器后流入进口。
在一些实施例中，流动反应器为偶联柱(即预装柱)。
在一些实施例中，偶联设备进一步包括选自下组中的至少一项：压力传感模块、流量检测模块、pH计量模块、电导计量模块和UV检测模块，压力传感模块、流量检测模块、pH计量模块、电导计量模块和UV检测模块。在一些实施例中，所述模块可分别设置在进口和/或出口处。
专利申请WO2022170676A或CN114480115A全文引入本申请中。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
仪器、材料与试剂
除非特别说明，实施例中所使用的仪器和试剂均为可商购的。试剂可以不经进一步纯化，直接使用。
Ni Sepharose 6 FF：金属螯合亲和层析介质，又称固定金属离子亲和色谱，其原理是利用蛋白质表面的一些氨基酸，如组氨酸、色氨酸、半胱氨酸等能和金属离子(Cu2+，Zn2+，Ni2+，Co2+，Fe3+)发生特殊的相互作用的原理，从而对蛋白质加以分 离。这些作用包括配价键结合、静电吸附、共价键结合，其中以配价键结合为主，而且这其中又以6组氨酸标签(His-Tag)应用最为广泛。
Q Sepharose FF/Capto S impact：离子交换对分子的分离是基于表面静电荷的差异。蛋白质有许多含有弱酸弱碱基团的不同氨基酸组成，表面静电荷会随着周围环境pH的改变而逐渐改变，即蛋白质分子为两性分子。在离子交换层析分离过程中，通过控制带电分子与带相反电荷的离子交换填料之间的可逆相互作用来实现特定分子的结合与洗脱，从而达到分离的效果。处于与等电点相同pH值环境中的蛋白质表面静电荷为零，不会与带电的填料发生相互作用。当所处环境pH值高于其等电点时，蛋白质会同带正电荷的填料，也就是阴离子交换剂相结合；而当所处环境pH值低于其等电点时，蛋白质会同带负电荷的填料，也就是阳离子交换剂相结合。
SDS-PGAE:十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)，SDS是阴离子去污剂，作为变性剂和助溶剂，能断裂分子内和分子间的氢键，去分子内疏水作用使分子去折叠，破坏蛋白分子的二、三级结构。SDS与变性的蛋白结合使其带负电荷，其结合SDS的量与分子量成正，在达到饱和的状态下，每克蛋白可与1.4g SDS结合，所带的负电荷大大超过了蛋白原由的电荷量，消除了不同分子间的电荷差异和结构差异，因此SDS-蛋白复合物在丙烯酰胺凝胶电泳中的迁移率只与蛋白的大小有关。强还原剂(如β-巯基乙醇，二硫苏糖醇)能打开分子内及分子间的二硫键，使蛋白分离更有效。
实施例1.内切糖苷酶融合蛋白的制备
1.1 Halo-EndoS2-His的氨基酸序列(Halo采用单下划线标出，His Tag采用斜体表示，GGGGSGGGGS为连接序列)

1.2 Halo-Endo S2-His克隆
编码Halo-Endo S2-His的核酸序列通过基因合成标准方法合成，并插入到pET表达载体中。将Halo-Endo S2-His表达质粒转化到大肠杆菌BL21(DE3)中。在LB(Luria-Bertani)培养基中加入对应抗生素，37℃培养至OD600＝0.5-1.0，进行保菌。
1.3 Halo-Endo S2-His Tag纯化(反应器)
取出甘油菌，接种到含有相应抗生素的LB液体培养基中培养，100-300rpm，37℃振荡培养2-8h；当OD600达到0.5～1.0时。接种到10L反应器中，培养5-10h后，加入0.2mM终浓度的IPTG，16摄氏度过夜诱导表达。2-8℃离心收集菌体沉淀10-30min，转速3000-5000rpm，加入平衡缓冲液重新悬浮细胞，高压破碎；2-8℃，5000-10,000rpm离心10-60min分离上清和沉淀；
Ni柱亲和纯化，Ni层析柱连接到蛋白纯化系统，先用纯化水和平衡缓冲液进行充分冲洗；低流速上清进行上样。分别使用平衡缓冲液清洗和80mM咪唑缓冲液洗涤；500mM咪唑缓冲液洗脱。SDS-PAGE检测分析。
如图1所示，Halo-Endo S2-His挂在Ni磁珠上，流穿中没有目的样品，Halo-Endo S2-His在500mM咪唑条件下洗脱下来。该结果说明Halo-Endo S2-His可以通过反应器进行放大培养，通过Ni层析柱实现线性生产放大。
实施例2.固定化Halo-Endo S2-His的制备
制备流程
2.1氯代烷基-连接子修饰的树脂(Chloro Resin)的制备
制备氯树脂的方法参见，例如，美国专利号7,429,472、7,888,086和8,202,700，其全部内容援引加入本文。用于制备氯树脂的树脂见表1。
表1.树脂
方法：
(1)预处理
对于NHS活化树脂(Bestchrom)和CNBr活化树脂(Bestchrom)：
用异丙醇过滤树脂，滤饼用DMF(N,N-二甲基甲酰胺)洗涤一次，然后抽吸干。用DMF将滤饼转移到烧瓶中并搅拌。接着，向混合物中加入乙二胺，并搅拌10到15小时。过滤并用DMF洗涤滤饼。然后沥干液体；
对于环氧活化树脂：
用异丙醇过滤树脂，滤饼用H2O洗涤一次，然后抽吸干。将滤饼转移到烧瓶中并搅拌。接着，向混合物中加入25％～28％的浓氨水，将体系缓慢加热至40-50℃，并在40-50℃搅拌下反应。将体系温度降至20-30℃，过滤混合物。用H2O洗涤滤饼直至滤液pH达到约7-8。接着，用DMF洗涤滤饼并沥干液体；
(2)将步骤(1)的滤饼转移到烧瓶中并搅拌。接着，将含有式(III-1)结构的氯代烷基底物的DMF和三乙胺依次加入体系中。搅拌反应。接着，过滤系统并用DMF洗涤滤饼，最后沥干液体；
(3)将步骤(2)的滤饼转移到烧瓶中。打开搅拌。接着，向混合物中依次加入Ac2O和三乙胺。搅拌反应。然后，过滤混合物并用DMF洗涤滤饼。然后用H2O洗涤滤饼并沥干液体。最后，用20％乙醇将混合物转移到容器中进行储存。
结果：得到具有式(IV-1)结构的氯树脂：
其中为高度交联的琼脂糖树脂或聚甲基丙烯酸甲酯树脂。
将Halo-Endo S2-His与氯树脂(Chloro resin)室温混合孵育10min-24h，用20mM Tris-HCl，150mM NaCl，pH 6.0-10.0缓冲液进行洗涤步骤，重复3次。固定化的糖苷内切酶融合蛋白(Halo-Endo S2-His与氯树脂共价连接在一起)进行活性检测，检测合格后，用20mM Tris-HCl，150mM NaCl清洗，最后2-8℃保存。
2.2固定化酶载量
1)取各种待测氯树脂250μl，投入过量糖苷酶，置于旋转混匀仪上，室温，固定化2h；
2)Halo-Endo S2-His固定化过程中，计时15min、30min、1h和2h取固定化酶上清，每次3000g室温离心3min，吸取上清，使用Nanodrop紫外分光光度计检测上清中酶的浓度；
3)计算每个时间点的糖苷内切酶浓度，通过酶的起始浓度减去每个时间点的浓度，计算酶的计时时刻的固定化量，绘出介质固定化Halo-Endo S2-His过程的载量变化曲线。
SDS-PAGE检测上清中蛋白趋势变化，如图2所示，结果发现随着时间延长，上清中的Halo-Endo S2-His越来越少，特异性固定到氯树脂上。
实施例3.连接子-负载物的制备
3.1二糖底物的制备
二糖底物化合物1的制备
采用以下步骤制备化合物1，其结构如下：
(1)化合物1c的制备
在油泵抽真空条件下，用热风枪烘烤100mL Schlenk反应瓶5分钟，冷却后向体系中加入活化的分子筛，再烘烤5分钟。抽真空置换氮气三次，氮气保护下向体系中加入化合物1a(4.41g,7.96mmol)，搅拌3分钟后，加入无水二氯甲烷(30mL)搅拌0.5小时。另外按照同样操作，向另一个已添加活化分子筛的50mL Schlenk反应瓶中加入化合物1b(1.89g,3.98mmol，溶于20mL无水二氯甲烷)，搅拌1小时预干燥以除去体系中残留的水分。
氮气保护下，室温下向上述干燥的化合物1a溶液中加入1-(苯基亚硫酰基)哌啶(BSP,1.37g,6.56mmol)、2,4,6-三叔丁基嘧啶(TTBP,2.94g,11.94mmol)，再搅拌20分钟。将反应瓶置于干冰/乙酸乙酯浴中，降至-65℃，向体系中加入三氟甲磺酸酐(1.2mL,7.16mmol)。2分钟后向该体系中加入此前预干燥的化合物1b的二氯甲烷溶液，所得反应混合液在-65℃下搅拌，直至TLC(展开剂：EtOAc/PE＝1/8)检测反应结束(约3h)。向体系中加入饱和碳酸氢钠溶液淬灭反应，二氯甲烷萃取分液(150mL×3)，合并的有机相依次用水、饱和食盐水洗涤，无水硫酸钠干燥，过滤、减压浓缩，湿法上样,柱层析分离(洗脱剂：EtOAc/PE＝1/12-1/10)，得化合物1c(2.72g，收率75.4％，无色粘稠油状液体)。1H NMR(400MHz,氯仿-d)δ7.55(dd,J＝7.6,2.1Hz,2H),7.51–7.28(m,28H),5.60(s,1H),5.14(d,J＝10.4Hz,1H),5.00(d,J＝12.0Hz,1H),4.94(d,J＝11.8Hz,1H),4.90–4.80(m,2H),4.76(d,J＝12.0Hz,1H),4.74–4.63(m,3H),4.57(s,1H,H1’),4.48(d,J＝12.1Hz,1H),4.37(d,J＝8.1Hz,1H，H1),4.22–4.10(m,2H),4.05(t,J＝9.3Hz,1H),3.80(d,J＝3.1Hz,1H),3.72(dd,J＝11.2,2.2Hz,1H),3.67–3.52(m,3H),3.49(dd,J＝9.8,3.1Hz,1H),3.41(t,J＝9.3Hz,1H),3.35(dt,J＝9.8,2.9Hz,1H),3.16(td,J＝9.7,4.8Hz,1H)。C54H56N3O10 +[M+H]+的MS(ESI)m/z，计算值906.4,实际值906.7。
(2)化合物1d的制备
室温下，向25mL Schlenk反应瓶中依次加入化合物1c(228mg,0.252mmol)、氯仿(1.2mL)、吡啶(1mL)、AcSH(1.2mL)溶解。封住翻口塞，体系在60℃下搅拌，直至HPLC检测反应基本结束，约需18小时。减压浓缩后除去大部分溶剂后，向体系中加乙酸乙酯(50mL)，依次用饱和碳酸氢钠溶液(30mL)、1M盐酸(10mL×4)和饱和碳酸氢钠溶液(30mL)洗涤,有机相用无水硫酸钠干燥，减压浓缩后经柱层析纯化(洗脱剂：EtOAc/PE＝1/10-1/1)，得到化合物1d(200mg,收率86％,白色固体)。1H NMR(400MHz,氯仿-d)δ7.57–7.49(m,2H),7.48–7.39(m,5H),7.39–7.24(m,23H),5.80(d,J＝8.1Hz,1H),5.59(s,1H),5.00(d,J＝6.7Hz,1H),4.97–4.87(m,3H),4.87–4.75(m,2H),4.69–4.59(m,4H),4.57(s,1H),4.45(d,J＝12.0Hz,1H),4.19–4.05(m,3H),3.96(t,J＝7.3Hz,1H),3.85–3.76(m,2H),3.73–3.59(m,4H),3.49(dd,J＝9.8,3.1Hz,1H),3.18(td,J＝9.7,4.8Hz,1H),1.77(d,J＝1.2Hz,3H).C56H60NO11 +[M+H]+的MS(ESI)m/z，计算值922.4,实际值922.4。
(3)化合物1e的制备
室温下，向100mL单口瓶中依次加入化合物1d(168mg,0.168mmol)、一水合对甲苯磺酸(35mg,0.168mmol)、甲醇(5mL)、四氢呋喃(5mL)，置换氮气三次，反应在室温下搅拌过夜，直至HPLC检测反应结束。反应体系用饱和碳酸氢钠溶液(20mL)淬灭，二氯甲烷(20mL×3)萃取分液，合并的有机相用无水硫酸钠干燥，过滤，减压浓缩后，经柱层析纯化(洗脱剂：MeOH/DCM＝1/20)，得到化合物1e(136mg,收率89％,白色固体)。1H NMR(400MHz,氯仿-d)δ7.43–7.24(m,29H),5.76(d,J＝7.9Hz,1H),5.01(d,J＝6.9Hz,1H),4.94(dd,J＝11.8,2.3Hz,2H),4.85(d,J＝11.7Hz,1H),4.77(d,J＝11.8Hz,1H),4.70–4.59(m,3H),4.58–4.47(m,3H),4.36(d,J＝11.7Hz,1H),4.20(t,J＝7.9Hz,1H),3.94(t,J＝7.5Hz,1H),3.91–3.80(m,3H),3.80–3.71(m,3H),3.63–3.50(m,2H),3.22–3.14(m,2H),1.78(s,3).C49H56NO11 +[M+H]+的MS(ESI)m/z，计算值834.4,实际值834.5。
(4)化合物1f的制备
室温下，向100mL单口瓶中依次加入化合物1e(130mg,0.156mmol)、二氯甲烷、 叔丁醇、水。向体系中加入碘苯二乙酸(0.5–10当量)、2,2,6,6-四甲基-1-哌啶氧化物(0.01–1当量)，反应在室温下搅拌过夜，直至TLC检测反应结束。二氯甲烷萃取分液，合并的有机相用无水硫酸钠干燥，过滤，浓缩后经柱层析纯化得到化合物1f(100mg,收率75.1％,白色固体)。1H NMR(400MHz,氯仿-d)δ7.42–7.31(m,18H),7.30–7.27(m,2H),7.25–7.20(m,3H),7.15(dd,J＝6.7,2.9Hz,2H),6.06(d,J＝8.3Hz,1H),4.96(d,J＝6.6Hz,1H),4.93(d,J＝11.9Hz,1H),4.86(d,J＝11.1Hz,1H),4.81(d,J＝12.1Hz,1H),4.73(d,J＝11.1Hz,1H),4.71–4.65(m,3H),4.65–4.57(m,3H),4.49(s,1H),4.46(d,J＝12.0Hz,1H),4.23(t,J＝7.1Hz,1H),4.15(t,J＝9.5Hz,1H),3.89(t,J＝6.5Hz,1H),3.86–3.65(m,6H),3.54(d,J＝9.7Hz,1H),3.30(dd,J＝9.3,2.8Hz,1H),1.65(s,3H).C49H52NO12 -[M-H+]-的MS(ESI)m/z，计算值846.3,实际值846.3。
