Reaction system for preparing bioconjugate and preparation method therefor

Abstract

A reaction system and a method for preparing a bioconjugate. The reaction system comprises a ligase, a targeting molecule, a linker-load, and Ni2+. The preparation method comprises: the ligase catalyzing the targeting molecule and the linker-load to generate the bioconjugate under the action of Ni2+. On the basis of a biological coupling method of a sortase, the reaction system and the preparation method can inhibit reversible reactions, thereby reducing coupling production costs.

Description

A reaction system and preparation method for preparing bioconjugates Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a reaction system and method for preparing bioconjugates. Background Technology

[0002] Enzymatic bioconjugation methods have been increasingly reported and applied (Debon A, Siirola E, Snajdrova R, Enzymatic Bioconjugation: A Perspective from the Pharmaceutical Industry, JACS Au 2023, 3, 1267-1283.). For example, microbial transglutaminase (MTG), glycosyltransferase, and sorting enzymes can all effectively achieve bioconjugation. Among them, sorting enzymes have received more attention and application due to their excellent coupling site selectivity, enzyme catalytic kinetics, wide substrate applicability, and convenient preparation of coupling substrates. However, these enzyme-catalyzed bioconjugation technologies also have certain common problems, such as the reversible control of enzyme-catalyzed reactions, the lack of economic efficiency of excessive substrate feed ratios, and the control of enzyme residues in the liquid phase. These issues will limit their further application (Morgan HE, Turnbull WB, Webb ME, Challenges in the use of sortase and other peptide ligases for site-specific protein modification, Chem. Soc. Rev., 2022, 51, 4121-4145.).

[0003] The coupling reaction mechanism catalyzed by sortase A is as follows: the LPXTGJ sequence (J is a short peptide composed of 0-20 amino acids) is introduced at the end of the biomolecule (the first reactant). This reaction is an equilibrium consisting of forward and reverse reactions. Taking the biocoupled reaction promoted by sortase A of biomolecules containing the common LPXTGG sequence as an example, the enzyme selectively cleaves the peptide bond between TG to form the byproduct GG (diglycine). This byproduct participates in the reversible reaction back to the starting material, which is why the reversible reaction of sortase A-promoted biocoupled reaction is difficult to control.

[0004] To encourage a greater forward reaction, current strategies typically employ an excess of the second reactant (linker-payload). According to Le Chatelier's principle, the reaction will shift towards the forward direction, and with more second reactant, more of the target product can be obtained. However, this method significantly increases the additional costs associated with the incremental second reaction, reducing the overall economics of the conjugation process. This is especially true in the conjugation production of antibody-drug conjugates, where this portion constitutes a significant portion of the cost.

[0005] Another strategy involves altering the short peptide sequence structure, employing the LPXT-G'J short peptide sequence. In this sequence, the T-G' bond is no longer the natural amide bond between T and G, but rather a glycine derivative containing a non-natural ester bond (Williamson DJ, Fascione MA, Webb ME, et al., Efficient N-Terminal Labeling of Proteins by Use of Sortase, Angew. Chem., Int. Ed., 2012, 51, 9377–9380.) or thioester bond, referred to here as G' (Zuo C, Ding R, Wu X, et al., Thioester-Assisted Sortase-A-Mediated). (Ligation, Angew. Chem. Int. Ed. 2022, 61, e202201887.). In this way, the first reactant can still be recognized and cleaved by the sorting enzyme, resulting in the removal of the G'J and successful coupling to the target product. However, the generated byproduct G'J is no longer a substrate for the sorting enzyme and cannot participate in the reversible reaction back to the first reactant, thus inhibiting the reversible reaction. However, this strategy is limited in its direct application to protein molecules because proteins are difficult to express through simple and direct translation to generate LPXT-G'J recognition tags containing non-natural ester or thioester bonds, thus greatly limiting its application.

[0006] This shows that there is an urgent need to find a new bioconjugation method based on sorting enzymes that can effectively inhibit reversible reactions. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, the present invention aims to provide a system and method for reducing coupling production costs while maintaining coupling efficiency, based on the bio-coupling method for sorting enzymes.

[0008] In a first aspect, the present invention provides a reaction system for preparing bioconjugates, wherein the reaction system comprises:

[0009] The ligase contains a sortase sequence.

[0010] Targeted molecules,

[0011] Connector or connector-load, and

[0012] Ni 2+ .

[0013] In some embodiments, the targeting molecule comprises a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence, and the linker or linker-load contains the corresponding ligase acceptor substrate recognition sequence or ligase donor substrate recognition sequence. In some preferred embodiments, the N-terminus or C-terminus of the targeting molecule comprises a ligase donor substrate recognition sequence, and the linker or linker-load contains a ligase acceptor substrate recognition sequence.

[0014] In some embodiments, the C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2; wherein J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H.

[0015] In some embodiments, the ligase is a sortase, a sortase-dehalogenase fusion protein (sortase-Halo), or a mutant thereof; preferably, the sortase is selected from one or more of the following group: sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), and sortase F (SrtF).

[0016] In some embodiments, the sortase enzyme is Ca 2+ Dependency or Ca 2+ Independent. In some embodiments, the sortase enzyme is Ca2+. 2+ The sortase is a sortase-dependent enzyme whose amino acid sequence is as shown in SEQ ID NO:20 or 23, or has at least 85%, 88%, 90%, or 95% sequence identity with SEQ ID NO:20 or 23. In some embodiments, the sortase enzyme is Ca2+. 2+ The independent type has amino acids as shown in SEQ ID NO:21 or 22, or has at least 85%, 88%, 90%, or 95% sequence identity with SEQ ID NO:21 or 22.

[0017] In some embodiments, the sortase enzyme is sortase A, sortase C, or sortase F. In some embodiments, the ligase donor substrate recognition sequence is LPXTGJ1HJ2 (SEQ ID NO:1); X is a natural or non-natural amino acid, preferably E, A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LPXTGGH (SEQ ID NO:2), LPXTGAH (SEQ ID NO:3), or LPXTGGHHHHHH (SEQ ID NO:4). In some preferred embodiments, the ligase donor substrate recognition sequence is LPETGGH (SEQ ID NO:5), LPETGAH (SEQ ID NO:6), or LPETGGHHHHHH (SEQ ID NO:7).

[0018] In some embodiments, the sortase enzyme is sortase B, and the ligase donor substrate recognition sequence is NPXTGJ1HJ2 (SEQ ID NO:8); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is NPXTGGH (SEQ ID NO:9), NPXTGAH (SEQ ID NO:10), or NPXTGGHHHHHH (SEQ ID NO:11).

[0019] In some embodiments, the sortase enzyme is sortase D, and the ligase donor substrate recognition sequence is LPXTAJ1HJ2 (SEQ ID NO:12); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LPXTAGH (SEQ ID NO:13), LPXTAAH (SEQ ID NO:14), or LPXTAGHHHHHH (SEQ ID NO:15).

[0020] In some embodiments, the sortase enzyme is sortase E, and the ligase donor substrate recognition sequence is LAXTGJ1HJ2 (SEQ ID NO:16); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LAXTGGH (SEQ ID NO:17), LAXTGAH (SEQ ID NO:18), or LAXTGGHHHHHH (SEQ ID NO:19).

[0021] In some embodiments, the ligase receptor substrate recognition sequence is (Gly). n G n n is an integer from 1 to 20 or from 2 to 20. In some implementations, n is an integer from 2 to 6.

[0022] In some embodiments, the sortase enzyme is immobilized on a support. In some embodiments, the sortase-Halo is immobilized on a support containing a haloalkyl linker (the sortase enzyme is immobilized on the support via a covalent interaction between the haloalkyl linker and Halo). In some embodiments, the support comprises a chloroalkyl linker, such that the sortase enzyme is immobilized on the support via a covalent interaction between the chloroalkyl linker and Halo.

[0023] In some embodiments, the support has the following structure:

[0024] Where 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; It can be a resin, bead, membrane, gel, matrix, thin film, plate, hole, tube, glass slide, or surface. In some embodiments, It is a resin; more preferably, It can be agarose resin, silicone resin, polymethyl methacrylate resin, or cellulose resin. In some embodiments, It is a highly cross-linked agarose resin or polymethyl methacrylate resin.

[0025] In some embodiments, the ligase immobilized on the support is packed in a pre-packed column. In some embodiments, sortase-Halo immobilized on resin microspheres is packed in a pre-packed column. In some embodiments, the target molecule and linker or linker-load material are passed through a pre-packed column under the catalysis of sortase enzyme to generate a target molecule-linker complex or bioconjugate. The target molecule-linker complex can be linked to the load material to form a bioconjugate through a chemical reaction (such as a click reaction).

[0026] In some embodiments, the Ni in the reaction system 2+ The concentration of Ni is 0.06–30 mM; in some preferred embodiments, the Ni in the reaction system 2+ The concentration is 0.06–15 mM. In some more preferred embodiments, the Ni in the reaction system 2+ The concentration is 0.06–10 mM or 0.1–5 mM.

[0027] In some embodiments, the Ni in the reaction system 2+ The concentration is 0.06–25 mM. In some embodiments, the Ni... 2+ The concentrations are 0.06 mM, 0.1 mM, 0.15 mM, 0.25 mM, 1.0 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 10 mM, 12 mM, 15 mM, 25 mM, or 30 mM, or any range of two values, such as 0.06–0.1 mM, 0.06–0.25 mM, 0.06–1.0 mM, 0.06–2.0 mM. 5mM, 0.06~5mM, 0.1~0.25mM, 0.1~1.0mM, 0.1~2.5mM, 0.1~5mM, 0.1~25mM, 0.25~1mM, 0. 25~2.5mM, 0.25~5mM, 0.25~25mM, 1.0~2.5mM, 1.0~5mM, 1.0~25mM, 2.5~5mM, 2.5~25mM, etc.

[0028] In some preferred embodiments, the Ni in the reaction system 2+ The concentration ranges from 0.25 mM to 5 mM.

[0029] In some implementations, Ni in the system 2+ It is derived from soluble nickel salts. In some preferred embodiments, the soluble nickel salts are nickel sulfate and / or nickel acetate.

[0030] In some embodiments, the targeting molecule is selected from one or more of the group consisting of ligands, aptamers, peptides, antibodies or antigen-binding fragments thereof or antibody mimics.

[0031] In some preferred embodiments, the targeting molecule is an antibody or its antigen-binding fragment. In some preferred embodiments, the targeting molecule is an anti-HER2 antibody or an anti-Trop2 antibody. In some preferred embodiments, the C-terminus and / or N-terminus of the heavy chain and / or light chain of the antibody contains a ligase donor substrate recognition sequence, and the linker or linker-load contains a ligase receptor substrate recognition sequence. In some preferred embodiments, the C-terminus and / or N-terminus of the heavy chain and / or light chain of the antibody contains a ligase receptor substrate recognition sequence, and the linker or linker-load contains a ligase donor substrate recognition sequence.

[0032] In some embodiments, the C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2; wherein J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H.

[0033] In some embodiments, the concentration of the target molecule in the reaction system is 1–60 mg / mL or 0.01–1 mM. In some embodiments, the concentration of the target molecule in the reaction system is 3–40 mg / mL or 0.03–0.6 mM. In some preferred embodiments, the concentration of the target molecule in the reaction system is 5–25 mg / mL or 0.034–0.17 mM.

[0034] In some preferred embodiments, the concentration of the target molecule in the reaction system is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 5.8 mg / mL, 10 mg / mL, 13 mg / mL, 15 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 34 mg / mL, 40 mg / mL, 43 mg / mL, 50 mg / mL, or 60 mg / mL, or any two of the above values, such as 5–10 mg / mL, 5–15 mg / mL, 5–20 mg / mL, 10–15 mg / mL, 10–20 mg / mL, 10–25 mg / mL, 15–20 mg / mL, 15–25 mg / mL, etc.