(5)化合物1的制备
室温下，向50mL单口瓶中依次加入化合物1f(160mg,0.189mmol)、四氢呋喃、甲醇、钯碳催化剂，反应体系在氢气气氛下搅拌，直到TLC检测原料全部消失。过滤，减压浓缩，油泵抽干得到化合物1(75mg,收率100％,白色固体)。C14H22NO12 -[M-H+]-的MS(ESI)m/z，计算值396.1,实际值396.1。
二糖底物化合物2的制备
采用以下步骤制备化合物2，其结构如下：
(1)化合物2-1的合成
Step A:化合物2-1b的合成
向单口瓶中加入化合物2-1a(1.0当量，商业可得，CAS：959153-39-0)、MeOH和甲醇钠(0.1当量,5mol/L in MeOH)，氮气气氛下室温搅拌，TLC监测反应进度，待反应完成后向其中1M盐酸淬灭反应并调节反应至中性，减压下除去溶剂，并加入少许甲苯，在减压旋转蒸发条件下利用共沸效应去除水分，得到粗产品2-1b浅黄色粘稠油状液体，无需进一步纯化，直接用于下一步反应。
Step B:化合物2-1c的合成
向单口瓶中依次加入2-1b粗产品(1当量)、乙腈、樟脑磺酸(0.1当量)、苯甲醛二甲缩醛(4当量)，反应体系在室温下搅拌过夜。TLC监测反应进度，待反应完成后(约24h)，向体系中加入饱和碳酸氢钠溶液淬灭反应，用乙酸乙酯萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样硅胶柱层析纯化(洗脱剂：PE/EtOAc＝5:1)得到2-1c(白色固体，83％收率)。1H NMR(400MHz,氯仿-d)δ7.51-7.49(m,2H,Ar-H),7.45(d,J＝8.0Hz,2H,Ar-H),7.42-7.37(m,5H,Ar-H),7.36-7.31(m,3H,Ar-H),7.15(d,J＝8.0Hz,2H,Ar-H),5.56(s,1H,PhCH),4.97(d,J＝11.6Hz,1H,PhCH2),4.81(d,J＝11.6Hz,1H,PhCH2),4.58(d,J＝9.6Hz,1H),4.40(dd,J＝10.2,4.8Hz,1H),3.80(dd,J＝10.0,10.4Hz,1H),3.70(dd,J＝9.2,9.2Hz,1H),3.65(dd,J＝9.2,9.2Hz,1H),3.53-3.48(m,2H),2.64(br s,1H,-OH),2.36(s,3H).C27H29O5S+[M+H]+的MS(ESI)m/z，计算值465.2,实际值465.3。核磁数据与文献报道一致，见Nature2007,446,896中化合物2b1.
Step C:化合物2-1d的合成
向单口瓶中依次加入2-1c(1当量)、二氯甲烷、三乙胺(5当量)，体系冷却至0℃，加入乙酸酐(2当量)，搅拌10min后，反应体系升至室温下搅拌，TLC监测反应进度，待反应完成后(约6h)，向体系中加入饱和碳酸氢钠溶液淬灭反应，用二氯甲烷萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样柱层析纯化(洗脱剂：PE/EtOAc＝5:1)得到2-1d(白色固体，89％收率)。1H NMR(400MHz,氯仿-d)δ7.51-7.48(m,2H,Ar-H),7.43-7.36(m,5H,Ar-H),7.34-7.25(m,5H,Ar-H),7.13(d,J＝8.0Hz,2H,Ar-H),5.58(s,1H,PhCH),5.00(dd,J＝8.0,8.0Hz,1H,H-2),4.87(d,J＝12.0Hz,1H,PhCH2),4.67(d,J＝12.0Hz,1H,PhCH2),4.64(d,J＝10.4Hz,1H,H-1),4.39(dd,J＝10.8Hz,J＝5.2Hz,1H),3.81(t,J＝10.4Hz,1H),3.78-3.70(m,2H),3.48(ddd,J＝9.6,5.4,10.0Hz,1H),2.35(s,3H,Ar-CH3),2.05(s,3H,OAc)。C29H31O6S+[M+H]+的MS(ESI)m/z，计算值507.2,实际值507.4。核磁数据与文献报道一致，见Nature 2007,446,896中化合物3b1.
Step D:化合物2-1e的合成
氮气气氛下向100mL两口瓶中依次加入化合物2-1d(1当量)、二氯甲烷、硼烷四氢呋喃(10当量，1M in THF)，将体系冷却至0℃，滴加三氟甲磺酸二丁硼(1.4当量，1.0M in DCM)，维持在0℃下直至TLC显示反应完成(约5小时)，随后在0℃下，向体系中加三乙胺溶液淬灭体系中的三氟甲磺酸二丁硼，再缓慢滴加甲醇淬灭硼烷四氢呋喃，待体系不再产生大量气泡后，再加水确保充分淬灭，然后用乙酸乙酯萃 取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样，快速柱层析(洗脱剂：洗脱剂：PE/EtOAc＝4:1)得到2-1e粗产品(白色固体)，LCMS确认产物结构无误，C29H33O6S+[M+H]+的MS(ESI)m/z，计算值509.2,实际值509.3，粗产品无需精细纯化直接用于下一步反应。
Step E/F:化合物2-1f的合成
Step E：向单口瓶中依次加入2-1e(1当量)、醋酸碘苯(3当量)、TEMPO(0.5当量)和叔丁醇/二氯甲烷/水(体积比4:4:1)，所得混合体系在室温下搅拌，TLC(展开剂：PE/EtOAc＝2/1,含1v/v％乙酸)监测反应进度，待反应完全后，向体系中加入饱和硫代硫酸钠淬灭反应，二氯甲烷萃取分液，合并的有机相，经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，所得羧酸中间体无需进一步纯化直接用于下一步反应。
Step F：将上一步所得羧酸中间体溶于DMF，加入碘甲烷(3当量)和碳酸钾(5当量)，室温下搅拌，TLC(展开剂：PE/EtOAc＝5/1)监测反应进度，反应完全后(约2小时)，向体系中加水，乙酸乙酯萃取分液，有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，柱层析纯化(洗脱剂：PE/EtOAc＝7:1)得到化合2-1f(白色固体,三步合计收率59％)。1H NMR(400MHz,氯仿-d)δ,7.42-7.26(m,12H,Ar-H),7.16(d,J＝7.6Hz,2H,Ar-H),5.02(dd,J＝8.4,8.0Hz,1H),4.83(d,J＝11.6Hz,1H,PhCH2),4.79(d,J＝11.2Hz,1H,PhCH2),4.70(d,J＝11.6Hz,1H),4.64(d,J＝11.2Hz,1H),4.62(d,J＝10.0Hz,1H),3.97(d,J＝10.0Hz,1H),3.90(dd,J＝8.4,8.0Hz,1H),3.79(s,3H,OMe),3.72(dd,J＝8.8,8.8Hz,1H),2.38(s,3H,Ar-CH3),2.04(s,3H,OAc)。C30H33O7S+[M+H]+的MS(ESI)m/z，计算值537.2,实际值537.4。
Step G:化合物2-1g的合成
向50mL单口瓶中依次加入2-1f(1.0当量)和丙酮，将体系降至0℃，加入N-溴代丁二酰亚胺(1.4当量)，0℃下搅拌约1小时，TLC(展开剂：PE/EtOAc＝4/1)显示反应完全。向体系中加入饱和硫代硫酸钠溶液淬灭反应，减压下旋转蒸发除去丙酮，加入水，乙酸乙酯分液萃取。合并有机相，经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，柱层析纯化(洗脱剂：PE/EtOAc＝6:1)得到化合物2-1g(白色固体，收率90％)。C23H27O8 +[M+H]+的MS(ESI)m/z，计算值431.2,实际值431.5。
Step H:化合物2-1的合成
在氮气气氛下，向50mL单口瓶中依次加入2-1g(1当量)、1,8-二氮杂双环[5.4.0]十一碳-7-烯(DBU，0.1当量)和二氯甲烷，体系降至0℃搅拌均匀，随后加入三氯乙腈(4当量)，撤去冰水浴自然升至室温下搅拌，TLC(展开剂：PE/EtOAc＝2/1)监测反应，反应完全后(约2小时)，减压下旋转蒸发除去溶剂，湿法上样柱层析纯化(洗脱剂：PE/EtOAc＝4:1)得化合物2-1(浅黄色粘稠油状液体，收率87％)。C23H25O7 +[M-Cl3CC(NH)O-]+的MS(ESI)m/z，计算值413.2,实际值413.2。
(2)化合物2-2的合成
Step A:化合物2-2a的合成
2-2a(CAS:1235137-45-7)商业可得，参考文献Carbohydr Res 2016,426,33中化合物2的合成方法进行制备。
室温下，向单口瓶中加入化合物2-2a(1当量)、甲醇和甲醇钠(0.1当量，5M in MeOH)，室温下搅拌，TLC(展开剂：PE/EtOAc＝2/1)监测反应，反应完全后(约1小时)，加入稀盐酸(1M)中和溶液至pH＝7，反应液经浓缩后，加入甲苯，减压下旋转蒸发利用共沸效应除去体系中残留水份，得到浅棕色油状粗品2-2b不经纯化，直接用于下一步反应。
Step B:化合物2-2c的合成
室温下，向上一步反应粗产品2-2b(1当量)中，依次加入一水对甲苯磺酸(0.2当量)，无水乙腈充分搅拌均匀，将体系置换氮气气氛，加入苯甲醛二甲缩醛(5当量)，所得反应液体在室温下搅拌过夜，直至TLC(展开剂：PE/EtOAc＝5/1)显示反应完全，然后向反应体系中加入饱和碳酸钠溶液淬灭反应，二氯甲烷萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥，过滤、浓缩，湿法上样，硅胶柱层析纯化(洗脱剂：PE/EtOAc＝5/1)得到化合物2-2c(白色固体，两步合计收率85％)。1H NMR(400MHz,氯仿-d)δ7.63–7.54(m,2H),7.52–7.39(m,5H),7.25–7.14(m,2H),5.60(s,1H),5.53(d,J＝5.4Hz,1H),4.52–4.39(m,1H),4.28(dd,J＝10.4,4.9Hz,1H),4.07(t,J＝9.5Hz,1H),3.92(dd,J＝10.0,5.6Hz,1H),3.79(t,J＝10.3Hz,1H),3.60(t,J＝9.3Hz,1H),3.08(br s,1H),2.40(s,3H).C20H22N3O4S+[M+H]+的MS(ESI)m/z，计算值400.1,实际值399.9。核磁数据与文献报道一致，见文献Angew.Chem.Int.Ed.2021,60,12413中化合物47。
Step C:化合物2-2d的合成
氮气气氛下，向干燥的两口瓶中依次加入化合物2-2c(1当量)、无水四氢呋喃(反应浓度0.2M)，将置于冰浴中降至0℃,加入氢化钠(1.2当量，60％含量，分散于矿物油中)，加毕，移除冰浴升至室温下搅拌，1小时后加入四丁基碘化铵(0.1当量)、苄溴(1.5当量)，所得反应体系在室温下搅拌，直至TLC(展开剂：PE/EtOAc＝8/1)显示反应完全(约6小时)，反应结束后滴加水淬灭，乙酸乙酯萃取分液，合并的有机相依次用水、饱和食盐水洗涤，无水硫酸钠干燥，过滤、浓缩，硅胶柱层析纯化(洗脱剂：PE/EtOAc＝5/1)得到化合物2-2d(白色固体,收率97％)。1H NMR(400MHz,氯仿-d)δ7.63–7.55(m,2H),7.53–7.33(m,10H),7.20(d,J＝8.4Hz,2H),5.67(s, 1H),5.56(d,J＝4.6Hz,1H),5.05(d,J＝10.9Hz,1H),4.90(d,J＝10.9Hz,1H),4.59–4.47(m,1H),4.31(dd,J＝10.4,4.9Hz,1H),4.10–3.97(m,2H),3.90–3.77(m,2H),2.41(s,3H)。C27H28N3O4S+[M+H]+的MS(ESI)m/z，计算值490.2,实际值490.5。核磁数据与文献报道一致，见参考文献Bioorg.Med.Chem.2011,19,30中化合物20。
Step D:化合物2-2e的合成
向单口瓶中加入化合物2-2d(1当量)、四氢呋喃/甲醇(v/v＝1:1，反应浓度0.5M)和一水合对苯甲磺酸(0.2当量)，室温下搅拌过夜。TLC(展开剂：PE/EtOAc＝8/1)监测反应进度，待反应完全后(约12小时)，向体系中加入饱和碳酸氢钠溶液淬灭反应，乙酸乙酯萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样，硅胶柱层析纯化(洗脱剂：EtOAc/PE＝1/1)得到化合物2-2e。1H NMR(400MHz,氯仿-d)δ7.41–7.34(m,7H),7.14(d,J＝8.0Hz,2H),5.49(d,J＝5.2Hz,1H),5.01(d,J＝11.2Hz,1H),4.77(d,J＝11.2Hz,1H),4.24–4.20(m,1H),3.89-3.85(m,1H),3.79-3.77(m,2H),3.69-3.65(m,2H),2.42(br s,1H),2.34(s,3H).C20H27N4O4S+[M+NH4]+的MS(ESI)m/z，计算值419.2,实际值419.2。
Step E:化合物2-2的合成
向单口瓶中依次加入化合物2-2e(1当量)、二氯甲烷(反应浓度0.5M)、咪唑(2当量)、叔丁基二苯基氯硅烷(1.5当量)，室温下搅拌，TLC(展开剂：EtOAc/PE＝1/12)监测反应进度，反应结束后(约6小时)，向体系中加入饱和氯化铵溶液淬灭反应，再用二氯甲烷萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样，硅胶柱层析纯化(洗脱剂：PE/EtOAc/DCM＝15/1/1)得到化合物2-2(黄色粘稠油状液体，收率66％)。1H NMR(400MHz,氯仿-d)δ7.73-7.68(m,4H),7.48-7.35(m,13H),7.06(d,J＝7.6Hz,1H),5.49(d,J＝5.2Hz,1H),4.94(ABq,J＝10.8Hz,2H),4.32-4.27(m,1H),3.96-3.90(m,2H),3.87(dd,J＝4.8,5.2Hz,1H),3.82(dt,J＝2.8,8.8Hz,1H),3.74-3.69(m,1H),2.70(d,J＝2.8Hz,OH),2.33(s,3H),1.09(s,9H).C36H42N3O4SSi+[M+H]+的MS(ESI)m/z，计算值640.3,实际值640.2。
(3)化合物2的合成
Step A:化合物2a的合成
向干燥的两口瓶中加入预先活化的分子筛，在油泵抽真空条件下，用热风枪烘烤，自然冷却后，置换氮气，重复上述抽真空-换氮气操作三次，随后在氮气保护下向体系中加入化合物2-1(2当量)、化合物2-2(1当量)和干燥的甲苯，所得体系在室温下搅拌0.5h以充分除去体系中残留的水分。随后将反应体系降至-40℃下搅拌，加入一定量TMSOTf，维持在该温度下搅拌，直至TLC显示检测反应完全(约3h)，向体系中加入三乙胺淬灭反应，过滤、浓缩，湿法上样，硅胶柱层析分离得化合物2a(白色固体，60％收率)。1H NMR(400MHz,氯仿-d)δ7.72(d,J＝6.4Hz,2H),7.64(d,J＝6.4Hz,2H),7.49–7.47(m,2H),7.44–7.23(m,21H),7.04(d,J＝7.8Hz,2H),5.57(d,J＝7.2Hz,1H),5.17(d,J＝7.2Hz,1H),5.10(dd,J＝9.7,8.1Hz,1H),4.93(d,J＝8.0Hz,1H),4.82(d,J＝11.4Hz,1H),4.74(d,J＝10.9Hz,1H),4.66–4.60(m,3H),4.20(dd,J＝9.2,9.2Hz,1H),4.05–4.02(m,2H),3.97(dd,J＝9.3,9.2Hz,1H),3.92–3.76(m,3H),3.74–3.67(m,1H),3.60(s,3H),3.51(dd,J＝9.2,9.2Hz,1H),2.32(s,3H),1.82(s,3H),1.07(s,9H).C59H66N3O11SSi+[M+H]+的MS(ESI)m/z，计算值1052.4,实际值1052.7。
Step B:化合物2b的合成
向反应瓶中加入化合物2a(1当量)、巯基乙酸(CAS：68-11-1)/吡啶/三氯甲烷(v/v/v＝1:1:1)，将体系升至60℃下搅拌，TLC监测反应进度，待反应完全后(约12h)，减压下浓缩除去大部分溶剂，加入适量乙酸乙酯，用饱和碳酸氢钠溶液洗涤，萃取分液后，用1M的盐酸溶液洗涤有机相，分液后再用饱和碳酸氢钠溶液洗涤，得到的有机相用无水硫酸钠干燥后，经过滤、浓缩柱层析纯化得化合物2b(白色固体，71％收率.)1H NMR(400MHz,氯仿-d)δ7.70(d,J＝6.8Hz,2H),7.66(d,J＝7.2Hz,2H),7.44–7.28(m,20H),7.26–7.21(m,3H),7.00(d,J＝7.9Hz,2H),5.68(d,J＝4.8Hz,1H),5.36–5.30(m,1H),5.09(dd,J＝9.5,8.1Hz,1H),4.92(d,J＝12.4Hz,1H),4.85(d,J＝11.5Hz,1H),4.75–4.66(m,3H),4.63–4.57(m,2H),4.29(ddd,J＝9.3,7.7,4.8Hz,1H),4.10(t,J＝7.8Hz,1H),4.01(dd,J＝11.5,3.2Hz,1H),3.94–3.89(m,2H),3.86–3.84(m,1H),3.80(dd,J＝11.5,2.5Hz,1H),3.67(s,3H),3.60–3.51(m,2H),2.28(s,3H),1.85(s,3H),1.82(s,3H),1.07(s,9H)。C61H70NO12SSi+[M+H]+的MS(ESI)m/z，计算值1068.4,实际值1068.4。
Step C:化合物2c的合成
向反应瓶中加入化合物2b(1当量)和四氢呋喃，随后向体系中加入TBAF(2当量，1M in THF)，室温下搅拌，TLC监测反应进度，待反应完全后(约10h)，向体系中加入饱和氯化铵溶液淬灭反应，再用乙酸乙酯萃取分液，合并的有机相经水洗、饱和食盐水洗、无水硫酸钠干燥、过滤、浓缩，湿法上样，硅胶柱层析初步纯化得到化合物2c(白色固体，56％粗收率)，C45H52NO12S+[M+H]+的MS(ESI)m/z，计算值830.3,实际值830.6。
Step D：化合物2d的合成
将化合物2c溶于THF/MeOH(v/v＝3:1)中，缓慢加入1M氢氧化钠水溶液调节体系至pH＝12，所得反应液在室温下搅拌，HPLC监测反应进度，待反应充分后(约12h)，向体系中加入1M的盐酸调节pH＝7～8，减压下浓缩除去大部分有机溶剂，所得粗产品经HPLC纯化得化合物2d(白色固体，82％收率)。1H NMR(400MHz,氯仿 -d)δ7.43–7.27(m,17H),7.07(d,J＝7.9Hz,2H),5.54(d,J＝5.0Hz,1H),5.33(d,J＝8.0Hz,1H),4.93(d,J＝12.1Hz,1H),4.86(d,J＝11.3Hz,1H),4.82–4.77(m,3H),4.74(d,J＝6.8Hz,1H),4.69(d,J＝12.2Hz,1H),4.36(ddd,J＝10.6,7.9,5.0Hz,1H),4.25–4.22(m,1H),4.13–4.02(m,3H),3.88(t,J＝8.6Hz,1H),3.81(dd,J＝12.7,2.2Hz,1H),3.74(dd,J＝10.7,8.5Hz,1H),3.68–3.59(m,2H),3.40(br s,1H),2.30(s,3H),1.77(s,3H).13C NMR(100MHz,氯仿-d)δ170.73,169.33,138.20,138.06,137.81,137.55,132.37,129.99,129.44,128.84,128.76,128.58,128.42,128.30,127.90,103.24,88.40,83.43,78.89,77.85,77.35,77.24,77.03,76.92,76.72,75.17,74.93,74.57,74.02,73.93,72.95,60.66,52.95,23.08,21.06(芳香区有三个碳信号重叠未出峰)。C42H46NO11S-[M-H]-的MS(ESI)m/z，计算值772.3,实际值772.2。
Step E：化合物2e的合成
称取化合物2d溶解于丙酮中，并在冰水浴中搅拌5min降温。称取NBS加入反应液，冰水浴中反应约1h。取样送测HPLC，确认反应结束。用硫代硫酸钠饱和溶液淬灭后，浓缩除丙酮，prep-HPLC制备、冻干得2e(白色固体，45％收率)。C35H40NO12 -[M-H]-的MS(ESI)m/z，计算值666.3,实际值666.4。
Step F：化合物2的合成
室温下，向50mL单口瓶中依次加入化合物2e、四氢呋喃、甲醇、钯碳催化剂，反应体系在氢气气氛下搅拌，直到TLC检测原料全部消失。过滤，减压浓缩，油泵抽干得到化合物2(白色固体,95％收率)。C14H22NO12 -[M-H+]-的MS(ESI)m/z，计算值396.1,实际值396.1。
二糖底物化合物3的制备
采用以下步骤制备化合物3，其结构如下：
化合物3的制备工艺参考化合物2的合成类似方法，具体路线如下：
终产物化合物3得到质谱验证，C14H22NO12 -[M-H+]-的MS(ESI)m/z，计算值396.1,实际值396.0。
二糖底物化合物4的制备
采用以下步骤制备化合物4，其结构如下：
化合物4的制备工艺参考化合物2的合成类似方法，具体路线如下：
终产物化合物4得到质谱验证，C14H22NO12 -[M-H+]-的MS(ESI)m/z，计算值396.1,实际值396.1。
二糖底物化合物5的制备
采用以下步骤制备化合物5，其结构如下：
化合物5的制备工艺参考化合物2的合成类似方法，具体路线如下：
终产物化合物5得到质谱验证，C14H22NO12 -[M-H+]-的MS(ESI)m/z，计算值396.1,实际值396.0。
实施例3.2连接子负载物(linker-payload，简写LP)的制备
LP-1的制备
连接子负载物1(LP-1)的结构如下：
(1)化合物LP-1b的制备
室温下，向10mL单口瓶中依次加入化合物1(0.5-5.0当量)、化合物LP-1a(1.0当量，GGG-VC-PAB-MMAE，CAS号：2684216-48-4，商业可得)、DMF、DIPEA(1-10当量)、HATU(0.5-10当量)，所得反应液在室温下搅拌，直至HPLC监测反应完全。反应液经半制备HPLC纯化，得到化合物LP-1b(白色固体,收率81.2％)。C78H126N14O26 2+[M+2H]2+的MS(ESI)m/z，计算值837.4,实际值837.9。
(2)化合物LP-1的制备
室温下，向10mL单口瓶中依次加入化合物LP-1b(21.7mg,0.013mmol，1当量)、H2O、Et3N(1–100当量)、DMC(2-氯-1，3-二甲基氯化咪唑啉鎓，CAS：37091-73-9,1–100当量)，所得反应液在室温下搅拌，HPLC监测反应，至反应完全。反应液经半制备HPLC纯化，得到化合物LP-1(15.3mg,收率71.3％，白色固体)。C78H124N14O25 2+[M+2H]2+的MS(ESI)m/z，计算值828.4,实际值828.6。
LP-2的制备
连接子负载物2(LP-2)的结构如下：
制备工艺如下：
20.1 Step A：化合物LP-2a的制备
室温下，向10mL单口瓶中依次加入化合物2(0.5-5.0当量)、化合物LP-1a(1.0当 量，GGG-VC-PAB-MMAE，CAS号：2684216-48-4，商业可得)、DMF、DIPEA(1-10当量)、HATU(0.5-10当量)，所得反应液在室温下搅拌，直至HPLC监测反应完全。反应液经半制备HPLC纯化，得到化合物LP-2a(白色固体，收率84％)。C78H126N14O26 2+[M+2H]2+的MS(ESI)m/z，计算值837.4,实际值837.8。
20.2 Step B：化合物LP-2的制备
室温下，向10mL单口瓶中依次加入化合物LP-2a(1当量)、H2O、Et3N(1–100当量)、DMC(2-氯-1，3-二甲基氯化咪唑啉鎓，CAS：37091-73-9,1–100当量)，所得反应液在0℃下搅拌，HPLC监测反应，至反应完全。反应液经半制备HPLC纯化，得到化合物LP-2(收率87％，白色固体)。C78H124N14O25 2+[M+2H]2+的MS(ESI)m/z，计算值828.4,实际值828.7。
LP-3，LP-4，LP-5的制备
采用和LP-2制备类似的步骤，制备以下结构的连接子负载物LP-3,LP-4,LP-5。
LP-6的制备
(1)LP-6-1的合成
Step A：中间体LP-6-1b的合成
向反应瓶中加入化合物LP-6-1a(1.0当量)和DMF(溶解浓度为1g/mL)，氮气气氛下搅拌溶解，降温到0-5℃后滴加DIEA(3当量)，随后所得体系在5℃下搅拌10min，再滴加溴化苄(1.3当量)，滴加完毕后让反应体系自然升至室温搅拌16小时。将反应液缓慢倒入冰水，加入甲基叔丁基醚搅拌后静置分液，水相用甲基叔丁基醚萃取4次，合并有机相，并用饱和食盐水洗涤、无水硫酸钠干燥、过滤、浓缩得黄色油状物粗产品，湿法上样，硅胶柱层析纯化(洗脱剂：PE/EA＝6:1)，得产物LP-6-1b(浅黄色油状物，定量收率)。
Step B：中间体LP-6-1d的合成
氮气保护下将中间体LP-6-1b(2.0当量)、化合物LP-6-1c(1.0当量)和THF(溶 解浓度为10g/mL)加入反应瓶中，搅拌溶解，取TsOH(0.1当量)加入反应，体系在室温下反应4h。将反应液缓慢倒入冰水中，乙酸乙酯萃取3次，合并的有机相依次用饱和碳酸氢钠水溶液、水和饱和食盐水洗涤，无水硫酸钠干燥，过滤浓缩得粗产品，硅胶柱层析纯化(洗脱剂：PE/EA＝1:1)，得产物LP-6-1d(白色固体，收率40％)。
Step C：中间体LP-6-1e的合成
氮气保护下，向反应瓶中加入化合物LP-6-1d和N,N-二甲基乙酰胺(DMAc，溶解浓度为10g/mL)，搅拌溶解，将体系降至14～18℃，滴加DBU(0.5当量),维持该温度反应，直到TLC显示反应完全(约1.5h)，得到中间体LP-6-1e，无需纯化直接用于下一步反应。
Step D：中间体LP-6-1g的合成