[0035] In some embodiments, the concentration of the target molecule in the reaction system is 0.01 mM, 0.034 mM, 0.068 mM, 0.102 mM, 0.136 mM, 0.17 mM, 0.2 mM, 0.3 mM, 0.32 mM, 0.41 mM, 0.46 mM, 0.5 mM, 0.55 mM, 0.59 mM, 0.6 mM, 0.7 mM, 0.8 mM, or 1 mM. M, or a range of any two of the above values, such as 0.034–0.068 mM, 0.034–0.102 mM, 0.034–0.136 mM, 0.068–0.102 mM, 0.068–0.136 mM, 0.068–0.17 mM, 0.102–0.17 mM, 0.136–0.17 mM, or 0.102–0.136 mM, etc. In some preferred embodiments, the concentration of the target molecule in the reaction system is 13 mg / mL. In some embodiments, the concentration of the target molecule is 0.088 mM.

[0036] In some embodiments, the loading material is selected from one or more of the group consisting of: small molecule compounds, peptides, polysaccharides, PEG, radionuclides, cytokines, immunomodulators, nucleic acids or analogues thereof, and tracer molecules.

[0037] In some preferred embodiments, the linker is a small molecule compound. In some embodiments, the small molecule compound is a cytotoxin. In some embodiments, the cytotoxin is selected from one or more of the group consisting of: taxanes; maytansines; olprestatins; epothilones; compressoritine A-4 phosphate, compressoritine A-4 and its derivatives; indole-sulfonamides; vinca alkaloids, such as vinca alkaloid, vincristine, vindesine, vinorelbine, vinflunine, vinca glycerol, anhydrous vinca alkaloid; salicylic acid 10 and analogues; leucocelein B; eribulin; indole-3-oxoacetamide; podophyllotoxins; 7- Diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC); Sponge lactone; Lelimycin; Camptothecins and their derivatives; Mitoxantrone; Mitoxantrone hydrazone; Nitrogen mustard; Nitrosoureas; Aziproidines; Benzodopa; Carboquinone; Metoprolol; Uretoprolol; Danonemycin; Esperamycin; Neocarcinone; Aclarubicin; Actinomycin; Atrazomycin; Bleomycin; Actinomycin C; Carrubicin; Erythromycin; Anticancermycin; Erythromycin; Actinomycin D; Normycin; Detoxin; Doxorubicin; Epirubicin; Isorubicin; Idarubicin; Methamphetamine; Mitomycin; Nopramine; Olivycin; Pelomycin; Porphyromycin; Purulentin; Ferrous Oxytocin; Rodoxin; Streptomycin; Streptozotocin; Netostatin; Levorubicin; Trichoderma; T-2 Toxin; Myxomycin A; Bacitracin A; Serpentin; Ubenimex; Azaserine; 6-Diazon-5-oxo-L-leucine; Dimethylfolate; Methotrexate; Pteroxate Trimethoprim; Idatracin; Fludarabine; 6-Mercaptopurine; Thiomipurine; Thioguanine; Ancitabine; Gemcitabine; Enoxabine; Azacitidine; 6-azauridine; Carmoflurane; Cytarabine; Dideoxyuridine; Deoxyfluorouridine; Fluorouracil; Dimethyltestosterone; Drotahistosterone Propionate; Cyclothothermone; Medanone; Testrolide; Aminoglutamate; Mitotan; Tralostan; Flutamide; Nilumet; Bicalutamide; Leuprorelin Acetate; Protein kinase inhibitors; and Proteasome inhibitors. In some embodiments, the cytotoxic agent is selected from vincristine alkaloids, colchicine alkaloids, taxanes, oliquistatins, maytansine, chalcogenide, doxorubicin, pyroxine, SN-38, nodularia cyclic peptide analogs, delutecan, docarmazine, chalcogenide, sintammycin, dolatamine, pyrrolobenzodiazepines, ethatecan, and their derivatives. In some embodiments, the cytotoxic agent is an oliquistatin (such as MMAE, MMAF, or MMAD), ethatecan and its derivatives (such as DX8951f), or a maytansine (such as DM1).

[0038] In some preferred embodiments, the connector-load has the following structure:

[0039] In some embodiments, the N3 and DBCO described above can be linked to the supported derivative via click chemistry. In some embodiments, the supported compound is derivatized to link the click chemical groups N3 or DBCO.

[0040] In some embodiments, the C-terminus of the targeted molecule contains a ligase donor substrate recognition sequence, and the linker or linker-load contains a ligase acceptor substrate recognition sequence. In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or linker-load in the target molecule is greater than or equal to 1:1 in the reaction system. In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or linker-load in the target molecule is (1:1.5) to (1:14). In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or linker-load in the target molecule is 1:1.5, 1:2, 1:3, 1:3.5, 1:3.9, 1:4, 1:5, 1:6, 1:7.2, 1:8, 1:10, 1:12, 1:13, or 1:14. In some embodiments, the molar ratio of the ligase donor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:4) to (1:8), (1:4) to (1:12), (1:4) to (1:20), (1:8) to (1:12), (1:8) to (1:20), or (1:12) to (1:20). In some embodiments, the molar ratio of the ligase donor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:2) to (1:6) or (1:7.5) to (1:14).

[0041] In some embodiments, the C-terminus of the targeted molecule contains a ligase receptor substrate recognition sequence, and the linker or linker-load contains a ligase donor substrate recognition sequence. In some embodiments, the molar ratio of the ligase receptor substrate recognition sequence in the targeted molecule to the linker or linker-load in the reaction system is greater than or equal to 1:1. In some embodiments, the molar ratio of the ligase receptor substrate recognition sequence in the targeted molecule to the linker or linker-load in the reaction system is (1:1.5) to (1:14). In some embodiments, the molar ratio of the ligase receptor substrate recognition sequence in the targeted molecule to the linker or linker-load is 1:1.5, 1:2, 1:3, 1:3.5, 1:3.9, 1:4, 1:5, 1:6, 1:7.2, 1:8, 1:10, 1:12, 1:13, or 1:14. In some embodiments, the molar ratio of the ligase receptor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:4) to (1:8), (1:4) to (1:12), (1:4) to (1:20), (1:8) to (1:12), (1:8) to (1:20), or (1:12) to (1:20). In some embodiments, the molar ratio of the ligase receptor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:2) to (1:6) or (1:7.5) to (1:14).

[0042] In some embodiments, when each target molecule contains one ligase donor substrate recognition sequence (e.g., the target molecule is an scFv, and each scFv is linked to one ligase donor substrate recognition sequence), the molar ratio of the target molecule to the linker or the linker-load is (1:1.5) to (1:14). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:2) to (1:6). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:2, 1:3, 1:3.5, 1:4, 1:5, or 1:6.

[0043] In some embodiments, when each target molecule contains two ligase donor substrate recognition sequences (e.g., the target molecule is an scFv, and each scFv is linked with two ligase donor substrate recognition sequences), the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:4, 1:4.2, 1:4.5, 1:5, 1:5.6, 1:6, 1:6.4, 1:7.3, 1:7.4, 1:8.4, 1:9.1, 1:10, or 1:12.

[0044] In some embodiments, when each target molecule contains two ligase donor substrate recognition sequences (e.g., the target molecule is a complete antibody in a "Y" shape containing two heavy chains and two light chains, each antibody being linked with two ligase donor substrate recognition sequences), the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:4, 1:4.3, 1:5, 1:5.6, 1:6, 1:6.4, 1:7, 1:7.1, 1:8, 1:9, 1:10, or 1:12.

[0045] In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (0.3–20):1. In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (0.5–14):1 or (0.58–12):1. In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (1–6):1. In some preferred embodiments, the molar ratio of ligase to target molecule in the reaction system is 1.45:1, 2.23:1, or 2.91:1. In some embodiments, the ligase is sortase-Halo.

[0046] In some preferred embodiments, the molar ratio of the ligase to the target molecule in the reaction system is 0.3:1, 0.4:1, 0.5:1, 0.58:1, 0.7:1, 0.8:1, 1:1, 1.2:1, 1.3:1, 1.4:1, 1.45:1, 1.6:1, 1.7:1, 1.86:1, 2.0:1, 2.23:1, 2.45:1, 2.91:1, 3.6:1, 4:1, 5:1, 5.82:1, 6:1, 8:1, 9.6:1, 10:1, 11.64:1, 12:1, 13:1, or 16:1. 20:1, or any range of two of the above ratios, such as (0.58:1)~(1.45:1), (0.58:1)~(2.91:1), (0.58:1)~(5.82:1), (0.58:1)~(11.64:1), (1.45:1)~(2.91:1), (1.45:1)~(5.82:1), (1.45:1)~(11.64:1), (2.91:1)~(5.82:1), (2.91:1)~(11.64:1) or (5.82:1)~(11.64:1), etc.

[0047] In a preferred embodiment, the molar ratio of ligase to target molecule in the reaction system is (1.45–11.64):1.

[0048] The ligase of this invention is divided into Ca 2+ Dependent or independent.

[0049] In some embodiments, when the ligase is Ca 2+ In the case of a calcium-dependent reaction, a soluble calcium salt needs to be added to the reaction system, and the Ca in the reaction system... 2+ The concentration is 0.05–20 mM; in some embodiments, the Ca in the reaction system 2+ The concentration is 1–10 mM or 3–8 mM. In some embodiments, the Ca in the reaction system 2+ The concentrations are 0.05 mM, 0.1 mM, 0.2 mM, 1 mM, 2 mM, 2.3 mM, 3 mM, 3.2 mM, 3.6 mM, 4 mM, 4.2 mM, 4.4 mM, 4.8 mM, 5 mM, 5.3 mM, 5.9 mM, 6 mM, 6.4 mM, 6.9 mM, 7 mM, 8 mM, 10 mM, 15 mM, 18 mM, or 20 mM. In some preferred embodiments, the Ca in the reaction system... 2+ The concentration was 5 mM.

[0050] When the ligase is Ca 2+ In the case of the independent reaction, there is no need to add soluble calcium salts to the reaction system.

[0051] In some embodiments, the soluble calcium salt is CaCl2.

[0052] In some implementations, the temperature of the reaction system is 4–40°C.

[0053] In some embodiments, the temperature of the reaction system is 4°C, 10°C, 20°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or 40°C, or any two of the above values, such as 4°C~10°C, 4°C~20°C, 4°C~30°C, 4°C~40°C, 10°C~20°C, 10°C~30°C, 10°C~40°C, 20°C~30°C, 20°C~40°C, or 30°C~40°C, etc.

[0054] In some preferred embodiments, the temperature of the reaction system is 18–26°C.

[0055] In some embodiments, the pH of the reaction system is 6.5 to 8.5.

[0056] In some embodiments, the pH value of the reaction system is 6.5, 6.6, 6.8, 7.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0, or 8.5, or a range of any two of the above values, such as 6.5–7.0, 6.5–7.5, 6.5–8.0, 6.5–8.5, 7.0–7.5, 7.0–8.0, 7.0–8.5, 7.5–8.0, 7.5–8.5, 8.0–8.5, or 7.1–7.8.

[0057] In some preferred embodiments, the pH value of the reaction is 7.5 to 8.0.

[0058] In some embodiments, the reaction system further includes an amino acid-based co-solvent. In a preferred embodiment, the amino acid-based co-solvent is arginine or histidine. In some embodiments, the concentration of arginine or histidine in the reaction system is 10–70 mM. In some embodiments, the concentration of arginine in the reaction system is 10 mM, 12 mM, 17 mM, 20 mM, 22 mM, 24 mM, 28 mM, 30 mM, 32 mM, 37 mM, 41 mM, 43 mM, 45.2 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 57 mM, 58 mM, or 60 mM, or a range of any two of the above values.