将上一步反应液冷却至0～5℃，依次加入4-甲基苯磺酸吡啶(PPTS,0.5当量)，1-(3-二甲胺基丙基)-3-乙基碳二亚胺(EDCI，1.0当量),1-羟基苯并三唑(HOBT，1.0当量)和LP-6-1f(0.85当量)，反应体系在0～10℃反应，直至LCMS显示反应完成(约4h)。将反应液加入到冰水中，加入2-甲基四氢呋喃萃取一次，水相再用2-甲基四氢呋喃萃取二次，合并有机相，依次用0.5M盐酸、饱和NaHCO3水溶液、水、饱和食盐水洗涤，无水硫酸钠干燥、过滤、浓缩，硅胶柱层析纯化(洗脱剂：DCM/MeOH)，得产品LP-6-1g(白色固体，收率78％)。
Step E：中间体LP-6-1h的合成
氮气保护下，将LP-6-1g和DMAc(溶解浓度为10g/mL)加入反应瓶中，搅拌溶解。降温到14～18℃，滴加DBU(0.5当量),并在该温度下搅拌反应1.5h，TLC监测反应进度，待反应完成后，得到中间体LP-6-1h，无需纯化直接用于下一步反应。
Step F：中间体LP-6-1j的合成
上一步LP-6-1h的反应溶液降温到0～5℃，加入PPTS(0.5当量)，EDCI(1当量)，HOBT(1当量)和化合物3i(0.85当量)，在0～10℃反应3～4h，LCMS监测反应进度，待反应完全后，将反应液加入到冰水中，加入2-甲基四氢呋喃萃取一次，水相再用2-甲基四氢呋喃萃取二次，合并有机相，依次用0.5M盐酸、饱和NaHCO3水溶液、水、饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，干法拌样，柱层析纯化(洗脱剂：DCM/MeOH)得到LP-6-1j(白色固体，收率50％)。
Step G：化合物LP-6-1的合成
氮气保护下，将中间体LP-6-1j溶解于DCM(15g/mL浓度)中，20℃下滴加DBU(0.5当量)，保持该温度搅拌反应直至HPLC显示反应完全。随后向体系中加入DCM稀释反应液，直接湿法上样，柱层析纯化(洗脱剂：DCM/MeOH)得化合物LP-6-1(白色固体,收率82％)。C34H47N6O10 +[M+H]+的MS(ESI)m/z，计算值699.4,实际值699.6。
(2)LP-6-2的合成
LP-6-2采用多肽固相合成法进行合成，步骤如下所示，
第一步：NH2-Asp(OtBu)-Rink酰胺树脂的制备
称取400g Rink酰胺树脂装入反应釜中，用2400mL DCM浸泡0.5h，使树脂充分溶胀，抽干。加入2400mL脱帽试剂洗涤，抽干。加入2400mL脱帽试剂鼓氮25±1℃搅拌反应0.5h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为蓝色。
称取88.87g Fmoc-Asp(OtBu)-OH，29.19g HOBT溶解于2000mL的DMF溶液和80ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干,Kaiser检测为淡蓝色。加入2400mL的DCM，再加入60ml的封头试剂鼓氮25±1℃搅拌反应1h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干。Kaiser检测为无色。
加入2400mL脱帽试剂洗涤，抽干。加入2400mL脱帽试剂鼓氮25±1℃搅拌反应0.5h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为蓝色。
第二步：NH2-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
称取131.64g Fmoc-PEG4-OH，48.64g HOBT溶解于2000mL的DMF溶液和80.0ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2-4h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干。Kaiser检测为无色。
加入2400mL脱帽试剂洗涤，抽干。再加入2400mL脱帽试剂鼓氮25±1℃搅拌反应0.5h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为蓝色。
第三步：NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
称取222.18g Fmoc-Asp(OtBu)-OH，72.96g HOBT溶解于2000mL的DMF溶液和80ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2-4h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干。Kaiser检测为无色。
加入2400脱帽试剂洗涤，抽干。再加入2400mL脱帽试剂鼓氮25±1℃搅拌反应0.5h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF 各洗涤二次，抽干，Kaiser检测为蓝色。
第四步：Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
称取191.75g Dde-Lys(Fmoc)-OH，48.64g HOBT溶解于2000mL的DMF溶液和80.0ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2-4h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干。Kaiser检测为无色。
加入2400±100mL脱帽试剂洗涤，抽干。再加入2400mL脱帽试剂鼓氮25±1℃搅拌反应0.5h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为蓝色。
第五步：Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
称取170.84g m-PEG12-CH2CH2COOH，48.64g HOBT溶解于2000mL的DMF溶液和80.0ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2-4h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干。Kaiser检测为无色。
第六步：NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
加入2400mL脱dde试剂鼓氮25±1℃搅拌反应10min，抽干，重复该操作3次后，依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为蓝色。
第七步：Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink酰胺树脂的制备
称取111.08g Fmoc-Gly-Gly-Gly-OH，48.64g HOBT溶解于2000mL的DMF溶液和80.0ml的DIC中，放置-10℃冰浴0.5h后缓慢加入反应釜中反应，室温鼓氮搅拌反应2-4h，抽干。依次用2400mL DMF，2400mL无水乙醇，2400mL DCM，2400mL DMF各洗涤二次，抽干，Kaiser检测为无色。树脂肽用2400mL无水乙醇洗涤三次，抽干，待切割。
第八步：LP-6-2的制备
在10L反应釜中加入10000mL切割试剂(TFA:TIS:H2O＝95:2.5:2.5)并冷却至-10±2℃，将干燥后称重的树脂加入其中后升温，在20±5℃通氮气搅拌2h。过滤，用100mL TFA洗涤树脂一次，滤液和洗涤液合并。
加入40L预冷(-10℃以下)的冷乙醚，搅拌10分钟后离心沉淀，离心沉淀后弃掉上清液，沉淀用冷乙醚混合摇匀后，再次离心沉淀(此步骤重复3次，每次用量为10L，10L，10L。每次离心转速设置为3600转，离心时间5分钟，离心机内腔温度为-5℃)。
将沉淀收集即为LP-6-2粗产品，其经Prep-HPLC纯化，冻干后得到LP-6-2。C74H121N9O31 2+[M+2H]2+的MS(ESI)m/z，计算值815.9,实际值816.3。
(3)LP-6-3的合成
Step A：中间体LP-6-3a的合成
向反应瓶中加入化合物LP-6-1(2.2当量)和化合物LP-6-2(1.0当量)，溶解于DMF中，随后加入DIPEA(5.0当量)搅拌均匀后，向体系中加入HATU(2.5当量)，常温下反应，HPLC监测直至反应完全(约2h)，反应体系直接经prep-HPLC制备，制备液冻干后得到化合物LP-6-3a(白色固体，收率52％)，C143H212O48N21 3+[M+3H]3+的MS(ESI)m/z，计算值997.2,实际值875.9(离子化后会脱落部分官能团)。
Step B：中间体LP-6-3b的合成
将化合物LP-6-3a溶解于纯化水中，加入一定量氢氧化钯(10wt％Pd(OH)2on carbon)，体系用氢气置换3次，常温搅拌1.5h，期间监控反应进程，原料消失后立刻停止反应，防止脱Fmoc产品增多。反应液过滤，prep-HPLC制备，得到化合物LP-6- 3b(白色固体，产率76％)，C128H200O48N21 3+[M+3H]3+的MS(ESI)m/z，计算值937.1,实际值875.9(离子化后会脱落部分官能团)。
Step C：中间体LP-6-3d的合成
称化合物LP-6-3b(1.0当量)和化合物LP-6-3c(2.2当量)溶解于DMF中，加入DIPEA(5.0当量)搅拌均匀，随后加入HATU(2.5当量)，常温下反应，HPLC监测直至反应完全(约16h)，反应体系直接经prep-HPLC制备，制备液冻干后得到化合物LP-6-3d(黄色固体，收率66％),C174H232O55Cl2F2N27 3+[M+3H]3+的MS(ESI)m/z，计算值1229.2,实际值1229.3。
Step D：化合物LP-6-3的合成
将化合物LP-6-3d溶解于DMF中，加入二乙胺，常温反应，HPLC监测直至反应完全(约0.5h)，反应完成后，调pH至中性，然后用prep-HPLC制备，冻干后得到化合物LP-6-3(黄色固体，产率73％)，C159H222O53Cl2F2N27 3+[M+3H]3+的MS(ESI)m/z，计算值1155.2,实际值1155.3。
其中，化合物LP-6-3c的合成请参考专利CN202211428194.6。
(4)LP-6的合成
LP-6a的合成
取化合物1(0.5-5.0当量)和化合物LP-6-3(1.0当量)溶解于DMF中，加入DIPEA(1-10当量)在0℃搅拌均匀，加入HATU(0.5-10当量)，反应体系在0℃下搅拌，HPLC监测反应进展直至反应完全(约2h)。反应体系直接经prep-HPLC制备，制备液冻干后得到化合物LP-6a(黄色固体，产率63％)，C173H243O64Cl2F2N28 3+ [M+3H]3+的MS(ESI)m/z，计算值1281.5,实际值1281.8。
LP-6的合成
称取化合物LP-6a溶解于纯化水中，在冰水浴中搅拌降温后，加入加入Et3N(1-100当量)、DMC(2-氯-1，3-二甲基氯化咪唑啉鎓，CAS：37091-73-9，1-100当量)，HPLC监测反应进度，反应完全后用prep-HPLC纯化，制备液冻干后得到化合物LP-6(浅黄色固体，产率74％)，C173H241O63Cl2F2N28 3+[M+3H]3+的MS(ESI)m/z，计算值1275.5,实际值1275.9。
LP-7的制备
LP-7的结构如下：
合成路线如下：
Step A:化合物LP-7b的合成
化合物LP-7a通过多肽固相合成的方法合成得到，参考LP-6-2的类似方法。
向反应瓶中加入化合物LP-7a(1.0当量)和化合物LP-6-1(3.6当量)，溶解于DMF中，随后加入DIPEA(6.0当量)搅拌均匀后，向体系中加入HATU(3.6当量)，常温下反应，HPLC监测直至反应完全(约2h)，反应体系直接经prep-HPLC制备， 制备液冻干后得到化合物LP-7b(白色固体，收率56％)，C191H280O66N29 3+[M+3H]3+的MS(ESI)m/z，计算值1345.3,实际值1345.8。
Step B：中间体LP-7c的合成
将化合物LP-7b溶解于纯化水中，加入一定量氢氧化钯(10wt％Pd(OH)2on activated carbon)，体系用氢气置换3次，常温搅拌1.5h，期间监控反应进程，反应液经过滤，直接用prep-HPLC制备，得到化合物LP-7c(白色固体，产率80％)，C170H262O66N29 3+[M+3H]3+的MS(ESI)m/z，计算值1255.3,实际值1255.9。
Step C：中间体LP-7d的合成
称化合物LP-7c(1.0当量)和化合物LP-6-3(1-10当量)溶解于DMF中，加入DIPEA(1-20当量)搅拌均匀，随后加入HATU(1-10当量)，常温下反应，HPLC监测直至反应完全(约2h)，反应直接用于下一步反应,C239H313O75Cl3F3N38 3+[M+3H]3+的MS(ESI)m/z，计算值1692.4,实际值1692.9。
Step D：化合物LP-7e的合成
将化合物LP-7d溶解于DMF中，加入二乙胺，常温反应，HPLC监测直至反应完全(约0.5h)，反应完成后，调pH至中性，然后用prep-HPLC制备，冻干后得到化合物LP-7e(黄色固体，产率73％)，C224H303O73Cl3F3N38 3+[M+3H]3+的MS(ESI)m/z，计算值1618.3,实际值1618.5。
Step E：化合物LP-7f的合成
取化合物1(0.5-5.0当量)和化合物LP-7e(1.0当量)溶解于DMF中，加入DIPEA(1-10当量)在0℃搅拌均匀，加入HATU(0.5-10当量)，反应体系在0℃下搅拌，HPLC监测反应进展直至反应完全(约2h)。反应体系直接经prep-HPLC制备，制备液冻干后得到化合物LP-7f(黄色固体，产率65％)，C238H325O84Cl3F3N39 4+[M+4H]4+的MS(ESI)m/z，计算值1308.8,实际值1309.0。
15.6Step F：化合物LP-7的合成
称取化合物LP-7f溶解于纯化水中，在冰水浴中搅拌降温后，加入Et3N(1-100当量)、DMC(2-氯-1，3-二甲基氯化咪唑啉鎓，CAS：37091-73-9，1-100当量)，HPLC监测反应进度，反应完全后用prep-HPLC纯化，制备液冻干后得到化合物LP-7(浅黄色固体，产率50％)。
实施例4.抗Her2抗体的制备
抗人ErbB2/Her2抗体T-LCCTL-HC(mAb-1)以及抗人TROP2抗体mAb-2的生产、纯化及鉴定参见专利CN106856656B的实施例1，在此将CN106856656B全文引入作为参考。
实施例5.混悬式偶联
ADC-1的固相制备
基于Halo-Endo S2-His催化抗体与连接子-负载物(Linker-payload)的偶联反应，制备ADC药物。在内切酶缓冲液中，将抗体与连接子-负载物按适当摩尔数比例(1:1至 1:100)充分混合，加入Halo-Endo S2-His介质混匀。混匀状态下的偶联反应在4-40℃进行0.5-20小时。反应结束后，离心，取出固相偶联反应混合物，经纯化、超滤或透析以除去未反应的药物中间体。纯化的ADC1储存于1×PBS pH7.4，存储于4℃或-80℃。
1)SDS-PAGE检测分析
偶联反应结束后，可通过SDS-PAGE法检测ADC-1的纯度和偶联效率。ADC1的SDS-PAGE检测结果如图3所示，偶联反应定点在抗体的重链上，(偶联上细胞毒素重链与未偶联细胞毒素的重链相比有了明显的分子量跃迁)；偶联产物中没有检测到未偶联细胞毒素的重链，偶联效率高达95％以上。
2)HIC-HPLC检测分析
HIC-HPLC：使用Proteomix HIC Butyl-NP5 4.6*100mm 5μm Non-Porous色谱柱；柱温30℃；流动相A：1.5M硫酸铵+20mM磷酸缓冲盐，pH7.0，流动相B：20mM磷酸缓冲盐：异丙醇＝7:3(v/v)；流速0.8mL/min；梯度方法：8分钟内B相从10％升到100％；选取280nm为检测波长，检测ADC1药物的DAR分布。
检测结果如图4和表2所示，未与化合物LP-1偶联的抗体＜1％；偶联产物以DAR2为主，ADC-1药物的DAR值为1.93。
表2.Halo-Endo S2-His偶联效率表
表2中偶联效率为1.93/2＝96.5％
3)质谱
ADC-1的高精度分子量质谱(ESI-MS)分析
利用高精度分子量质谱对ADC-1进行分析检测，结果表明其表观分子量为150539.27，理论分子量为150538.78，符合预期，确证了每条重链Fc端偶联了一个细胞毒素分子。
实施例6.柱式偶联
分别配制抗体和小分子毒素(LP-1)反应液，抗体反应液浓度为1-100mg/ml，小分子反应液浓度为0.1-50mg/ml。经过处理的固定化酶(Halo-Endo S2-His)介质进行装柱，通过空气或者水浴传导加热至10-40℃，提前预热＞30min。将抗体反应液和小分子反应液按照固定比例混合均匀，通过偶联柱进行反应，流出液即为ADC药物，偶联柱留时间为5min至24h。偶联反应液样品进行SDS-PAGE，HIC-HPLC和RP-HPLC检测。
1)SDS-PAGE检测分析
偶联反应结束后，可通过SDS-PAGE法检测ADC-1的纯度和偶联效率。ADC-1的SDS-PAGE检测结果显示，偶联反应定点发生抗体的重链上，偶联产物中没有检测到未偶联细胞毒素的重链，偶联效率高达95％以上。
2)HIC-HPLC检测分析
检测结果显示，偶联反应后的ADC-1的DAR为1.93，偶联效率为96.5％。
柱式偶联和混悬式偶联的效率都可以达成95％以上，柱式偶联的结果用于支持ADC药物线性生产放大。
柱式偶联的具体操作流程如下：将合适的管路(例如蠕动泵硅胶管)安装到合适的泵头(例如蠕动泵泵头(重庆杰恒，BT-600CA/DG4(10)))，进行管路平衡。然后将管路出口端和偶联柱进口端相连接，用另外一段管路与偶联柱下端连接。将连接好的偶联柱置于温度设置为10-40℃的温控装置(例如杂交炉(UVP/HB-1000Hybridizer)内，偶联柱下端管路出口端置于温控装置外。将管路进口端置于偶联反应液中，开启泵速进行偶联反应，收集偶联反应液，偶联反应液上样结束后，用偶联平衡缓冲液冲洗偶联柱，按照同样的收集体积进行收集；流出液即为ADC药物。收集结束后，用紫外分光光度计检测收集液浓度，经纯化、超滤或透析以除去未反应的药物中间体。纯化的ADC储存于1×PBS pH7.4，存储于4℃或-80℃。偶联反应液样品进行SDS-PAGE，HIC-HPLC或RP-HPLC检测。
固相偶联柱反应能够实现线性放大，在保证关键工艺参数(例如保留时间)相同条件下，根据偶联柱大小调整相应的工艺(如泵流速)，实现偶联柱线性放大到几百毫升(500mL)、几升(3L)或更大体积的偶联柱。实验室规模实现了糖苷酶偶联柱催化，离线进行DAR检测；采用体积更大的偶联柱通过更高流速实现偶联药物的线性放大，以及在线DAR值检测。专利申请WO2022170676A中偶联装置适用于本发明的糖基化工艺。本实施例验证了不同规模的偶联设备应用于糖基化偶联平台的可行性。
实施例7.Halo-Endo S2-His与ADC的分离
当所处环境pH值高于其等电点时，蛋白质会同带正电荷的填料，也就是阴离子交换剂相结合；而当所处环境pH值低于其等电点时，蛋白质会同带负电荷的填料，也就是阳离子交换剂相结合。Halo-Endo S2-His等电点为5.5，在pH6.0-pH8.0条件下，Halo-Endo S2-His蛋白表面带负电荷；Halo-Endo S2-His蛋白质会同带负电荷的填料，也就是阳离子交换剂流穿模式；Halo-Endo S2-His蛋白质会同带正电荷的填料，也就是阴离子交换剂流相结合。ADC等电点常规为8-9，在pH6.0-pH8.0条件下，ADC蛋白表面带正电荷；ADC会同带负电荷的填料，也就是阳离子交换剂相结合；ADC会同带正电荷的填料，也就是阴离子交换剂流穿模式。Halo-Endo S2-His与ADC等电点有大于2的显著差异，选择pH6.0-pH8.0/缓冲液，可以通过离子交换层析可以有效分离ADC和Halo-Endo S2-His。即用Halo-Endo S2-His定点催化ADC，即使柱上固定的Halo-Endo S2-His蛋白少量脱落在ADC产物中，后续的阴阳离子层析也可以有效清除Halo-Endo S2-His。
1)阴离子层析(AEX)
选用Q Sepharose FF填料装柱，用20mM Tris-HCl pH7.5平衡层析柱，进行上样。上样完成后，继续用20mM Tris-HCl pH7.5平衡至基线，最后用20mM Tris-HCl,1M NaCl pH7.5洗脱样品，用1M NaOH进行CIP(清洗)。用相同方法和填料分别进行ADC和Halo-Endo S2-His纯化，以及将ADC-1和Halo-Endo S2-His混合后纯化。
结果如图5A所示：ADC-1样品在pH 7.5条件下，过Q FF层析柱流穿模式。
Halo-Endo S2-His样品在pH 7.5条件下，过Q FF层析柱挂柱模式，20mM Tris-HCl,1M NaCl pH7.5洗脱样品；如图5B所示，ADC-1和Halo-Endo S2-His混合上样样品，经过Q FF层析柱后，可实现有效分离，ADC-1流穿，Halo-Endo S2-His挂柱。
2)阳离子层析(CEX)
选用Capto S impact阳离子填料装柱，用20mM柠檬酸/柠檬酸钠(Citric acid/Citric sodium,)pH6.2平衡层析柱，进行上样。上样完成后，继续用20mM柠檬酸/柠檬酸钠,pH6.2平衡至基线，最后用20mM柠檬酸/柠檬酸钠,160mM NaCl pH6.2洗脱样品。用20mM柠檬酸/柠檬酸钠,1M NaCl pH6.2进行重生层析柱，用1M NaOH进行层析柱CIP。用相同方法和填料分别进行ADC和Halo-Endo S2-His纯化，以及将ADC和Halo-Endo S2-His混合后纯化。
结果如图6A所示：ADC-1样品在pH 6.2条件下，过Capto S impact阳离子层析柱挂柱模式。
Halo-Endo S2-His样品在pH 7.5条件下，过Capto S impact阳离子层析柱流穿模式；如图6B所示：ADC-1与Halo-Endo S2-His混合上样样品，经过Capto S impact阳离子层析柱后，可实现有效分离，ADC-1挂柱，Halo-Endo S2-His流穿。
实施例8 ADC-1对细胞表面ErbB2/Her2的亲和力检测
取Her2 ECD，用CBS包被液(0.1M的碳酸盐缓冲液，pH 9.6)配成1μg/ml，100μl/孔，25℃包被60min。包被完成后用PBST洗板3次，用封闭液(5％脱脂奶粉)封闭(300μl/孔)，25℃，200rpm，孵育60min。封闭完成后，用PBST洗板3次，加入抗体T-LCCTL-HC及ADC-1(100μl/孔)，放置于25℃，在200rpm条件下孵育60min。与样品孵育完成后，用PBST洗板3次，加入抗人IgG Fc-HRP 100μl/孔，25℃，200rpm，孵育60min。60min孵育结束后再用TMB(100μl/孔)显色5min，然后用终止液终止反应，OD450下读数。
Elisa测试结果表明T-LCCTL-HC裸单抗与ADC-1对Her2 ECD的抗原亲和力无明显差异，见图7。
实施例9.ADC-1对ErbB2/Her2不同表达水平的肿瘤细胞增殖的影响
1)ErbB2/Her2阳性人乳腺癌细胞BT-474、ErbB2/Her2阳性人胃癌细胞NCI-N87、ErbB2/Her2阴性人肝癌细胞HepG2以每孔100μl(含有1000～10000个细胞)接种入96孔细胞板内，37℃，5％CO2，100％湿度，细胞培养箱内培养过夜。
2)向培养过夜的ErbB2/Her2阳性细胞分别加入不同浓度(10、3.3、1.1、0.37、0.12、0.041、0.014、0.0046、0.0015、0.00051nM)的ADC-1或抗体T-LCCTL-HC或不同浓度(30、10、3.3、1.1、0.37、0.12、0.041、0.014、0.0046、0.0015nM)的MMAE(一甲基奥瑞他汀E)；向培养过夜的ErbB2/Her2阴性细胞分别加入不同浓度(100、10、3.3、1.1、0.37、0.12、0.041、0.014、0.0046、0.0015nM)的ADC-1或抗体T-LCCTL-HC或不同浓度(30、10、3.3、1.1、0.37、0.12、0.041、0.014、0.0046、0.0015nM)的MMAE；向对照 组加入浓度为50μM的嘌呤霉素(Puromycin)。37℃继续温育72～120h。
3)自37℃细胞培养箱取出细胞板，平衡约30分钟至室温。每孔添加100μl CellTiter Glo试剂，振荡器震荡2min后室温避光静置10min，用MD M4酶标仪测量发光值(RLU)。
4)不同药物对肿瘤细胞增殖抑制作用的结果如表3及图8-10所示，其中ADC1和MMAE小分子毒素对ErbB2/Her2阳性细胞均有明显的增殖抑制作用，抗体T-LCCTL-HC单抗对ErbB2/Her2阳性细胞有轻微的增殖抑制作用，且ADC-1明显优于T-LCCTL-HC单抗和MMAE小分子毒素。ADC-1和T-LCCTL-HC单抗对ErbB2/Her2阴性细胞则没有抑制作用。
表3.不同药物对肿瘤细胞增殖的抑制作用(IC50,nM)
实施例10：ADC-1的体内活性测试(NCI-N87CDX小鼠模型)
按照以下方法测试ADC-1对ErbB2/HER2 NCI-N87CDX小鼠模型肿瘤生长的影响
1)细胞培养:收集对数生长期NCI-N87人胃癌肿瘤细胞(ATCC,Manassas,VA,cat#CRL-5822)，用基质胶缓冲液(PBS:Matrigel＝1:1)调整细胞密度，于7～9周龄SPF级雌性BALB/c裸鼠右肩胛部位经皮下注射0.2mL配制好的NCI-N87细胞悬液，细胞接种量为10×106/只。
2)用游标卡尺测量肿瘤直径，并按公式V＝0.5a x b2计算肿瘤体积(其中，a为肿瘤的最长直径，b为肿瘤的最短直径)。接种细胞5天后，当平均肿瘤体积范围处于100-300mm3时，将动物随机分为溶媒对照组和ADC-1 3mg/kg组，每组5只。动物经尾静脉注射给药，对照组给予等体积溶媒(Vehicle)，分组给药当天定义为Day0。给药后35天内每周两次测量各组动物的肿瘤体积，将第35天的动物肿瘤体积进行组间比较，用肿瘤体积计算T/C值和TGI值。计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：溶媒对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(Relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组时测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。TGI(％)的计算：TGI(％)＝[1-(某治疗组治疗结束时平均瘤体积-该治疗组开始给药时平均瘤体积)/(溶媒对照组治疗结束时平均瘤体积-溶媒对照组开始治疗时平均瘤体积)]×100％。