[0059] In a second aspect, the above-described reaction system is provided for use in the preparation of bioconjugates, wherein the coupling efficiency of the bioconjugates is at least 75%. In some embodiments, the coupling efficiency of the bioconjugates is at least 82.5%, 85%, or 90%.

[0060] In a third aspect, a method for preparing a bioconjugate is provided, comprising: a ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker or linker-loaded material are used to generate a target molecule-linker complex or bioconjugate. The target molecule-linker complex can be linked with the load to form a bioconjugate. The ligase contains a sortase enzyme sequence. The target molecule contains a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence. The linker or linker-loaded material contains a corresponding ligase acceptor substrate recognition sequence or a ligase donor substrate recognition sequence. The C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2, where J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H.

[0061] The Ni in the reaction system 2+ The concentration of Ni is 0.06–30 mM; preferably, the Ni in the reaction system 2+ The concentration of Ni is 0.06–15 mM; more preferably, the Ni in the reaction system 2+ The concentration is 0.06–10 mM or 0.1–5 mM.

[0062] In some embodiments, the coupling efficiency of the bioconjugate is at least 75%. In some preferred embodiments, the coupling efficiency of the bioconjugate is at least 82.5%, 85%, or 90%.

[0063] In some embodiments, the ligase is a sortase, a sortase-dehalogenase fusion protein (sortase-Halo), or a mutant thereof; preferably, the sortase is selected from one or more of the following group: sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), and sortase F (SrtF).

[0064] In some embodiments, the sortase enzyme is Ca 2+ Dependency or Ca 2+ Independent. In some embodiments, the sortase enzyme is Ca2+. 2+ The sortase is a type of amino acid sequence as shown in SEQ ID NO:20 or 23, or has at least 85%, 88%, 90%, or 95% sequence identity with SEQ ID NO:20 or 23. In some embodiments, the sortase enzyme is Ca2+. 2+The independent type has amino acids as shown in SEQ ID NO:21 or 22, or has at least 85%, 88%, 90%, or 95% sequence identity with SEQ ID NO:21 or 22.

[0065] In some embodiments, the sortase enzyme is sortase A, sortase C, or sortase F. In some embodiments, the ligase donor substrate recognition sequence is LPXTGJ1HJ2 (SEQ ID NO:1); X is a natural or non-natural amino acid, preferably E, A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LPXTGGH (SEQ ID NO:2), LPXTGAH (SEQ ID NO:3), or LPXTGGHHHHHH (SEQ ID NO:4). In some preferred embodiments, the ligase donor substrate recognition sequence is LPETGGH (SEQ ID NO:5), LPETGAH (SEQ ID NO:6), or LPETGGHHHHHH (SEQ ID NO:7).

[0066] In some embodiments, the sortase enzyme is sortase B, and the ligase donor substrate recognition sequence is NPXTGJ1HJ2 (SEQ ID NO:8); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is NPXTGGH (SEQ ID NO:9), NPXTGAH (SEQ ID NO:10), or NPXTGGHHHHHH (SEQ ID NO:11).

[0067] In some embodiments, the sortase enzyme is sortase D, and the ligase donor substrate recognition sequence is LPXTAJ1HJ2 (SEQ ID NO:12); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LPXTAGH (SEQ ID NO:13), LPXTAAH (SEQ ID NO:14), or LPXTAGHHHHHH (SEQ ID NO:15).

[0068] In some embodiments, the sortase enzyme is sortase E, and the ligase donor substrate recognition sequence is LAXTGJ1HJ2 (SEQ ID NO:16); X is a natural or non-natural amino acid, preferably A, R, N, D, Q, I, L, or K; J1 is any one amino acid, preferably G or A; J2 is 0 to 10 amino acids, preferably 0 to 10 H. In some preferred embodiments, the ligase donor substrate recognition sequence is LAXTGGH (SEQ ID NO:17), LAXTGAH (SEQ ID NO:18), or LAXTGGHHHHHH (SEQ ID NO:19).

[0069] In some embodiments, the ligase receptor substrate recognition sequence is (Gly). n G n n is an integer from 1 to 20 or from 2 to 20. In some implementations, n is from 3 to 6.

[0070] In some embodiments, the sortase enzyme is immobilized on a support. In some embodiments, the sortase-Halo is immobilized on a support containing a haloalkyl linker (the sortase enzyme is immobilized on the support via a covalent interaction between the haloalkyl linker and Halo). In some embodiments, the support comprises a chloroalkyl linker, such that the sortase enzyme is immobilized on the support via a covalent interaction between the chloroalkyl linker and Halo.

[0071] In some embodiments, the support has the following structure:

[0072] Where 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; It can be a resin, bead, membrane, gel, matrix, thin film, plate, hole, tube, glass slide, or surface. In some embodiments, It is a resin; more preferably, It can be agarose resin, silicone resin, polymethyl methacrylate resin, or cellulose resin. In some embodiments, It is a highly cross-linked agarose resin or polymethyl methacrylate resin.

[0073] In some embodiments, the ligase immobilized on the support is packed in a pre-packed column. In some embodiments, sortase-Halo immobilized on resin microspheres is packed in a pre-packed column. In some embodiments, the target molecule and linker or linker-load material are passed through a pre-packed column under the catalysis of sortase enzyme to generate a target molecule-linker complex or bioconjugate, which can then be linked to the load to form a bioconjugate.

[0074] In some embodiments, the Ni in the reaction system 2+ The concentration of Ni is 0.06–30 mM; in some preferred embodiments, the Ni in the reaction system 2+ The concentration is 0.06–15 mM. In some more preferred embodiments, the Ni in the reaction system 2+ The concentration is 0.06–10 mM or 0.1–5 mM.

[0075] In some embodiments, the Ni in the reaction system 2+ The concentration is 0.06–25 mM. In some embodiments, the Ni... 2+ The concentrations are 0.06 mM, 0.1 mM, 0.15 mM, 0.25 mM, 1.0 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 10 mM, 12 mM, 15 mM, 25 mM, or 30 mM, or any range of two values, such as 0.06–0.1 mM, 0.06–0.25 mM, 0.06–1.0 mM, 0.06–2.0 mM. 5mM, 0.06~5mM, 0.1~0.25mM, 0.1~1.0mM, 0.1~2.5mM, 0.1~5mM, 0.1~25mM, 0.25~1mM, 0. 25~2.5mM, 0.25~5mM, 0.25~25mM, 1.0~2.5mM, 1.0~5mM, 1.0~25mM, 2.5~5mM, 2.5~25mM, etc.

[0076] In some preferred embodiments, the Ni in the reaction system 2+ The concentration ranges from 0.25 mM to 5 mM.

[0077] In some implementations, Ni in the system 2+It is derived from soluble nickel salts. In some preferred embodiments, the soluble nickel salts are nickel sulfate and / or nickel acetate.

[0078] In some embodiments, the targeting molecule is selected from one or more of the group consisting of ligands, aptamers, peptides, antibodies or antigen-binding fragments thereof or antibody mimics.

[0079] In some preferred embodiments, the targeting molecule is an antibody or its antigen-binding fragment. In some preferred embodiments, the targeting molecule is an anti-HER2 antibody or an anti-Trop2 antibody. In some preferred embodiments, the C-terminus and / or N-terminus of the heavy chain and / or light chain of the antibody contains a ligase donor substrate recognition sequence, and the linker or linker-load contains a ligase acceptor substrate recognition sequence.

[0080] In some embodiments, the C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2; wherein J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H.

[0081] In some embodiments, the concentration of the target molecule in the reaction system is 1–60 mg / mL or 0.01–1 mM. In some embodiments, the concentration of the target molecule in the reaction system is 3–40 mg / mL or 0.03–0.6 mM. In some preferred embodiments, the concentration of the target molecule in the reaction system is 5–25 mg / mL or 0.034–0.17 mM.

[0082] In some preferred embodiments, the concentration of the target molecule in the reaction system is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 5.8 mg / mL, 10 mg / mL, 13 mg / mL, 15 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 34 mg / mL, 40 mg / mL, 43 mg / mL, 50 mg / mL, or 60 mg / mL, or any two of the above values, such as 5–10 mg / mL, 5–15 mg / mL, 5–20 mg / mL, 10–15 mg / mL, 10–20 mg / mL, 10–25 mg / mL, 15–20 mg / mL, 15–25 mg / mL, etc.

[0083] In some embodiments, the concentration of the target molecule in the reaction system is 0.01 mM, 0.034 mM, 0.068 mM, 0.102 mM, 0.136 mM, 0.17 mM, 0.2 mM, 0.3 mM, 0.32 mM, 0.41 mM, 0.46 mM, 0.5 mM, 0.55 mM, 0.59 mM, 0.6 mM, 0.7 mM, 0.8 mM, or 1 mM. M, or a range of any two of the above values, such as 0.034–0.068 mM, 0.034–0.102 mM, 0.034–0.136 mM, 0.068–0.102 mM, 0.068–0.136 mM, 0.068–0.17 mM, 0.102–0.17 mM, 0.136–0.17 mM, or 0.102–0.136 mM, etc. In some preferred embodiments, the concentration of the target molecule in the reaction system is 13 mg / mL. In some embodiments, the concentration of the target molecule is 0.088 mM.

[0084] In some embodiments, the loading material is selected from one or more of the group consisting of: small molecule compounds, peptides, polysaccharides, PEG, radionuclides, cytokines, immunomodulators, nucleic acids or analogues thereof, and tracer molecules.

[0085] In some preferred embodiments, the linker is a small molecule compound. In some embodiments, the small molecule compound is a cytotoxin. In some embodiments, the cytotoxin is selected from one or more of the following: taxanes; maytansines; olprestatins; epothilones; compressoretin A-4 phosphate, compressoretin A-4 and its derivatives; indole-sulfonamides; vinca alkaloids, such as vinca alkaloid, vincristine, vindesine, vinorelbine, vinflunine, vinca glycerol, anhydrous vinca alkaloid; salicylate 10 and analogues; leucocelein B; eribulin; indole-3-oxoacetamide; podophyllotoxins; 7-diethyltoxins. 3-(2'-benzoxazolyl)-coumarin (DBC); spherulone; lelimycin; camptothecins and their derivatives; mitoxantrone; mitoxantrone hydrazone; nitrogen mustards; nitrosoureas; aziridines; benzodopa; carboquinone; metoprolol; urotepiperazine; danendomycin; esperamycin; neomycin; aclarubicin; actinomycin; atrazomycin; bleomycin; actinomycin C; carrubicin; erythromycin; anticancer mycin; erythromycin; actinomycin D; [unclear - possibly a brand name] Mycin; Detoxin; Doxorubicin; Epirubicin; Isorubicin; Idarubicin; Methamphetamine; Mitomycin; Nopramine; Olivycin; Pelomycin; Porphyromycin; Purulin; Ferrous Oxytocin; Rodoxin; Streptomycin; Streptozotocin; Netostatin; Levorubicin; Trichoderma; T-2 Toxin; Myxomycin A; Bacitracin A; Serpentin; Ubenimex; Azaserine; 6-Diazon-5-oxo-L-leucine; Dimethylfolate; Methotrexate; Pteroxate; Trimethoprim; Idatracin; Fludarabine; 6-Mercaptopurine; Thiomipurine; Thioguanine; Ancitabine; Gemcitabine; Enoxabine; Azacitidine; 6-azauridine; Carmoflurane; Cytarabine; Dideoxyuridine; Deoxyfluorouridine; Fluorouracil; Dimethyltestosterone; Drotahistosterone Propionate; Cyclothothermone; Medanone; Testrolide; Aminoglutamate; Mitotan; Tralostan; Flutamide; Nilumet; Bicalutamide; Leuprorelin Acetate; Protein kinase inhibitors; and Proteasome inhibitors. In some embodiments, the cytotoxic agent is selected from vincristine alkaloids, colchicine alkaloids, taxanes, oliquistatins, maytansine, chalcogenide, doxorubicin, pyroxine, SN-38, nodularia cyclic peptide analogs, delutecan, docarmazine, chalcogenide, sintammycin, dolatamine, pyrrolobenzodiazepines, ethatecan, and their derivatives. In some embodiments, the cytotoxic agent is an oliquistatin (such as MMAE, MMAF, or MMAD), ethatecan and its derivatives (such as DX8951f), or a maytansine (such as DM1).