3)使用独立样本t检验对组间数据进行统计分析，所有分析使用SPSS17.0进行。P<0.05认为差异具有统计学意义。
4)表4和图11显示：与溶媒对照组相比，ADC-1能够显著抑制NCI-N87CDX小鼠模型中的肿瘤生长。
表4.ADC-1对NCI-N87CDX小鼠模型的抑制效果
1.平均值±SEM；
2.T/C％＝TRTV/CRTV x 100％(TRTV：治疗组RTV；CRTV：溶媒对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(Relative tumor volume，RTV)，计算公式为RTV＝V35/V0，其中V0是分组给药时(即D0)测量所得平均肿瘤体积，V35为给药后第35天时的平均肿瘤体积，TRTV与CRTV取同一天数据；
3.TGI(％)＝[1-(T35-T0)/(V35-V0)]×100)
4.P值根据肿瘤体积计算。
实施例11：ADC-2的制备和表征
参考上述制备方法制备ADC-2，其与ADC-1区别为：linker-payload是LP-6，抗体偶联药物ADC-2的表征数据如以下所述。
抗体偶联药物ADC-2的SDS-PAGE检测分析：偶联反应结束后，通过SDS-PAGE法检测ADC-2的纯度和偶联效率。ADC-2的SDS-PAGE检测结果显示，偶联反应定点发生抗体的重链上，且偶联上Linker-payload的ADC-2重链与切掉糖链的单抗重链相比有了明显的分子量跃迁，证明Linker-payload成功定点偶联到单抗重链分子上；偶联产物中基本未观察到未偶联Linker-payload的抗体，偶联效率高达95％以上，偶联产物的纯度符合预期。
ADC-2的HIC-HPLC检测分析,检测结果如图12所示，未偶联细胞毒素抗体＜5％；偶联产物以DAR4为主，总体上ADC-2药物的DAR值为3.8左右。
ADC-2的血浆稳定性测试
试验设计：
取混合健康人血浆(5男5女等体积混合)，添加ADC-2至特定终浓度，分装成4份，每份450μL，于37℃培养箱进行孵育，样本采集时间为0h、24h、48h、96h，采集后于-60～-90℃冰箱保存，以检测游离载荷脱落率及DAR变化率。
小分子毒素(此实施例中涉及的LP-6的小分子毒素以payload表示)的LC-MS检测
每份样品(除Double Blank)取40μL加入一定量内标沉淀剂(1ng/mL DXd)，摇振至少10min；Double Blank取40μL空白基质加入120μL不含内标沉淀剂，摇振至少10min，3600g，4℃，15min离心，吸取上清60μL至进样瓶中，加入一定量0.1％FA超纯水，摇振至少3min，HPLC检测。
杂交LC-MS法DAR检测
称取CNBr-活化琼脂糖(Sigma，Cat#C9142)1g至50mL离心管，加入50mL预冷1mM HCl，放置在旋转混合器上孵育至少30min，孵育条件为4℃，10RPM，离心 去除1mM HCl，离心一定时间。使用5-10倍体积的去离子水清洗溶胶1次，再使用0.1M NaHCO3清洗3次。取适量HER2 ECD，使用30kD超滤管置换(Buffer为0.1M NaHCO3)，然后将置换后的HER2 ECD与填料混合，置于旋转混合器，25℃、10RPM孵育2h±10min偶联。再加入0.1M NaHCO3缓冲液5mL，离心清洗去除未偶联的蛋白，离心一定时间，清洗3次。取一定量甘氨酸(pH 8.0)加入填料中，2～8℃静置封闭16h，封闭填料上未反应化学基团。首先使用5mL 0.1M NaHCO3清洗填料，离心一定时间，然后使用一定量醋酸缓冲液清洗填料，离心一定时间，重复该清洗循环若干次。分别取0.1mL样品于1.5mL EP管中，加入HER2 ECD偶联的填料0.1mL，25℃ 10RPM孵育2h，1mL PBST洗涤3次，离心一定时间。洗脱离心取上清测定浓度并进行LC-MS检测。
结果分析：
ADC-2在健康混合人血浆中孵育0，24，48和96h，DAR变化率为100.00％，106.7％，104.2％和106.5％；小分子毒素的脱落比仅分别为0.006％，0.179％，0.373％，1.07％。上述结果表明，ADC-2在健康混合人血浆中37℃孵育96h后，其毒素脱落量极低，DAR保持稳定，低毒素脱落预示着临床上因游离毒素导致的毒副作用显著降低，高DAR稳定性预试着抗体分子可靶向递送更多的毒素分子到达肿瘤部位，从而提高药效。两者协同可显著拓宽ADC-2的治疗窗口。
实施例13：ADC-2的体外活性测试(SK-BR-3、HCC1954，MDA-MB-468)
按照以下方法测试ADC-2对ErbB2/HER2不同表达水平的肿瘤细胞增殖的影响
参考实施例9中所描述的方法，对抗体偶联药物ADC-2在各种不同ErbB2/HER2表达水平的癌细胞增殖作用进行测试。例如，选取了SK-BR-3、HCC1954等ErbB2/HER2阳性人肿瘤细胞，及MDA-MB-468ErbB2/HER2阴性人肿瘤细胞.不同药物对肿瘤细胞增殖抑制作用的结果如表5及图13-15所示，其中ADC-2和小分子毒素对ErbB2/HER2阳性细胞均有明显的增殖抑制作用，抗体mAb-1单抗对ErbB2/HER2阳性细胞有一定增殖抑制作用，且ADC-2明显优于mAb-1。ADC-2和mAb-1单抗对ErbB2/HER2阴性细胞则没有抑制作用，体现出良好的靶向性。
表5.不同药物对肿瘤细胞增殖的抑制作用(IC50,nM)
实施例14：ADC-3的制备和表征
参考上述制备方法制备抗体偶联药物ADC-3，其与ADC-1的区别为：所用的抗体是抗TROP2抗体即mAb-2，所用的linker-payload是LP-6，抗体偶联药物ADC-3的表征数据如以下所述。
ADC-3的SEC-HPLC显示，高分子量聚体<5％，ADC样品主要以单体形式存在，偶联反应对抗体的损伤可以忽略不计。
抗体偶联药物ADC-3的高分辨率质谱(ESI-MS)DAR值分析
通过高分辨质谱仪对ADC-3的分子量进行解析，去卷积后的质谱图见图16，根据测得分子量信息，通过与理论分子量进行比对，可对各主要分子量变体进行归属，并利用各主要分子量变体的质谱丰度进行DAR值分析，计算出其平均DAR值为3.93。
实施例15：ADC-3的体外活性测试(BxPC-3、FaDu、HepG2)
参考实施例9中所描述方法的类似操作，对抗体偶联药物ADC-3在不同浓度梯度下针对各种不同TROP2表达水平的癌细胞增殖抑制作用进行测试。选取了BxPC-3、FaDu、HepG2等人肿瘤细胞。不同药物对肿瘤细胞增殖抑制作用的结果如表6及图17-19所示，其中ADC-3和小分子毒素对TROP2阳性细胞均有明显的增殖抑制作用，TROP2抗体即mAb-2对TROP2阳性细胞无明显增殖抑制作用，ADC-3明显优于单抗和小分子毒素。ADC-3和mAb-2单抗对TROP2阴性细胞均无抑制作用，展示出良好的靶向性。
表6.不同药物对肿瘤细胞增殖的抑制作用(IC50,nM)
实施例16：ADC-3的体内活性测试(NCI-N87CDX小鼠模型)
参照实施例10中描述的类似方法，评估ADC-3在NCI-N87CDX小鼠模型中肿瘤生长抑制方法的作用，给药后肿瘤生长曲线和体重变化曲线如图20-21所示，ADC-3展示出良好的肿瘤生长抑制作用和良好的安全性，实验小鼠未表现出体重下降相关毒性。
实施例17：ADC-4的制备和表征
参考上述制备方法制备抗体偶联药物ADC-4，其与ADC-1的区别为：所用的抗体是抗TROP2抗体即mAb-2，抗体偶联药物ADC-4的表征数据如以下所述。
ADC-4的HIC-HPLC检测分析，检测结果如图22所示，未偶联细胞毒素抗体＜5％；偶联产物以DAR2为主，总体上ADC-4药物的DAR值为1.89。ADC-4的SEC-HPLC检测结果显示，其中ADC药物中高分子量聚体＜5％，ADC样品主要以单体形式存在。
实施例18：ADC-5的制备和表征
参考上述制备方法制备抗体偶联药物ADC-5，其与ADC-1的区别为：抗体是抗TROP2抗体mAb-2,linker-payload用的是LP-2，抗体偶联药物ADC-5的表征数据如以下所述。
ADC-5的HIC-HPLC检测分析，检测结果如图23所示，未偶联细胞毒素抗体＜5％；偶联产物以DAR2为主，总体上ADC-5药物的DAR值为1.87。ADC-5的SEC-HPLC检测显示，ADC药物中高分子量聚体＜5％，ADC样品主要以单体形式存在。
实施例19：ADC-4和ADC-5的体外活性比较(BxPC-3，FaDu，HepG2)
参考实施例9中所描述的方法，对抗体偶联药物ADC-4和ADC-5在各种不同TROP2表达水平的癌细胞增殖作用进行测试。选取了BxPC-3(人胰腺癌细胞)、FaDu(人咽喉癌细胞)等TROP2阳性人肿瘤细胞，及HepG2(人肝癌细胞)TROP2阴性肿瘤细胞。不同药物对肿瘤细胞增殖抑制作用的结果如表7及图24-26所示，其中ADC-4、ADC-5和MMAE小分子毒素对阳性细胞均有明显的增殖抑制作用，且ADC-4与ADC-5活性无明显差异，单抗对TROP2阳性细胞无明显增殖抑制作用。ADC-4、ADC-5和单抗对抗原阴性细胞则没有抑制作用，体现出良好的靶向性。
表7.不同药物对肿瘤细胞增殖的抑制作用(IC50,nM)
实施例20：ADC-6的制备和表征
参考上述制备方法制备抗体偶联药物，其与ADC-1的区别为：所用的抗体是mAb-3(Trastuzumab)、linker-payload用的是LP-6，抗体偶联药物ADC-6的表征数据如以下所述。ADC-6的HIC-HPLC检测分析，检测结果如图27所示，未偶联细胞毒素抗体＜5％；偶联产物以DAR4为主，总体上ADC-6药物的DAR值为3.93。ADC-6的SEC-HPLC检测显示，ADC药物中高分子量聚体＜5％，ADC样品主要以单体形式存在
实施例21：ADC-6的体外活性测试(SK-BR-3，NCI-N87)
参考实施例9中所描述的方法，对抗体偶联药物ADC-6在各种HER2不同表达水平的癌细胞增殖作用进行测试。选取了SK-BR-3和NCI-N87两组HER2细胞系，比较ADC-2(含Sortase recognition sequence)与ADC-6(不含Sortase recognition sequence)的活性差异,结果表明有无Srt A酶的特异性识别短肽序列(Sortase recognition sequence)，目标ADC在上述两种细胞系的细胞杀伤活性方面没有差异(参见图28-29)。
本申请所涉及的序列如下所述：
SEQ ID No:1(Halo-Endo S2-His)：
SEQ ID No:2(mAb-1轻链)：
SEQ ID No:3(mAb-1重链)：
SEQ ID No:4(mAb-2轻链)：
SEQ ID No:5(mAb-2重链)：
SEQ ID No:6(mAb-3轻链)：
SEQ ID No:7(mAb-3重链)：
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (53)

  1. 一种制备偶联药物的方法，其特征在于，所述偶联药物是基于Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
    (1)提供含有噁唑啉低聚糖的供体、含有Fc区的蛋白以及固定化的具有糖苷转移活性的糖苷内切酶，所述Fc含有GlcNAc基序；
    (2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有Fc基序的蛋白。
  2. 根据权利要求1所述的方法，其特征在于，所述含有Fc区的蛋白为抗体或Fc融合蛋白。
  3. 一种制备抗体偶联药物的方法，其特征在于，所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
    (1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；
    (2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体。
  4. 根据权利要求1-3任一项所述的方法，其中所述含有噁唑啉低聚糖的供体还含有负载物。
  5. 根据权利要求4所述的方法，所述负载物选自下组：小分子化合物、激动剂、核酸、核酸类似物、荧光分子、放射性核素和免疫调节蛋白；或
    所述负载物选自由小分子化合物(例如，各种作用机制的小分子药物，包括各种传统小分子药物、光声动力疗法药物、光热疗法药物等，例如化疗药、小分子靶向药、免疫激动剂等，例如传统细胞毒性药物，如顺铂、紫杉醇、5-氟尿嘧啶、环磷酰胺和苯达莫司汀等；小分子靶向药，如甲磺酸伊马替尼、吉非替尼和安罗替尼等；免疫激动剂，如STING激动剂、TLR激动剂等)、核酸及核酸类似物、示踪分子(包括荧光分子、生物素、荧光团、发色团、自旋共振探针及放射性标记等)、短肽、多肽、拟肽和蛋白质组成的组。
  6. 根据权利要求1-3任一项所述的方法，其特征在于，所述噁唑啉低聚糖选自以下组中的一种或多种：二糖噁唑啉、三糖噁唑啉、四糖噁唑啉、五糖噁唑啉、六糖噁唑啉、七糖噁唑啉、八糖噁唑啉、九糖噁唑啉、十糖噁唑啉和十一糖噁唑啉。
  7. 根据权利要求1-3任一项所述的方法，其特征在于，所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-(第二六碳糖基或其衍生物)f-β-D-吡喃葡萄糖噁唑啉，f为0、1、2、3、4、5或6；β-D-吡喃葡萄糖噁唑啉的结构为：
  8. 根据权利要求7所述的方法，其特征在于，所述第一六碳糖基或其衍生物选自葡萄糖基、甘露糖基、半乳糖基、果糖基、古洛糖基、艾杜糖基或它们的衍生物；和/或
    其6位碳呈-C(O)-形式；和/或
    所述第二六碳糖基或其衍生物在每次出现时独立地选自葡萄糖基、甘露糖基、半乳糖基、果糖基或它们的衍生物；和/或
    低聚糖结构中的各个单糖部分通过β-(1→4)糖苷键相连；和/或
    第一六碳糖基衍生物和第二六碳糖基衍生物独立地选自糖醛酸或单糖的羟基被酰氨基替换的衍生物。
  9. 根据权利要求7或8所述的方法，其特征在于，所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为甘露糖基或其衍生物；或
    所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为半乳糖基或其衍生物；或
    所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为葡萄糖基或其衍生物；或
    所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为果糖基或其衍生物；或
    所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为古洛糖基或其衍生物；或
    所述噁唑啉低聚糖具有如下结构：第一六碳糖基或其衍生物-β-D-吡喃葡萄糖噁唑啉，所述第一六碳糖基或其衍生物为艾杜糖基或其衍生物。
  10. 根据权利要求7或8所述的方法，其特征在于，所述噁唑啉低聚糖的结构为：
  11. 一种制备偶联药物的方法，其特征在于，所述抗体偶联药物是基于Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
    (1)提供含有噁唑啉低聚糖的供体、含有Fc区的蛋白以及固定化的具有糖苷转移活性的糖苷内切酶，所述Fc区包含GlcNAc基序；
    其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
    (2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有Fc区的蛋白；
    其中，药物偶联药物结构如式(II)所示：
    其中，P为负载物；
    D-C(O)-L-为连接子；
    第一六碳糖基或其衍生物部分的6位碳呈-C(O)-形式，f为0、1、2、3、4、5或6；
    L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
    R为氢或α-L-岩藻糖基；
    q为1或2；
    Protein为含有Fc区的蛋白。
  12. 根据权利要求11所述的方法，其特征在于，所述第一六碳糖基或其衍生物选自葡萄糖基、甘露糖基、半乳糖基、果糖基、古洛糖基、艾杜糖基或它们的衍生物；和/或
    所述第二六碳糖基或其衍生物在每次出现时独立地选自葡萄糖基、甘露糖基、半乳糖基、果糖基或它们的衍生物；和/或
    低聚糖结构中的各个单糖部分通过β-(1→4)糖苷键相连；和/或
    第一六碳糖基衍生物和第二六碳糖基衍生物独立地选自糖醛酸或单糖的羟基被酰氨基替换的衍生物。
  13. 根据权利要求11所述的方法，其特征在于，所述含有Fc区的蛋白为抗体或Fc融合蛋白。
  14. 一种制备抗体偶联药物的方法，其特征在于，所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
    (1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；
    其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
    (2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体；
    其中，抗体偶联药物结构如式(II-1)，(II-2)，(II-3)，(II-4)或(II-5)所示：
    其中，P为负载物；
    D-C(O)-L-为连接子；
    D-C(O)-为二糖结构；
    L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
    R为氢或α-L-岩藻糖基；
    q为1或2；
    Ab为抗体或其抗原结合片段。
  15. 一种制备抗体偶联药物的方法，其特征在于，所述抗体偶联药物是基于抗体Fc区N-糖基化位点进行定点偶联，所述方法包括以下步骤：
    (1)提供含有噁唑啉低聚糖的供体、含有GlcNAc基序的抗体以及固定化的具有糖苷转移活性的糖苷内切酶；
    其中，含有噁唑啉低聚糖的供体为式(I)的连接子-负载物化合物：
    (2)通过所述糖苷内切酶的催化作用将所述含有噁唑啉低聚糖的供体共价连接到所述含有GlcNAc基序的抗体；
    其中，抗体偶联药物结构如式(II)所示：
    其中，P为负载物；
    D-C(O)-L-为连接子；
    D-C(O)-为二糖结构
    L为接头，且L经由其中的-NH-与D-C(O)-中的羰基直接相连，其中当L为无分支接头时，其与1个P连接，且t为1，而当L为有分支接头时，每个分支可与1个P连接，且t为大于1的整数；
    R为氢或α-L-岩藻糖基；
    q为1或2；
    Ab为抗体或其抗原结合片段。
  16. 根据权利要求11-15任一项所述的方法，其特征在于，其中-L-(P)t为-L2-L1-B-P，即式(I)为：
    其中
    B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
    L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
    L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
    1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、-C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
    2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
    R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
    P是与B部分，或L1部分，或L2部分连接的负载物。
  17. 如权利要求11-15任一项所述的连接子-负载物化合物，其中-L-(P)t为
    即式(I)为：
    其中，
    Ld2和每个Ld1独立地是键；或选自-NH-C1-20亚烷基-(CO)-、-NH-(PEG)i-(CO)-；或为侧链上各自独立地未取代或被-CO-(PEG)j-R11取代的天然氨基酸或聚合度为2-10(即2、3、4、5、6、7、8、9或10)的寡聚天然氨基酸；R11是C1-10烷基；
    d是0、1、2、3、4、5或6；
    -(PEG)i-和-(PEG)j-各自为PEG片段，包含指定数量的连续-(O-C2H4)-结构单元或连续-(C2H4-O)-结构单元，任选地在一个末端附加C1-10亚烷基；每个i独立地为1-100的整数，每个j独立地为1-100的整数；
    M是氢或LKa-L2―L1―B―P；
    Q是NH2或L2―L1―B―P；
    条件是不包括以下情况：M是氢且同时Q是NH2；
    每个LKa独立选自
    opSu为或其混合物；其中，*表示和L2的连接部分；
    B独立地不存在，或为以下1)，或为以下2)，或以下1)和2)的组合：1)自切除间隔子Sp1；2)一个二价基团，或两个或更多个二价基团的组合，其中所述二价基团选自：-CR1R2-、C1-10亚烷基、C4-10亚环烷基、C4-10亚杂环基和-(CO)-；
    L1独立地不存在；或是不可裂解序列；或是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸；
    L2独立地不存在；或为以下1)；或为以下2)；或为以下1)和2)的组合：
    1)-NH-C2-20亚烷基，其中亚烷基中的一个或多个-CH2-结构任选地被以下基团代替：-CR3R4-、-O-、-(CO)-、-S-、-S(＝O)2-、-NR5-、-N⊕R6R7-、C4-10亚环烷基、C4-10亚杂环基、亚苯基，其中亚环烷基、亚杂环基和亚苯基各自独立地未被取代或被选自卤素、-C1-10烷基、-C1-10卤代烷基、-C1-10亚烷基-NH-R8和-C1-10亚烷基-O-R9的至少一个取代基取代；
    2)氨基酸残基序列，即-*(AA)n**-，n为1、2、3、4、5、6、7、8、9或10-100的 整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键；
    R1、R2、R3、R4、R5、R6、R7、R8、R9各自独立地选自氢、卤素、取代或未被取代的-C1-10烷基、C4-10亚环烷基；或R1和R2及与它们连接的碳原子一起形成3-6元亚环烷基，和/或R3和R4及与它们连接的碳原子一起形成3-6元亚环烷基；
    P是与B部分，或L1部分，或L2部分连接的负载物。
  18. 根据权利要求16或17所述的方法，其特征在于，L2为氨基酸残基序列，即-*(AA)n**-，n为1-100的整数，AA在每次出现时独立地为氨基酸残基，*表示相应氨基酸的N-端，**表示相应氨基酸的C-端，且在一个氨基酸的氨基和α-碳之间任选存在-(C2H4-O)m-(CH2)p-，其中m为1、2、3、4、5、6、7、8、9或10；p为0、1、2或3，并且*端与二糖结构中的羰基形成酰胺键。
  19. 根据权利要求18所述的方法，其特征在于，AA在每次出现时独立地为Phe、Lys、Gly、Ala、Leu、Asn、Val、Ile、Pro、Trp、Ser、Tyr、Cys、Met、Asp、Gln、Glu、Thr、Arg、His中的任意一种或其任意地组合。
  20. 根据权利要求16或17所述的方法，其特征在于，n为1-100的整数，优选为1-50的整数，优选为1-30的整数，优选为1-20的整数，优选为1-10的整数，优选为1、2、3、4、5、6、7、8、9、10。
  21. 根据权利要求16或17所述的方法，其特征在于，L1是包含可被酶裂解的氨基酸序列的可裂解序列，所述可被酶裂解的氨基酸序列包含1、2、3、4、5、6、7、8、9或10个氨基酸。
  22. 根据权利要求21所述的方法，其特征在于，其中L1为Val、Cit、Phe、Lys、Gly、Ala、Leu、Asn中的任意一种或其任意地组合，优选地，为-Gly-Gly-Phe-Gly-、-Phe-Lys-、-Val-Cit-、-Val-Lys-、-Gly-Phe-Leu-Gly-、-Ala-Leu-Ala-Leu-、-Ala-Ala-Ala-及其组合。
  23. 根据权利要求16或17所述的方法，其特征在于，L1为-Val-Cit-。
  24. 根据权利要求16或17所述的方法，其特征在于，其中B选自：(-PABC-)，-NH-CH2-U-，或-NH-CH2-U-(CH2)g-(CO)-；其中g为1、2、3、4、5或6；U不存在，或是CH2、O、S或NH，优选O或S。
  25. 根据权利要求16或17所述的方法，其特征在于，其中-L1-B-表示-Val-Cit-PABC-。
  26. 根据权利要求16或17所述的方法，其特征在于，其中-L2-L1-B-表示-Gly-Gly-Gly-Val-Cit-PABC-或-HN-(C2H4-O)m-(CH2)p-Gly-Gly-Phe-Gly-。
  27. 根据权利要求11-15任一项所述的方法，其特征在于，所述负载物P选自由小分子化合物(例如，各种作用机制的小分子药物，包括各种传统小分子药物、光声动力疗法药物、光热疗法药物等，例如化疗药、小分子靶向药、免疫激动剂等，例如传统细胞毒性药物，如顺铂、紫杉醇、5-氟尿嘧啶、环磷酰胺和苯达莫司汀等；小分子靶向药，如甲磺酸伊马替尼、吉非替尼和安罗替尼等；免疫激动剂，如STING激动剂、TLR激动剂等)、核酸及核酸类似物、示踪分子(包括荧光分子、生物素、荧光团、发色团、自旋共振探针及放射性标记等)、短肽、多肽、拟肽和蛋白质组成的组。
  28. 根据权利要求11-15任一项所述的方法，其特征在于，所述负载物P是细胞毒素或其片段，具有任选的衍生化以连接到式(I)化合物中的L部分；