[0086] In some preferred embodiments, the connector-load has the following structure:

[0087] In some embodiments, the N3 and DBCO described above can be linked to the supported derivative via click chemistry. In some embodiments, the supported compound is derivatized to link the click chemical groups N3 or DBCO.

[0088] In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or the linker-load in the target molecule is greater than or equal to 1:1. In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or the linker-load in the target molecule is (1:1.5) to (1:14). In some embodiments, the molar ratio of the ligase donor substrate recognition sequence to the linker or the linker-load in the target molecule is 1:1.5, 1:2, 1:3, 1:3.5, 1:3.9, 1:4, 1:5, 1:6, 1:7.2, 1:8, 1:10, 1:12, 1:13, or 1:14. In some embodiments, the molar ratio of the ligase donor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:4) to (1:8), (1:4) to (1:12), (1:4) to (1:20), (1:8) to (1:12), (1:8) to (1:20), or (1:12) to (1:20). In some embodiments, the molar ratio of the ligase donor substrate recognition sequence in the targeting molecule to the linker or the linker-load is (1:2) to (1:6) or (1:7.5) to (1:14).

[0089] In some embodiments, when each target molecule contains one ligase donor substrate recognition sequence (e.g., the target molecule is an scFv, and each scFv is linked to one ligase donor substrate recognition sequence), the molar ratio of the target molecule to the linker or the linker-load is (1:1.5) to (1:14). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:2) to (1:6). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:2, 1:3, 1:3.5, 1:4, 1:5, or 1:6.

[0090] In some embodiments, when each target molecule contains two ligase donor substrate recognition sequences (e.g., the target molecule is an scFv, and each scFv is linked with two ligase donor substrate recognition sequences), the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:4, 1:4.2, 1:4.5, 1:5, 1:5.6, 1:6, 1:6.4, 1:7.3, 1:7.4, 1:8.4, 1:9.1, 1:10, or 1:12.

[0091] In some embodiments, when each target molecule contains two ligase donor substrate recognition sequences (e.g., the target molecule is a complete antibody in a "Y" shape containing two heavy chains and two light chains, each antibody being linked with two ligase donor substrate recognition sequences), the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). In some embodiments, the molar ratio of the target molecule to the linker or the linker-load is 1:4, 1:4.3, 1:5, 1:5.6, 1:6, 1:6.4, 1:7, 1:7.1, 1:8, 1:9, 1:10, or 1:12.

[0092] In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (0.3–20):1. In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (0.5–14):1 or (0.58–12):1. In some embodiments, the molar ratio of ligase to target molecule in the reaction system is (1–6):1. In some preferred embodiments, the molar ratio of ligase to target molecule in the reaction system is 1.45:1, 2.23:1, or 2.91:1.

[0093] In some preferred embodiments, the molar ratio of the ligase to the target molecule in the reaction system is 0.3:1, 0.4:1, 0.5:1, 0.58:1, 0.7:1, 0.8:1, 1:1, 1.2:1, 1.3:1, 1.4:1, 1.45:1, 1.6:1, 1.7:1, 1.86:1, 2.0:1, 2.23:1, 2.45:1, 2.91:1, 3.6:1, 4:1, 5:1, 5.82:1, 6:1, 8:1, 9.6:1, 10:1, 11.64:1, 12:1, 13:1, or 16:1. 20:1, or any range of two of the above ratios, such as (0.58:1)~(1.45:1), (0.58:1)~(2.91:1), (0.58:1)~(5.82:1), (0.58:1)~(11.64:1), (1.45:1)~(2.91:1), (1.45:1)~(5.82:1), (1.45:1)~(11.64:1), (2.91:1)~(5.82:1), (2.91:1)~(11.64:1) or (5.82:1)~(11.64:1), etc.

[0094] In a preferred embodiment, the molar ratio of ligase to target molecule in the reaction system is (1.45–11.64):1.

[0095] The ligase of this invention is divided into Ca 2+ Dependent or independent.

[0096] In some embodiments, when the ligase is Ca 2+ In the case of a calcium-dependent reaction, a soluble calcium salt needs to be added to the reaction system, and the Ca in the reaction system... 2+ The concentration is 0.05–20 mM; in some embodiments, the Ca in the reaction system 2+ The concentration is 1–10 mM or 3–8 mM. In some embodiments, the Ca in the reaction system 2+ The concentrations are 0.05 mM, 0.1 mM, 0.2 mM, 1 mM, 2 mM, 2.3 mM, 3 mM, 3.2 mM, 3.6 mM, 4 mM, 4.2 mM, 4.4 mM, 4.8 mM, 5 mM, 5.3 mM, 5.9 mM, 6 mM, 6.4 mM, 6.9 mM, 7 mM, 8 mM, 10 mM, 15 mM, 18 mM, or 20 mM. In some preferred embodiments, the Ca in the reaction system... 2+ The concentration was 5 mM.

[0097] When the ligase is Ca 2+ In the case of the independent reaction, there is no need to add soluble calcium salts to the reaction system.

[0098] In some embodiments, the soluble calcium salt is CaCl2.

[0099] In some implementations, the temperature of the reaction system is 4–40°C.

[0100] In some embodiments, the temperature of the reaction system is 4°C, 10°C, 20°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or 40°C, or any two of the above values, such as 4°C~10°C, 4°C~20°C, 4°C~30°C, 4°C~40°C, 10°C~20°C, 10°C~30°C, 10°C~40°C, 20°C~30°C, 20°C~40°C, or 30°C~40°C, etc.

[0101] In some preferred embodiments, the temperature of the reaction system is 18–26°C.

[0102] In some embodiments, the pH of the reaction system is 6.5 to 8.5.

[0103] In some embodiments, the pH value of the reaction system is 6.5, 6.6, 6.8, 7.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0, or 8.5, or a range of any two of the above values, such as 6.5–7.0, 6.5–7.5, 6.5–8.0, 6.5–8.5, 7.0–7.5, 7.0–8.0, 7.0–8.5, 7.5–8.0, 7.5–8.5, 8.0–8.5, or 7.1–7.8.

[0104] In some preferred embodiments, the pH value of the reaction is 7.5 to 8.0.

[0105] In some embodiments, the reaction time of the coupling reaction is 10–150 min. In some preferred embodiments, the reaction time of the coupling reaction is 15–90 min. In some more preferred embodiments, the reaction time of the coupling reaction is 20–60 min. In some most preferred embodiments, the reaction time of the coupling reaction is 20–40 min.

[0106] In some embodiments, the reaction system further includes an amino acid-based co-solvent. In a preferred embodiment, the amino acid-based co-solvent is arginine or histidine. In some embodiments, the concentration of arginine or histidine in the reaction system is 10–70 mM. In some embodiments, the concentration of arginine in the reaction system is 10 mM, 12 mM, 17 mM, 20 mM, 22 mM, 24 mM, 28 mM, 30 mM, 32 mM, 37 mM, 41 mM, 43 mM, 45.2 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 57 mM, 58 mM, or 60 mM, or a range of any two of the above values.

[0107] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0108] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 85%. The coupling reaction must be carried out under the following conditions:

[0109] a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM;

[0110] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1;

[0111] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0112] d) The temperature of the reaction system is 4–40℃;

[0113] e) The pH value of the reaction system is 7.0–8.5;

[0114] f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM.

[0115] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0116] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:5) to (1:12) to form a bioconjugate with a coupling efficiency of at least 87.5%. The coupling reaction must be carried out under the following conditions:

[0117] a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM;

[0118] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1;

[0119] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0120] d) The temperature of the reaction system is 4–40℃;

[0121] e) The pH value of the reaction system is 7.3–8.5;

[0122] f) The Ni in the reaction system 2+ The concentration is 0.25–3 mM.

[0123] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0124] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:7) to (1:12) to form a bioconjugate with a coupling efficiency of at least 90%. The coupling reaction must be carried out under the following conditions:

[0125] a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM;

[0126] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–11.64):1;

[0127] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0128] d) The temperature of the reaction system is 10–40℃;

[0129] e) The pH value of the reaction system is 7.3–7.8;

[0130] f) The Ni in the reaction system 2+ The concentration ranges from 0.25 to 1.9 mM.

[0131] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0132] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 82.5%. The coupling reaction must be carried out under the following conditions:

[0133] a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM;

[0134] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1;

[0135] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0136] d) The temperature of the reaction system is 4–40℃;

[0137] e) The pH value of the reaction system is 7.0–8.5;

[0138] f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM.

[0139] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+ Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0140] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 85%. The coupling reaction must be carried out under the following conditions:

[0141] a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM;

[0142] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1;

[0143] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0144] d) The temperature of the reaction system is 4–40℃;

[0145] e) The pH value of the reaction system is 7.0–8.5;

[0146] f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM;

[0147] g) The reaction system contains 20-70 mM arginine.

[0148] In some specific embodiments, the method for preparing the bioconjugate of the present invention includes: ligase in Ni 2+Under the action of catalysis, the target molecule and the linker-supported material generate bioconjugates, in which,

[0149] The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:8) to (1:12) to form a bioconjugate with a coupling efficiency of at least 89.5%. The coupling reaction must be carried out under the following conditions:

[0150] a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM;

[0151] b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1;

[0152] c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system;

[0153] d) The temperature of the reaction system is 4–40℃;

[0154] e) The pH value of the reaction system is 7.0–8.5;

[0155] f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM;

[0156] g) The reaction system contains 20-70 mM arginine.

[0157] The reaction system of this invention, based on the bioconjugation method of sorting enzymes, effectively inhibits reversible reactions, reduces the production cost of conjugation while maintaining high conjugation efficiency, and has a broader application prospect in industry. Detailed Implementation

[0158] the term

[0159] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. Furthermore, terms and experimental methods relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are widely used terms and common practices in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference. Meanwhile, for a better understanding of this invention, definitions and explanations of relevant terms are provided below.

[0160] As used herein, the expressions “at least one” or “one or more” mean one, two, three, four, five, six, seven, eight, nine or more, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred or more, etc. As used herein, unless explicitly stated otherwise, “a” and “an” shall be understood as “at least one”.

[0161] When a specific quantity, concentration, or other value or parameter is described as a range, preferred range, or preferred upper or lower limit of a numerical value, it should be understood as equivalent to specifically disclosing any range by combining any upper or preferred numerical value with any lower or preferred numerical value, regardless of whether the range is explicitly listed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range. For example, the statement "i" is an integer from 2 to 20 should be understood as i being any integer from 2 to 20, such as i being 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar statements should be understood in a similar manner.

[0162] The terms “about” and “approximately”, when used in conjunction with numerical variables such as concentration, isoelectric point (pI), pH, temperature, or a specific range, typically refer to the fact that the value of the variable and all values ​​of the variable are within the experimental error range (e.g., within the 95% confidence interval of the mean) or within ±10% of the specific value, or a wider range.

[0163] The term “optional” or “optionally” means that the event described thereafter may occur but is not certain to occur; the statement includes the possibility that the event or situation may or may not occur.

[0164] The expression "comprising," or similar expressions such as "including," "containing," and "having," are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus any optional elements, steps, or components, without materially affecting the key and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0165] As used in this article, the definition of "biomolecule" encompasses proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids and their derivatives.

[0166] As used herein, “nucleic acid” or “polynucleotide” refers to a polymer formed by linking at least two nucleotides or nucleotide derivatives together by phosphodiester bonds (such as PO and / or PS), including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0167] As used herein, a nucleic acid “vector” is a medium for transferring exogenous nucleic acids into host cells, whereby the exogenous nucleic acids are amplified or expressed. As used herein, the definition of a nucleic acid “vector” encompasses plasmids (such as linearized plasmids), viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. As used herein, a nucleic acid “vector” capable of expression and / or replication within a host cell means that the vector is capable of expressing RNA polynucleotides or polypeptides and / or producing multiple copies of the vector within the host cell. For it to be “expressible” or “replicable,” a vector may contain nucleic acid sequences or elements operatively linked to a promoter. As used herein, “operatively linked” to a nucleic acid sequence or element means that these nucleic acid sequences are functionally related. For example, a promoter may be operatively linked to a nucleic acid sequence encoding a polypeptide, thereby regulating or mediating the transcription of the nucleic acid. Those skilled in the art can select and use appropriate vectors for specific purposes.

[0168] As used herein, “peptide,” “polypeptide,” or “protein” refers to two or more amino acids covalently linked. Unless otherwise specified, these terms are used interchangeably.

[0169] As used herein, “sequence identity” has the generally accepted meaning in the art, and the percentage of sequence identity between two peptides can be calculated by aligning the two sequences using publicly available algorithms, such as the Local Alignment Search Tool (BLAST) and the Fast Adaptive Shrink / Threshold Algorithm (FASTA) (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994). Although there are various methods to measure the identity between two peptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0170] As used herein, the term "variant" refers to a protein that has one or more residues substituted, deleted, or inserted compared to a reference protein or nucleic acid. The reference protein or nucleic acid can be a naturally occurring protein (i.e., a wild-type protein) or nucleic acid that can be isolated from natural resources, or an engineered protein or nucleic acid. As used herein, the function or activity of a variant, such as a sortase A variant or a Halo variant, is substantially similar to, comparable to, or greater than that of the reference sortase A or Halo, respectively.

[0171] As used herein, the sorting enzymes include, but are not limited to, sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE) or sortase F (SrtF). In this article, “sortase” or “sortase enzyme” refers to an enzyme with sortase activity that catalyzes transpeptidation reactions, including, for example, sortases of class A, B, C, D, E, and F of the sortase superfamily (see, for example, Dramsi, et al., Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria, Research in Microbiology, (2005), 156:289–297; Bradshaw, et al., Molecular features of the sortase enzyme family, FEBS Journal, (2015), 282:2097–2114; Malik and Kim, A comprehensive in silico analysis of sortase superfamily, J Microbiol., (2019), 57(6):431-443; and EP3647419A1), but not limited thereto. Such enzymes may be called SrtA, SrtB, SrtC, SrtD, SrtE, or SrtF, but are not limited thereto. Sortases can be naturally occurring or engineered. Naturally occurring sortases can be found in a variety of Gram-positive bacteria, such as any strain, species, or subspecies of the following genera: Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus argenteus and Staphylococcus aureus), Bacillus (e.g., Bacillus anthracis), and Listeria (e.g., Listeria monocytogenes), but are not limited thereto.Engineered sortases, such as sortase variants having one or more amino acid residue substitutions, deletions, or insertions, can be obtained from their natural counterparts by methods known in the art, such as protein engineering and chemical synthesis. Other variants of any wild-type sortase known in the art (such as those having one or more active groups or tags) are also considered. The prerequisite is that the variant has the same or similar function as the wild-type sortase. Those skilled in the art will be able to readily identify sortases and classify them into specific categories based on their sequence and other characteristics. However, the definition of a sortase is not limited to any classification method or nomenclature system. In some embodiments, sortase enzyme examples include the sortase enzymes disclosed in WO2022160156A or the sortase enzymes shown in Table 1; in Table 1, HHHHHH (SEQ ID NO:24) is the Ni purification tag, GGGGSGGGGS (SEQ ID NO:25) is the adapter sequence, and WSHPQFEKWSHPQFEK (SEQ ID NO:26) is the Strep purification tag.

[0172] Table 1. Sortase enzymes

[0173] In some embodiments, sortase and Halo form a fusion protein via a linker sequence. In some embodiments, the linker sequence is GA, GGGGS (SEQ ID NO:27), or GGGGSGGGGS. In some embodiments, the C-terminus or N-terminus of the target molecule of this application is ligated to a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence via a linker sequence (such as GA, GGGGS, or GGGGSGGGGS).

[0174] As used herein, “Halo” refers to a haloalkane dehalogenase or a variant thereof, which dehalogenates from a haloalkyl substrate (e.g., containing a haloalkyl moiety -(CH2)). 2-30-X reagents, where X is a halogen such as F, Cl, Br, I, especially Cl or Br), remove the halogen and form a covalent bond with the remainder of the substrate. Haloalkane dehalogenases have been described, for example, in WO2006093529 and WO2008 / 054821, the contents of which are incorporated herein by reference. The haloalkane dehalogenases that can be used in this invention may include, but are not limited to, mutants of Xanthobacter dehalogenases (such as Xanthobacter autotrophicus dehalogenase (DhIA)) or Rhodococcus dehalogenases (such as Rhodococcus rhodochrous dehalogenase (DhaA)), such as those containing one or more substitutions at the catalytic triplet residues, such as replacing His272 with Phe / Ala / Gly / Gln / Asn or replacing Asp106 with Cys or other substitutions, as described in WO2008054821. The prerequisite is that the haloalkane dehalogenase or its variants are capable of forming covalent bonds with the haloalkyl substrate.

[0175] In some embodiments, the Halo examples of this application comprise the dehalogenases or variants thereof disclosed in WO2006093529A and WO2008054821A. In some embodiments, the amino acid sequence of Halo is as shown in SEQ ID NO:28 or has at least 85%, 88%, 90%, or 95% identity with SEQ ID NO:28.

[0176] In some embodiments, the amino acid sequence of the sortase-Halo fusion protein is as shown in SEQ ID NO:29 or has at least 85%, 88%, 90%, or 95% identity with SEQ ID NO:29.

[0177] In this article, sortase enzyme, sortase-Halo fusion protein and immobilization can be referenced to patent WO2022160156A. The entire contents of patent WO2022160156A can be incorporated into this article by reference.

[0178] As used herein, the term "targeting molecule" refers to a molecule that has an affinity for a specific target (e.g., receptor, cell surface protein, cytokine, etc.). Targeting molecules are capable of delivering a payload to a specific site in the body via targeted delivery. Targeting molecules can recognize one or more targets. A specific target site is defined by the target it recognizes. For example, a targeting molecule that targets a receptor can deliver a cytotoxin to a site containing a large number of said receptors. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins to a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0179] As used herein, a targeting molecule is a molecule that specifically binds to one or more targets from the following group: PDL1, FRα, CD19, CD20, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KITk, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRvⅢ, SLC44A4 / AGS-5 , CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, E phA2, 5T4, FOLR1, LAMP1, Cadherin6, FGFR2, FGFR3, CA6, CanAg, integrinαV, TDGF1, EphrinA4, TROP2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, Tissue Factor or ROR1 / 2.

[0180] In some preferred embodiments, the targeting molecule is an antibody or its antigen-binding fragment; preferably, the targeting molecule is a monoclonal antibody, a secondary antibody, or a triclonal antibody. In some specific embodiments, the targeting molecule is selected from one or more combinations of the following: anti-PDL1 antibody, anti-FRα antibody, 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 / K antibody. ITk 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-CEACAM antibody 5 Antibodies, including anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-Nectin4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-Mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-Cadherin6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrinαV antibody, anti-TDGF1 antibody, anti-EphrinA4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, and anti-Tissue antibody. Factor antibody or anti-ROR1 / 2 antibody.

[0181] As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. Antibody encompasses antibody fragments. As used herein, the term “antibody” includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), fully humanized antibodies, non-human antibodies, humanized antibodies, single-domain antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, and anti-idiotypic (anti-Id) antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0182] As used herein, an “antibody fragment” or “antigen-binding fragment” refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antigen-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as the derived antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires an antibody that specifically binds to the antigen.

[0183] As used herein, "immunoglobulin single variable domain" or "single variable domain" refers to a single variable region (variable domain) with antigen-binding activity. Unlike conventional antibodies, which consist of a pair of VH and VL to form a functional antigen-binding unit, a single variable domain can form a functional antigen-binding unit independently. Single variable domains can be derived from naturally occurring light-chain-free antibodies, such as the variable domain of heavy chain of heavy-chain antibody (VHH) from camels (e.g., camels and alpacas) and the single variable domain of shark neoantigen receptors (IgNAR variable single-domain, VNAR). They can also be obtained through screening of full-length antibodies, such as the light chain variable domain and heavy chain variable domain with antigen-binding activity in human antibodies. A VHH typically contains three highly variable "complementarity-determining regions (CDRs)" and four relatively conserved "framework regions (FRs)," linked from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0184] As used herein, "single-domain antibody (sdAb)" or "nanobody" refers to an antibody containing a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable region of a full-length antibody, a single variable domain typically includes CDR1, CDR2, and CDR3, which form the antigen-binding site, as well as a supporting framework region. Unlike full-length antibodies, which typically contain two heavy chains and two light chains, single-domain antibodies usually consist of a single peptide chain composed of a single variable domain, with a molecular weight of only about 15 kDa. Examples of single variable domains include the variable domain of a heavy-chain antibody (VHH) from alpacas, the variable domain of IgNAR from sharks, or the variable domain of a human light-chain antibody.

[0185] As used herein, the terms "single-chain antibody," "single-chain Fv," or "scFv" refer to a molecule containing a variable domain (VH) of the antibody heavy chain and a variable domain (VL) of the antibody light chain linked by a linker. Such scFv molecules can have the universal structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0186] In this invention, the amino acid sequences of CDRs are all derived according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242) based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH). Verlag, pp. 95–118), Contact (MacCallum, R.M et al., (1996) J.Mol.Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev.Comp.Immunol., 27, 55-77(2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. In this paper, multiple CDR numbering systems, such as Chothia, Abm, Kabat, Contact, and IMGT, can be used for the same variable region. Those skilled in the art should understand that although CDRs defined by different numbering systems may be different, CDRs corresponding to the same numbering system represent effective antigen-binding sites that can bind antigenic epitopes.Unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described above by any of the known schemes described in this invention. While the scope of protection claimed in the claims of this invention is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0187] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0188] As used herein, the terms “frame region” and “architecture region” are used interchangeably. As used herein, the terms “frame region,” “architecture region,” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR sequence as defined above.

[0189] As used herein, the term "payload" refers to the functional portion contained in a conjugate, for example, linked by a linker. Examples of payloads include, but are not limited to, small molecule compounds (containing small molecule drugs, such as inhibitors and toxins (e.g., cytotoxins), or other non-pharmaceutical small molecules such as click chemical groups, biotin, and other functional molecules), radionuclides (e.g., 18 F, 77 Br、 131 I, 125 I, 43 Sc、 44 Sc、 47 Sc、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 90 In、 111 In、 177 Lu、 94 Tc, 99 Tc, 153 Sm、 89 Sr、 223 Ra、 151 Tb, 166 Ho、 186 Re、 188 Re、 212 Pb, 213 Bi、212 Bi、 225 Ac、 227 Th、 55 Co、 57 Co、 152 Gd, 153 Gd, 157 Gd, 166 Dy、 89 Zr or 211 At; more preferred 68 Ga、 64 Cu or 177 Lu), glycans, PEG moieties, nucleic acids and analogues (e.g., interfering RNA), tracer molecules (e.g., fluorophores and fluorescent molecules), peptides (e.g., protein tags, bioactive peptides, enzymes, antibodies and antibody fragments, and protein toxins), and peptide mimics.