    优选地，细胞毒素选自由紫杉烷类、美登木素类、奥利斯他汀类、埃博霉素类(epothilones)、康普瑞丁A-4磷酸盐(combretastatin A-4 phosphate)、康普瑞丁A-4(combretastatin A-4)及其衍生物、吲哚-磺胺类、长春碱类如长春碱(vinblastine)、长春新碱(vincristine)、长春地辛(vindesine)、长春瑞滨(vinorelbine)、长春氟宁(vinflunine)、长春甘酯(vinglycinate)、脱水长春碱(anhy-drovinblastine)、尾海兔素10(dolastatin 10)及其类似物、软海绵素B、艾瑞布林(eribulin)、吲哚-3-氧乙酰酰胺类、鬼臼毒素类、7-二乙氨基-3-(2'-苯并噁唑基)-香豆素(DBC)、圆皮海绵内酯(discodermolide)、laulimalide、喜树碱类及其衍生物、米托蒽醌、米托胍腙、氮芥类、亚硝基脲类、氮杂环丙烷类、苯并多巴、卡波醌、美妥替哌、乌瑞替哌、达内霉素(dynemicin)、埃斯培拉霉素(esperamicin)、新制癌菌素、阿克拉霉素、放线菌素、安曲霉素、博来霉素、放线菌素C、卡拉比星、洋红霉素、抗癌霉素、洋红霉素、放线菌素D、柔红霉素、地托比星、阿霉素、表柔比星、依索比星、依达比星、麻西罗霉素、丝裂霉素类、诺拉霉素、橄榄霉素、培洛霉素、泊非霉素、嘌罗霉素、铁阿霉素、罗多比星、链黑霉素、链佐星、净司他丁、佐柔比星、单端孢霉烯类、T-2毒素、verracurin A、杆孢菌素A、安归啶(anguidine)，乌苯美司、重氮丝氨酸、6-重氮基-5-氧代-L-正亮氨酸，二甲叶酸、甲氨蝶呤、蝶罗呤、三甲曲沙、依达曲沙、氟达拉滨、6-巯基嘌呤、硫咪嘌呤、硫鸟嘌呤、安西他滨、吉西他滨、依诺他滨、阿扎胞苷、6-氮尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、氟尿苷、卡普睾酮、丙酸屈他雄酮、环硫雄醇、美雄烷、睾内酯、氨鲁米特、米托坦、曲洛司坦、氟他胺、尼鲁米特、比卡鲁胺、醋酸亮丙瑞林、蛋白激酶抑制剂和蛋白酶体抑制剂组成的组；和/或
    选自长春碱类、秋水仙碱类、紫杉烷类、奥利斯他汀类、美登木素类、calicheamicin、doxonubicin、duocarmucin、SN-38、念珠藻素类似物(cryptophycin analog)、deruxtecan、duocarmazine、calicheamicin、centanamycin、dolastansine、pyrrolobenzodiazepine和exatecan及其衍生物；和/或
    选自奥利斯他汀(auristatin)，尤其是MMAE、MMAF或MMAD；和/或
    选自exatecan及其衍生物，例如DX8951f；和/或
    选自DXd-(1)和DXd-(2)；优选DXd-(1)。
  29. 根据权利要求11-14任一项所述的方法，其特征在于，所述连接子-负载物化合物为：

  30. 根据权利要求1-29中任意一项所述的方法，其特征在于，所述偶联药物中含Fc区的蛋白为抗体，所述偶联药物或所述抗体偶联药物中所述抗体为抗CD19抗体、抗CD20抗体、抗CD22抗体、抗CD25抗体、抗CD30/TNFRSF8抗体、抗CD33抗体、抗CD37抗体、抗CD44v6抗体、抗CD56抗体、抗CD70抗体、抗CD71抗体、抗CD74抗体、抗CD79b抗体、抗CD117/KITk抗体、抗CD123抗体、抗CD138抗体、抗CD142抗体、抗CD174抗体、抗CD227/MUC1抗体、抗CD352抗体、抗CLDN18.2 抗体、抗DLL3抗体、抗ErbB2/HER2抗体、抗CN33抗体、抗GPNMB抗体、抗ENPP3抗体、抗Nectin-4抗体、抗EGFRvⅢ抗体、抗SLC44A4/AGS-5抗体、抗CEACAM5抗体、抗PSMA抗体、抗TIM1抗体、抗LY6E抗体、抗LIV1抗体、抗Nectin4抗体、抗SLITRK6抗体、抗HGFR/cMet抗体、抗SLAMF7/CS1抗体、抗EGFR抗体、抗BCMA抗体、抗AXL抗体、抗NaPi2B抗体、抗GCC抗体、抗STEAP1抗体、抗MUC16抗体、抗间皮素(Mesothelin)抗体、抗ETBR抗体、抗EphA2抗体、抗5T4抗体、抗FOLR1抗体、抗LAMP1抗体、抗Cadherin 6抗体、抗FGFR2抗体、抗FGFR3抗体、抗CA6抗体、抗CanAg抗体、抗整合素αV抗体、抗TDGF1抗体、抗肝配蛋白(Ephrin)A4抗体、抗TROP2抗体、抗PTK7抗体、抗NOTCH3抗体、抗C4.4A抗体、抗FLT3抗体、抗B7H3/4抗体、抗TF(组织因子(Tissue Factor))抗体、抗ROR1/2/抗体；优选为抗CD19抗体、抗ErbB2/HER2抗体、抗CLDN18.2抗体、抗Nectin-4抗体、抗FGFR3抗体、抗Trop2抗体。
  31. 根据权利要求1-30中任意一项所述的方法，其特征在于，所述具有糖苷转移活性的糖苷内切酶为N-乙酰葡萄糖胺内切水解酶。
  32. 根据权利要求31所述的方法，其特征在于，所述N-乙酰葡萄糖胺内切水解酶包括选自Endo S(酿脓链球菌内切糖苷酶-S)、Endo F3(米尔伊丽莎白菌(Elizabethkingia miricola)内切糖苷酶-F3)、Endo S2(Endoglycosidase-S2，酿脓链球菌内切糖苷酶-S2)、Endo Sd(Endoglycosidase-Sd，酿脓链球菌内切糖苷酶-Sd)和Endo CC(Endoglycosidase-CC，酿脓链球菌内切糖苷酶-CC)中的至少一种；或
    所述N-乙酰葡萄糖胺内切水解酶包括选自Endo H、Endo D、Endo F2、Endo F3、Endo M、Endo CC1、Endo CC2、Endo Om、Endo S和Endo S2中的至少一种。
  33. 根据权利要求1-30中任意一项所述的方法，其特征在于，具有糖苷转移活性的糖苷内切酶共价连接有Halo标签，并通过Halo标签固定在含卤代烷基连接子的支持物上，所述Halo标签为脱卤素酶或其变体或其截短的功能活性部分。
  34. 根据权利要求33所述的方法，其特征在于，所述糖苷内切酶的一端共价连接有Halo标签，另一端共价连接有His标签；或
    所述糖苷内切酶的氨基端共价连接有Halo标签，羧基端共价连接有His标签，即Halo-糖苷内切酶-His；或
    所述糖苷内切酶的氨基端共价连接有Halo标签，羧基端共价连接有His标签，所糖苷内切酶为Endo-S2，即Halo-Endo S2-His。
  35. 根据权利要求33所述的方法，其特征在于，所述支持物包含氯代烷基连接子，使得所述糖苷内切酶通过氯代烷基连接子和Halo标签之间的共价相互作用固定在所述支持物上。
  36. 根据权利要求35所述的方法，其特征在于，所述氯代烷基连接子由具有式(III)结构的氯代烷基底物产生：
    其中，u为1-20的整数，v为0-20的整数，w为1-19的整数。
  37. 根据权利要求33所述的方法，其特征在于，所述支持物具有式(IV)的结构：
    其中u为1-20的整数，v为0-20的整数，w为1-19的整数；
    为树脂、珠子、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面，优选树脂，更优选琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂，最优选高度交联的琼脂糖树脂或聚甲基丙烯酸甲酯。
  38. 一种糖苷内切酶融合蛋白，其特征在于，包含共价连接的糖苷内切酶和Halo标签，所述Halo标签为脱卤素酶或其变体或其截短的功能活性部分。
  39. 根据权利要求38所述的糖苷内切酶融合蛋白，其特征在于，所述糖苷内切酶的氨基端端共价连接有Halo标签，所述糖苷内切酶的羧基端共价连接有His标签。
  40. 根据权利要求38或39所述的糖苷内切酶融合蛋白，其特征在于，所述糖苷内切酶选自Endo S(酿脓链球菌内切糖苷酶-S)、Endo F3(米尔伊丽莎白菌(Elizabethkingia miricola)内切糖苷酶-F3)、Endo S2(Endoglycosidase-S2，酿脓链球菌内切糖苷酶-S2)、Endo Sd(Endoglycosidase-Sd，酿脓链球菌内切糖苷酶-Sd)和Endo CC(Endoglycosidase-CC，酿脓链球菌内切糖苷酶-CC)中的至少一种；或
    所述糖苷内切酶选自Endo H、Endo D、Endo F2、Endo F3、Endo M、Endo CC1、Endo CC2、Endo Om、Endo S和Endo S2中的至少一种。
  41. 根据权利要求38或39所述的糖苷内切酶融合蛋白，其特征在于，所述糖苷内切酶融合蛋白包含如SEQ ID NO:1所示的氨基酸序列，或与SEQ ID NO:1相比具有至少80％同一性、至少90％同一性，或与SEQ ID NO:1相比具有1个或多个保守氨基酸取代。
  42. 根据权利要求38或39所述的糖苷内切酶融合蛋白，其特征在于，所述糖苷内切酶融合蛋白的PI约为4-7。
  43. 一种固定化的糖苷内切酶融合蛋白，其特征在于，包含固定在支持物上的权利要求38-42中任意一项的糖苷内切酶融合蛋白。
  44. 根据权利要求43所述的固定化的糖苷内切酶融合蛋白，其特征在于，所述支持物包含卤代烷基连接子，使得所述糖苷内切酶融合蛋白通过所述卤代烷基连接子和 Halo标签之间的共价相互作用固定在所述支持物上，优选所述卤代烷基连接子为氯代烷基连接子。
  45. 根据权利要求44所述的固定化的糖苷内切酶融合蛋白，其特征在于，所述氯代烷基连接子由具有式(III)结构的氯代烷基底物产生：
    其中，u为1-20的整数，v为0-20的整数，w为1-19的整数。
  46. 根据权利要求44所述的固定化糖苷内切酶融合蛋白，其特征在于，所述支持物具有式(IV)的结构：
    其中u为1-20的整数，v为0-20的整数，w为1-19的整数；
    为树脂、珠子、膜、凝胶、基质、薄膜、板、孔、管、载玻片或表面，优选树脂，更优选琼脂糖树脂、有机硅树脂、聚甲基丙烯酸甲酯树脂或纤维素树脂，最优选高度交联的琼脂糖树脂或聚甲基丙烯酸甲酯。
  47. 一种预装柱，其特征在于，所述预装柱填充有根据权利要求43-46中任意一项所述的固定化的糖苷内切酶融合蛋白。
  48. 权利要求43-46中任意一项所述的固定化的糖苷内切酶融合蛋白和/或权利要求47所述的预装柱在偶联药物或抗体偶联物的制备和/或纯化中的应用。
  49. 偶联设备在实施根据权利要求1-37中任一项所述的方法中的用途，所述偶联设备包括流动反应器和流体输送单元，所述流动反应器填充有固定化的糖苷内切酶，所述流体输送单元与流动反应器的进口流体连通，并且向流动反应器输送含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体。
  50. 根据权利要求44所述的用途，其特征在于，所述偶联设备包括：
    至少一个流动反应器，其具有进口和出口，所述流动反应器中填充有介质，并且糖苷内切酶固定到所述介质上；
    流体输送单元，其与所述流动反应器的进口流体连通，并且被配置为根据偶联过程的不同阶段，向流动反应器连续提供至少一种反应流体，该至少一种反应流体包括含有噁唑啉低聚糖的供体和含有GlcNAc基序的抗体或含有Fc区的蛋白；和
    流体收集单元，其与流动反应器的出口流体连通，并且被配置为根据偶联过程的不同阶段，控制对流出流动反应器的出口的流体的收集。
  51. 根据权利要求49或50所述的用途，其特征在于，所述至少一种反应流体包含第一反应流体和第二反应流体，缓冲液、第一反应流体和第二反应流体分别被储存在第一容器、第二容器和第三容器中，所述流体输送单元包括第一输送泵和第二输送泵，其中所述第一容器和第二容器分别经由第一容器出口管路和第二容器出口管路连接至第一输送泵，所述第三容器经由第三容器出口管路连接至第二输送泵，所述第一输送泵和第二输送泵分别经由第一进口支管和第二进口支管连接至进口主管，所述进口主管连接至流动反应器的进口，
    而且其中，在反应前平衡、反应后回收和回收后冲洗期间，所述第一输送泵将所述第一容器中的缓冲液泵送到所述进口主管中，在偶联反应期间，所述第一输送泵将所述第二容器中的第一反应流体泵送到所述进口主管中，所述第二输送泵将所述第三容器中的第二反应流体泵送到所述进口主管中。
  52. 根据权利要求49-51中任一项所述的用途，其特征在于，所述偶联设备还包括：取样检测单元，其与所述流动反应器的出口流体连通，并且被配置为：根据预设的取样时间从流出所述流动反应器的出口的流体中采集样品流体；以及对所述样品流体中的偶联物进行检测，以获得检测结果，检测结果指示偶联物是否达到预设标准。
  53. 根据权利要求52所述的用途，其特征在于，所述偶联设备还包括：循环回收单元，其设置在所述流动反应器的进口与出口之间；当检测结果指示偶联物未达到预设标准时，流体收集单元被配置为停止对流出所述流动反应器的出口的流体的收集，并且，所述循环回收单元被配置为控制流出所述流动反应器的出口的流体重新进入所述流动反应器的进口中，以在所述流动反应器中再次进行偶联反应；和/或
    所述取样检测单元还包括取样泵、第一切换阀、洗脱泵、至少一个分析柱和检测器，所述取样泵经由取样管路连接至所述流动反应器的出口，所述第一切换阀上设有样品环，并且其中，所述第一切换阀根据预设的取样时间在第一状态与第二状态之间切换，在所述第一切换阀处于所述第一状态时，所述取样泵与所述样品环流体连通，并经由取样管路从流出所述流动反应器的出口的流体中采集样品流体并泵送至所述样品环中，并且在所述第一切换阀处于所述第二状态时，所述洗脱泵、样品环、至少一个分析柱和检测器经由检测管路流体连通，所述洗脱泵将洗脱液泵送至所述检测管路中，使所述洗脱液流经所述样品环，从而带动样品环中的样品流体流过所述至少一个分析柱中的一个分析柱后进入所述检测器。
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