[0190] In some embodiments of the invention, the loading material may be one or more combinations of the following: small molecule drugs, toxins (e.g., cytotoxins), cytokines, immunomodulators (e.g., immune agonists TLR7 / 8, STING), nucleic acids and their analogues (e.g., siRNA, miRNA, ASO, and hnRNA), tracer molecules (e.g., fluorophores and fluorescent molecules), radionuclides, glycans, PEG moieties, macromolecules (e.g., protein toxins and enzymes), etc.

[0191] In some preferred embodiments, the loading material may be one or a combination of the following: cytotoxins and their fragments (e.g., drugs targeting the microtubule cytoskeleton), radionuclides, cytokines, immunomodulators, etc.

[0192] As used herein, the term “coupling” refers to a covalent connection of at least two parts (e.g., at least two molecules or at least two ends of the same molecule).

[0193] As used herein, a “conjugate” can be prepared by covalently linking at least two parts (e.g., at least two molecules or at least two terminal / side chains of the same molecule).

[0194] As used herein, “bioconjugate (XDC)” refers to a conjugate in which at least one of the conjugated parts is a biomolecule. Examples of bioconjugates include therapeutic molecules conjugated to polymers, lipids, antibodies, peptides, aptamers, or small molecule ligands, such as siRNA conjugates, peptide-hormone conjugates, peptide-peptide conjugates, peptide-drug conjugates, antibody-drug conjugates, and multispecific antibodies, etc. In some embodiments, the bioconjugate is an antibody-drug conjugate (ADC) (consisting of a monoclonal antibody with a small molecule cytotoxic drug), an immunostimulant conjugate (AIAC) (consisting of a monoclonal antibody with an immunostimulant), a radionuclide conjugate (RDC) (consisting of a monoclonal antibody with a radionuclide), or an antibody-oligonucleotide conjugate (AOC) (consisting of a monoclonal antibody with an oligonucleotide). As used herein, the terms “activity,” “enzyme activity,” and “catalytic activity” for enzymes (e.g., sortase) refer to the enzyme’s ability to catalyze the conjugation reaction and are used interchangeably.

[0195] In this article, the "molar ratio of the ligase donor substrate recognition sequence to the linker or linker-load material in the target molecule" is m. T / m L Wherein, the number of moles of the ligase donor substrate recognition sequence in the target molecule = the number of moles of the target molecule × the ligase donor substrate recognition sequence carried by each target molecule.

[0196] In some embodiments, the targeting molecule is an scFv, each scFv being linked to one sortase donor substrate recognition sequence. In some embodiments, the targeting molecule is an scFv, each scFv being linked to two sortase donor substrate recognition sequences. In some embodiments, the targeting molecule is a complete antibody in a "Y" shape containing two heavy chains and two light chains, each antibody being linked to two sortase donor substrate recognition sequences. In some embodiments, the targeting molecule is a complete antibody in a "Y" shape containing two heavy chains and two light chains, each antibody being linked to four sortase donor substrate recognition sequences. In some embodiments, the targeting molecule is a complete antibody in a "Y" shape containing two heavy chains and two light chains, each antibody being linked to one sortase donor substrate recognition sequence.

[0197] As used in this text, the conjugation efficiency is the conjugation efficiency of the loading, and the conjugation efficiency = (DAR value / theoretical number of loadings carried by each antibody) × 100%; where DAR is the average number of loadings conjugated to each target molecule in the bioconjugate.

[0198] In some embodiments, the connector-load structure of this application is as shown in equation (I):

[0199] in,

[0200] Q stands for hydrogen, -C2H4-(PEG). t -(CO)NH2 or LKb―P;

[0201] M is hydrogen or LKa-LKb―P; where

[0202] Each LKa is selected independently.

[0203] opSu is or mixtures thereof;

[0204] Each LKb is an independent L 2 ―L 1 ―B;

[0205] Each B is either absent independently, or a combination of the following 1) and 2): 1) self-cleavage of the spacer Sp1; and 2) a chemical bond, or a combination of one, two, or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclic group and -(CO)-; preferably, B is -NH-CH2-U-, or absent, or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-; U is not present, or it is O, S or NH, preferably O or S;

[0206] The condition is that Q and M are not both hydrogen;

[0207] P is the load, which is related to part B or L. 1 Partial connection;

[0208] Each L 1 Independently, it is a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1-10 amino acids;

[0209] Each L 2 Independently, it is a chemical bond; or C 2-20 Alkylenes, wherein one or more -CH2- structures in the alkylene group are optionally modified by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、-N⊕R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10Heterocyclic group, phenylene substitution; wherein the alkylene group, heterocyclic group and phenylene group are each independently unsubstituted or substituted by at least one substituent, the substituent being selected from halogens, -C 1-10 Alkyl, -C 1-10 Halogenated groups, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ;

[0210] Ld2 and each Ld1 are independently chemical bonds; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid or an oligomeric natural amino acid with a degree of polymerization of 2-10, wherein the natural amino acid or oligomeric natural amino acid is independently unsubstituted or has -(PEG) on its side chain. j -R 11 replace;

[0211] -(PEG) t -、-(PEG) i - and -(PEG) j Each of these is a PEG fragment containing a specified number of continuous -(O-C2H4)- or continuous -(C2H4-O)- structural units, each structural unit having an optional additional C at one end. 1- 10 Alkylene;

[0212] R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Each is independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Halogenated, C 4-10 Cycloalkylene; or

[0213] R 1 and R 2 Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl group; or

[0214] R 3 and R 4 Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl group;

[0215] R 11 It is C 1-10 alkyl;

[0216] m is any integer from 1 to 3;

[0217] n is any integer from 2 to 20;

[0218] d is 0, or any integer from 1 to 6;

[0219] Each i is an independent integer from 0 to 100, preferably an integer from 0 to 20; more preferably, each i is an independent integer from 0 to 12; more preferably, an integer from 0 to 8; especially 4;

[0220] Each j is an integer from 1 to 100, preferably an integer from 1 to 20; more preferably, each j is an integer from 1 to 12; more preferably, an integer from 8 to 12; especially 8 or 12.

[0221] Each t is an integer from 1 to 100, preferably an integer from 1 to 20; more preferably, each t is an integer from 1 to 12; more preferably, an integer from 8 to 12; especially 8 or 12.

[0222] The aforementioned connectors and loads can be referenced in application WO2024012566A, the entire contents of which are incorporated herein by reference.

[0223] As used herein, the term "linker" is used to connect a target molecule and a load, for example, to conjugate an antibody to a toxin to form an ADC. Linkers known in the art can be used in this invention.

[0224] The details of the connectors or connector-loads used in this invention can be found in CN115990269A (or WO2023088235A), the entire contents of which are incorporated herein by reference. In some preferred embodiments herein, the connectors may be HX20113 or HX20111 as specified in CN115990269A. In some preferred embodiments herein, the connectors may be connectors H0019, H0005, H0013, H0021, H0034, or H0035 as specified in CN115990269A.

[0225] As used in this article, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge a second thiol group.

[0226] As used herein, the terms "percentage (%) sequence identity," "sequence identity," or "sequence uniformity" have a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences (or nucleic acid sequences) determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0227] Non-critical regions of peptides can be modified, for example by substituting, adding, and / or deleting one or more amino acids, without altering the peptide's function. Suitable conserved amino acid substitutions in peptides or proteins (such as enzymes and antibodies) are known to those skilled in the art and can generally be performed without changing the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a peptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0228] As used in this article, the term "amino acid" includes both "natural amino acids" and "non-natural amino acids".

[0229] The term "natural amino acid" refers to amino acids, which are the building blocks of proteins. These include the twenty common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine ​​and pyrrolidone.

[0230] As used herein, the term "non-natural amino acid" refers to an amino acid that is not a constituent amino acid of a protein. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.

[0231] "Affinity" or "binding affinity" measures the strength of the non-covalent binding between an antibody and an antigen. Affinity can be determined using conventional techniques known in the art, such as biomembrane interferometry (using, for example, the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS). Binding affinity is typically measured by the equilibrium dissociation constant (KD), which is the ratio of the "off-rate" (koff) to the "on-rate" (kon), used to assess and grade the strength of bimolecular interactions. The "on-rate" (kon) characterizes the rate at which the ligand binds to its target, while the "off-rate" (koff) characterizes the rate at which the ligand dissociates from its target. KD (Koff / Kon) and binding affinity are inversely proportional.

[0232] "Specific binding" generally refers to a binding molecule (or targeting molecule), such as an antibody or its fragments, variants, or derivatives, binding to an epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to it more readily through its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to qualitatively analyze the relative affinity of an antibody for a given epitope. For example, binding molecule "A" can be considered to have higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" binds to epitope "C" with higher specificity than it does to related epitope "D."

[0233] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0234] Unless otherwise specified, all raw materials or reagents used in the embodiments and comparative examples of this invention are commercially available products.

[0235] The antibody sequence used is shown below:

[0236] Table 2 Some of the antibodies used in this invention

[0237] Synthesis Examples

[0238] Example 1: Preparation of linker-supported material (compound 1)

[0239] The structure of compound 1 is as follows (its preparation is based on patent CN115990269A):

[0240] Example 2: Preparation of linker-supported material (compound 2)

[0241] The structure of compound 2 is as follows:

[0242] (1) Preparation of compound 2-1

[0243] 2-1 was synthesized using a solid-phase polypeptide synthesis method, with synthetic steps similar to those for LP-6-2 in patent (WO2023232144A). The crude product after cleavage was prepared by Prep-HPLC. After lyophilization, 2-1 was obtained. Mass spectrometry confirmed the structure, C2-1. 76 H 125 N9O 31 2+ [M+2H] 2+ MS(ESI) of 2: Calculated value 829.92, actual value 830.56.

[0244] (2) Preparation of compound 2-2

[0245] Step A: Preparation of compounds 2-2c

[0246] At room temperature, 2-2a (1 g, 1.662 mmol, CAS No.: 159858-22-7) was weighed and placed in a 250 mL round-bottom flask. DMA (N,N-dimethylacetamide, 50 mL) was added, and after dissolution, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) was added, and the mixture was stirred for 2.5 h. HPLC monitoring showed that the reaction of the starting materials was complete. PPTS (pyridine 4-methylbenzenesulfonic acid) was added to the reaction system, and after stirring for 10 min, EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), 2-2b (0.1-10 equivalents of 2-2a, CAS No.: 557756-85-1), and HOBT (1-hydroxybenzotriazole) were added sequentially to the reaction solution, and the mixture was stirred for 12 h until HPLC detected that the reaction was complete. The reaction solution was purified by semi-preparative HPLC to give compound 2-2c (1.3 g, 92% yield, white solid). 44 H 61 N6O 11 + [M+H] + MS(ESI): Calculated value 849.44, actual value 849.33.

[0247] Step B: Preparation of compound 2-2d

[0248] At 0 °C, 2-2c (1.3 g, 1.531 mmol) was placed in a 100 mL round-bottom flask, and DMF (N,N-dimethylformamide, 25 mL) was added. After dissolution, p-(dinitrophenyl) carbonate and DIEA (diethylenetriamine) were added. The mixture was stirred at 0 °C for 0.5 h, then the ice-water bath was removed and the mixture was stirred at room temperature for 12 h. TLC (thin-layer chromatography) monitoring showed that the reaction was complete. Water (200 mL) was added to the reaction system, and the mixture was extracted with EA (ethyl acetate) (150 mL × 3). The organic phase was collected, washed with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, filtered, concentrated, slurried with diethyl ether, filtered again, and the solid was dried under oil pump to give compound 2-2d (1.43 g, 92% yield, white solid). 51 H 64 N7O 15 + [M+H] + MS(ESI): Calculated value 1014.45, actual value 1014.30.

[0249] Step C: Preparation of Compound 2-2

[0250] At room temperature, 2-2d (1.43 g, 1.417 mmol) was placed in a 100 mL round-bottom flask, and DMF (25 mL) was added. After dissolution, HOBT and DIEA were added, and the mixture was stirred for 5 min. Then, 2-2e (0.1-10 equivalents of 2-2d, CAS No.: 474645-27-7) was added, and the mixture was stirred at room temperature for 12 h until the reaction was complete as detected by HPLC. Diethylamine (2.5 mL) was added to the reaction mixture, and the mixture was stirred for 0.5 h until the reaction was complete as detected by HPLC. The reaction mixture was purified by semi-preparative HPLC to give compound 2-2 (856.6 mg, yield 44%, white solid). 69 H 117 N 11 O 17 2+ [M+2H] 2+ MS(ESI) of / 2: Calculated value 685.93, actual value 686.20.

[0251] (3) Preparation of compound 2

[0252] At room temperature, 2-1 (873 mg, 0.282 mmol) and 2-2 (0.1-10 equivalents of 2-1) were placed in a 25 mL round-bottom flask, DMF (8 mL) was added, and the mixture was brought to 0 °C on ice. DIEA was then added, and the mixture was stirred for 5 minutes. HATU was then added, and the mixture was stirred at room temperature for 2 hours until the reaction was complete as detected by HPLC. Diethylamine (800 μL) was added to the reaction mixture, and the mixture was stirred for 0.5 hours until the reaction was complete as detected by HPLC. The reaction mixture was purified by semi-preparative HPLC to give compound 2 (1.2 g, 88.8% yield, white solid). 199 H 346 N 32 O 61 4+ [M+3H+NH4] 4+ MS(ESI) of / 4: Calculated value 1040.13, actual value 1040.89.

[0253] Example 3: Preparation of linker-support (compound 3) (compound P31-DM1)

[0254] The structure of compound 3 is as follows (its preparation is based on patent WO2015165413A):

[0255] Example 4: Preparation of linker-supported material (compound 4)

[0256] The structure of compound 4 is as follows:

[0257] Preparation of compound 4

[0258] At room temperature, 4-1 (100 mg, 0.459 mmol) and 4-2 (1.2 equivalents of 4-1) were placed in a 25 mL round-bottom flask, and DMF (3 mL) was added, followed by DIPEA and HATU. The mixture was reacted at room temperature for 2 h until HPLC detected complete reaction. Diethylamine was then added to the reaction mixture, and the mixture was stirred at room temperature for 0.5 h until HPLC detected complete reaction. The reaction mixture was purified by semi-preparative HPLC to give compound 4 (133 mg, 74.5% yield, colorless oil). 14 H 28 N7O6[M+H] + MS(ESI): Calculated value 390.21, actual value 390.25.

[0259] Example 5: Preparation of linker-supported material (compound 5)

[0260] The structure of compound 5 is as follows:

[0261] (1) Preparation of compound 5-1

[0262] Compound 5-1 was synthesized using a solid-phase polypeptide synthesis method. The crude product after cleavage was purified by semi-preparative HPLC. Compound 5-1 (2.98 g, 90% yield, white solid) was obtained. 32 H 43 N4O 11 [M+H] + MS(ESI): Calculated value 659.29, actual value 659.21.

[0263] (2) Preparation of compound 5

[0264] At room temperature, 5-2 (139 mg, 0.503 mmol) and 5-1 (1.3 equivalents of 5-2) were placed in a 25 mL round-bottom flask, and DMF (4 mL) was added, followed by DIPEA and HATU. The mixture was reacted at room temperature for 2 h until HPLC detected complete reaction. Diethylamine was then added to the reaction mixture, and the mixture was stirred at room temperature for 0.5 h until HPLC detected complete reaction. The reaction mixture was purified by semi-preparative HPLC to give compound 5 (159 mg, 46% yield, white solid). 35 H 47 N6O9[M+H] + MS(ESI): Calculated value 695.34, actual value 695.22.

[0265] Screening Examples

[0266] Screening Example 1: Optimization of Metal Ions

[0267] Antibody sample Ab1 (IgG1) was concentrated to 21.39 mg / mL using a 30 kDa ultrafiltration concentrator, and compound 1 was dissolved to a concentration of 20 mg / mL. The coupling equilibration solution was: 20 mM Tris, 150 mM NaCl, pH 7.2; the calcium chloride stock solution was 0.1 M CaCl2; and the pH adjustment solution was 1 M Tris. 2+ The mother liquor was 10 mM NiSO4, Cu 2+ The mother liquor was 10 mM CuSO4, Fe 3+ The mother liquor was 10 mM FeCl3, Mg 2+ The mother liquor was 10 mM MgSO4, Zn 2+ The mother liquor is 10 mM ZnCl2, Mn 2+ The mother liquor was 10 mM MnCl2.

[0268] Seven experimental groups were set up (group 7 was a negative control). The concentration, temperature, time, pH and reaction volume of each group in the reaction system are shown in Table 3. The antibody concentration was 13 mg / mL (0.088 mmol / L), and the molar ratio of antibody Ab1 to compound 1 was 1:8. The molar ratio of sortase enzyme to antibody was 2.23:1. The concentration of calcium chloride was 5 mM, the concentration of metal ions was 0.2 mM, the pH was 7.2, the temperature was 20-22℃, and the reaction volume was 40 μL. DAR (Drug to Antibody Ratio, the ratio of small molecules to antibodies in the conjugated drug, which measures the conjugation efficiency) was detected by HIC-HPLC. Among them, the sortase enzyme was the sortae-Halo fusion protein shown in SEQ ID NO:29, and sortase-Halo was covalently linked to chlorinated resin (the same as in Example 2 of patent WO2022160156A).

[0269] Table 3 DAR values ​​for different ionic reaction systems

[0270] Based on the experimental results of different metal ions, the DAR values ​​of metal ions Fe, Mg, Zn, and Mn were comparable to those of the negative control group in group 7, with no significant increase. Among them, Zn had a certain inhibitory effect, Cu ions had a certain promoting effect on DAR, and Ni ions significantly improved the coupling reaction efficiency.

[0271] Screening Example 2: pH Optimization

[0272] Five experimental groups were set up, with the formulation referenced to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 13 mg / mL (0.088 mmol / L), the molar ratio of antibody to compound 1 was 1:8; the molar ratio of enzyme to antibody was 2.23:1; the concentration of calcium chloride was 5 mM; the concentration of NiSO4 was 2.5 mM; the temperature was 20–22 °C; and the reaction volume was 400 μL. The reaction DAR value was detected in real time using HIC-HPLC to explore the reaction kinetics under each reaction condition. The results are shown in Table 4.

[0273] Table 4. DAR values ​​of reaction systems at different pH levels

[0274] Based on the pH optimization experiments, within the pH range of 6.5–8.5, the DAR value of this coupling reaction can reach above 3.0 (i.e., coupling efficiency greater than 75%, where coupling efficiency is DAR value / theoretical saturation DAR value (theoretical saturation DAR value is 4)), meeting the quality standards within the ADC drug industry; within the pH range of 7.0–8.5, the coupling efficiency is greater than 85%.

[0275] (DAR value reaches 3.4 or higher); the coupling reaction has the highest DAR value in the pH range of 7.5 to 8.5. Therefore, the preferred pH range is 7.5 to 8.5.

[0276] Screening Example 3: Multifactor Screening Experiment

[0277] Based on the DOE principle, optimization experiments were designed for the main factors. After one round of screening, the relevant factor design ranges are shown in Table 5 below.

[0278] Table 5. Range of multifactor correlation factors

[0279] Sixteen experimental groups were designed using response surface methodology (group 17 was a control experiment). The reaction solution was prepared according to the volumes specified in Table 6 and adjusted to pH 7.2 with pH adjustment solution. The concentration of calcium chloride was 5 mM. The temperature was 20–22 °C, the reaction solution volume was 40 μL, and the enzyme to antibody molar ratio was 2.23:1. This example is the same as the sortase enzyme used in screening example 1.

[0280] Table 6 Multifactor Screening

[0281] The reaction was started and DAR was detected by HIC-HPLC after 120 min. The experimental data were fitted using a model, and DOE analysis showed that the model had a good fit and small error, indicating that the model is highly representative.

[0282] Screening Example 4: Ni Concentration Optimization

[0283] Seven experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: antibody concentration was 13 mg / mL (0.088 mmol / L), the molar ratio of antibody Ab1 to compound 1 was 1:8, the molar ratio of enzyme to antibody was 2.23:1, the concentration of calcium chloride was 5 mM, the pH was 7.5, the temperature was 20–22 °C, and the reaction volume was 400 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 7.

[0284] Table 7 DAR values ​​for different Ni concentrations

[0285] Based on the results of Ni concentration optimization experiments, within the range of 0.1 mM to 25 mM, the DAR value of the coupling reaction can reach above 3.0 (coupling efficiency greater than 75%), meeting the quality standards in the ADC drug industry; within the range of 0.25 mM to 5 mM, the DAR value of the coupling reaction can reach above 3.4 (coupling efficiency greater than 85%). Therefore, Ni... 2+ The preferred range is 0.25mM to 5mM.

[0286] Screening Example 5: Enzyme to Antibody Ratio

[0287] Five experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 13 mg / mL (0.088 mmol / L), the molar ratio of antibody to compound 1 was 1:8, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 1 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 400 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 8.

[0288] Table 8. DAR values ​​for different enzyme to antibody ratios.

[0289] Based on the optimization results of the enzyme-to-antibody ratio experiment, within the molar ratio range of (0.58–11.64):1, the DAR value of the coupling reaction can reach above 3.6 (coupling efficiency greater than 90%), meeting the quality standards in the ADC drug industry; within the range of (1.45–11.64):1, the coupling efficiency is even higher. Therefore, the preferred range for the enzyme-to-antibody molar ratio is (1.45–11.64):1.

[0290] Screening Example 6: Optimization of Different Antibody Concentrations

[0291] Five experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the molar ratio of antibody Ab1 to compound 1 was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 1 mM, the temperature was 20–22 °C, and the pH was 7.5. DAR was detected using HIC-HPLC, and the detection results are shown in Table 9.

[0292] Table 9. DAR values ​​for different antibody concentrations

[0293] Based on the optimization results of different antibody concentrations, within the antibody range of 5–25 mg / mL (0.034–0.170 mmol / L), the DAR value of the coupling reaction gradually increased, and the coupling efficiency was greater than 83.7%; within the antibody range of 10–25 mg / mL (0.068–0.170 mmol / L), the coupling efficiency was greater than 90%. Therefore, the preferred antibody range is 10–25 mg / mL (0.068–0.170 mmol / L).

[0294] Screening Example 7: Optimization of Different Reaction Temperatures

[0295] Five experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody Ab1 to compound 1 was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 1 mM, the pH was 7.5, and the reaction volume was 533.3 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 10.

[0296] Table 10 DAR values ​​at different reaction temperatures

[0297] Based on the results of optimization experiments at different temperatures, the coupling efficiency is greater than 88% in the temperature range of 4 to 40℃, which meets the quality standards in the ADC drug industry; the temperature in the GMP workshop is 18 to 26℃, and the optimal temperature range is 10 to 30℃.

[0298] Screening Example 8: Optimization Experiment of Different Molar Ratios of Antibody to Linker-Loaded Material

[0299] 1) Five experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: antibody Ab1 concentration was 15 mg / mL (0.102 mmol / L), enzyme to antibody molar ratio was 1.45:1, calcium chloride concentration was 5 mM, NiSO4 concentration was 1 mM, temperature was 20–22 °C, pH was 7.5, and reaction volume was 533.3 μL. DAR was detected using HIC-HPLC, and the detection results are shown in Table 11.

[0300] Table 11 DAR values ​​for different molar ratios of antibody to compound 1

[0301] Based on experimental results of different molar ratios of antibody to linker-load material, within the range of (1:3) to (1:20), the DAR value of the coupling reaction was higher than 3.0 (coupling efficiency greater than 75%), meeting the quality standards in the ADC drug industry; within the range of (1:4) to (1:20), the coupling effect was greater than 85%. To reduce the cost of linker-load material, the preferred molar ratio of antibody to linker-load material is (1:4) to (1:8) (i.e., the molar ratio of LPETGGH to linker-load material is (1:2) to (1:4)).

[0302] 2) Three sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody Ab1 to compound 1 was 1:8, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 0.5 mM, the temperature was 18–26 °C, and the pH was 7.5. The DAR values ​​corresponding to reaction times of 20 min and 30 min were 3.7 and 3.8, respectively.

[0303] 3) Prepare the sample for Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system are as follows: the concentration of antibody Ab1 is 15 mg / mL (0.102 mmol / L), the molar ratio of antibody Ab1 to compound 1 is 1:6, the concentration of calcium chloride is 5 mM, the concentration of NiSO4 is 0.5 mM, the temperature is 18–26 °C, and the pH is 7.5. When the reaction time is 20 min, the corresponding DAR value is 3.6.

[0304] Screening Example 9: Testing Coupling Efficiency After Optimization Conditions

[0305] 1) Two sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody Ab1 to compound 1 was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 533.3 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 12.

[0306] Table 12 Comparison of experiments with and without NiSO4

[0307] After multiple rounds of screening, even with a reduced feed ratio (i.e., the molar ratio of target molecules to linker-load material), high coupling efficiency (greater than 90%) can still be obtained.

[0308] 2) Two sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentration, temperature, time, pH, and reaction volume of each group in the reaction system were as follows: the concentration of antibody Ab1 was 23.0 mg / mL, the molar ratio of antibody Ab1 to compound 6 (the difference between compound 6 and compound 1 is that a different topoisomerase is used instead of toxin DXD) was 1:8, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 0.5 mM or 0, the temperature was 18–26 °C, and the pH was 7.5. When the concentration of NiSO4 was 0.5 mM, the DAR value was 3.7; when the concentration of NiSO4 was 0, the DAR value was 2.7.

[0309] Detection Examples

[0310] Detection Example 1: Validation of Different Small Molecules

[0311] Eight experimental groups were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody 2 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody to linker-loaded material was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 533.3 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 13.

[0312] Table 13 DAR values ​​of different small molecules

[0313] Different antibodies and linkers-loaded materials are assembled to carry out bioconjugation reactions with a conjugation efficiency of >90%, which can achieve high conjugation efficiency with low feed ratio.

[0314] Example 2: Validation of different Ni salts

[0315] Two sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody to compound 1 was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 533.3 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 14.

[0316] Table 14 DAR values ​​of different nickel salts

[0317] The type of nickel salt has little effect on coupling efficiency; different sources of Ni... 2+ All can achieve high coupling efficiency with low feed ratios. Example 3: The effect of different sortase enzymes on biocoupling.

[0318] Six experimental groups were set up, with the preparation method referring to Screening Example 1. The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of antibody Ab1 to compound 1 was 1:8, the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 400 μL; among them, groups 1 and 2 used Ca... 2+ The sortase-independent sequence is shown in SEQ ID NO:22 (non-sortase fusion protein). Groups 3 and 4 use Ca... 2+ The sortase-independent sequence is shown in SEQ ID NO:21 (non-sortase fusion protein). Groups 5 and 6 use Ca... 2+ The sortase-dependent enzyme sequence is shown in SEQ ID NO:23 (non-sortase fusion protein). DAR was detected by HIC-HPLC, and the results are shown in Table 15.

[0319] Table 15 DAR values ​​of different sortase enzymes

[0320] The above results confirm that different types of Sortase enzymes, in Ni 2+ High coupling efficiency with low feed ratio can be achieved in the presence of these components.

[0321] Detection Example 4: Ni 2+ The effect of antibody to linker-support molar ratio on coupling efficiency

[0322] Two sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab1 was 15 mg / mL (0.102 mmol / L), the molar ratio of enzyme to antibody was 1.45:1, the concentration of calcium chloride was 5 mM, the temperature was 20–22 °C, the pH was 7.5, and the reaction volume was 533.3 μL. DAR was detected by HIC-HPLC, and the detection results are shown in Table 16.

[0323] Table 16 Ni 2+ The effect of antibody to linker-support molar ratio on coupling efficiency

[0324] The above results indicate that: 1) When each antibody contains two LPETGGH sequences, the conjugation efficiency is greater than 75% when the molar ratio of antibody to linker-loaded material is greater than or equal to 1:3 (i.e., the molar ratio of LPETGGH to linker-loaded material is greater than or equal to 1:1.5); 2) Bioconjugation reactions with a conjugation efficiency of at least 85% require the following conditions: the molar ratio of antibody to linker-loaded material is (1:4) to (1:12) (i.e., the molar ratio of LPETGGH to linker-loaded material is (1:2) to (1:6)), Ni 2+ The concentration is 0.15–5 mM.

[0325] Example 5: Effect of nanobody Ab3 on conjugation efficiency

[0326] 1) Multiple sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme used in this example is the same as that in Screening Example 1). The concentration, temperature, time, pH, and reaction volume of each group in the reaction system were as follows: antibody concentration was 1.44 mg / mL (0.102 mmol / L), enzyme to antibody molar ratio was 1.02:1, calcium chloride concentration was 5 mM, temperature was 22–23 °C, and reaction volume was 280 μL. DAR was detected by LC-MS, and the detection results are shown in Table 17.

[0327] Table 17 Results of the effect of nanobody Ab3 on conjugation efficiency

[0328] Group 3 showed slight turbidity, which could be alleviated by increasing the pH. In Group 4, the addition of arginine not only eliminated turbidity but also improved the coupling efficiency. These results indicate that: 1) When each nanobody contains one LPETGGH sequence, the coupling efficiency is greater than 75% when the molar ratio of antibody to linker-loaded material is greater than or equal to 1:2 (i.e., the molar ratio of LPETGGH to linker-loaded material is greater than 1:1.5); 2) Bioconjugation reactions with a coupling efficiency of at least 82.5% require the following conditions: a molar ratio of antibody to linker-loaded material of (1:4) to (1:12) (i.e., a molar ratio of LPETGGH to linker-loaded material of (1:4) to (1:12)), Ni 2+ The concentration is 0.15–5 mM; 3) Arginine helps alleviate the effect of Ni ions on protein solubility and can also improve coupling efficiency.

[0329] 2) Two sets of experiments were set up, with the preparation method referring to Screening Example 1 (the sortase enzyme in this example is the same as that in Screening Example 1). The concentrations, temperatures, times, pH, and reaction volumes of each group in the reaction system were as follows: the concentration of antibody Ab3 was 1.45 mg / ml, the molar ratio of antibody Ab3 to compound 5 was 1:8, the concentration of calcium chloride was 5 mM, the concentration of NiSO4 was 0.5 mM or 0, the temperature was 18-26℃, and the pH was 8.0. When the concentration of NiSO4 was 0.5 mM, the DAR value was 0.97; when the concentration of NiSO4 was 0, the DAR value was 0.54.

[0330] Comparative Example

[0331] In another embodiment, the coupling reaction is carried out with LPETGG conjugated to the antibody-conjugated terminal, and the coupling efficiency can be improved by adding Tween.

[0332] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.

[0333] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is only defined by the claims.

Claims

A reaction system for preparing bioconjugates, wherein, The reaction system includes: The ligase contains a sortase sequence. Targeted molecules, Connector or connector-load, and In 2+ ; The targeting molecule contains a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence, and the linker or linker-load contains the corresponding ligase acceptor substrate recognition sequence or ligase donor substrate recognition sequence; the C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2, where J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H; The Ni in the reaction system 2+ The concentration of Ni is 0.06–30 mM; preferably, the Ni in the reaction system 2+ The concentration of Ni is 0.06–15 mM; more preferably, the Ni in the reaction system 2+ The concentration is 0.06–10 mM or 0.1–5 mM. According to the reaction system of claim 1, wherein, The target molecule is selected from one or more of the following group: ligand, aptamer, peptide, antibody or antigen-binding fragment thereof or antibody mimic; and / or The loading material is selected from one or more of the group consisting of: small molecule compounds, peptides, polysaccharides, PEG, radionuclides, cytokines, immunomodulators, nucleic acids or analogues thereof, and tracer molecules; and / or The ligase is a sortase or a sortase-dehalogenase fusion protein (sortase-Halo); and / or Ni in the reaction system 2+ Derived from soluble nickel salts; preferably, the nickel salts are nickel sulfate and / or nickel acetate. The reaction system as described in claim 2, wherein, The sortase enzyme is immobilized on a support; preferably, the sortase-Halo is immobilized on a support containing a haloalkyl linker; more preferably, the support contains a chloroalkyl group, such that the sortase enzyme is immobilized on the support through a covalent interaction between the chloroalkyl group and the dehalogenase (Halo). The reaction system according to any one of claims 1 to 3, wherein, The targeting molecule contains a ligase donor substrate recognition sequence at its N-terminus or C-terminus, and the linker or linker-load contains a ligase acceptor substrate recognition sequence; preferably, the ligase donor substrate recognition sequence is LPXTGJ1HJ2, where X is a natural or non-natural amino acid, J1 is any one amino acid, preferably G or A, and J2 consists of 0 to 10 amino acids, preferably 0 to 10 H atoms; preferably, the ligase donor substrate recognition sequence is LPXTGGH, LPXTGAH, or LPXTGGHHHHHH; the ligase acceptor substrate recognition sequence is (Gly). n n is an integer from 2 to 20. The reaction system as described in claim 4, wherein, The molar ratio of the ligase donor substrate recognition sequence in the target molecule to the linker or the linker-load is (1:1.5) to (1:14); preferably, the molar ratio of the ligase donor substrate recognition sequence in the target molecule to the linker or the linker-load is (1:2) to (1:6) or (1:7.5) to (1:14). The reaction system as described in claim 4, wherein, When each of the target molecules contains one ligase donor substrate recognition sequence, the molar ratio of the target molecule to the linker or the linker-load is (1:1.5) to (1:14); preferably, the molar ratio of the target molecule to the linker or the linker-load is (1:2) to (1:6); or When each of the target molecules contains two ligase donor substrate recognition sequences, the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28); preferably, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). The reaction system as described in claim 4, wherein, The concentration of the target molecule in the reaction system is 1–60 mg / mL or 0.01–1 mM; preferably, the concentration of the target molecule in the reaction system is 3–40 mg / mL or 0.03–0.6 mM; more preferably, the concentration of the target molecule in the reaction system is 5–25 mg / mL or 0.034–0.17 mM. The reaction system as described in claim 4, wherein, The molar ratio of ligase to target molecule in the reaction system is (0.3–20):1; preferably, the molar ratio of ligase to target molecule in the reaction system is (0.5–14):1 or (0.58–12):1; more preferably, the molar ratio of ligase to target molecule in the reaction system is (1–6):

1. The reaction system as described in claim 4, wherein, When the ligase is Ca 2+ In the case of a calcium-dependent reaction, a soluble calcium salt needs to be added to the reaction system, and the Ca in the reaction system... 2+ The concentration of Ca is 0.05–20 mM; preferably, the concentration of Ca in the reaction system is... 2+ The concentration is 1–10 mM or 3–8 mM; and / or The temperature of the reaction system is 4–40°C; preferably, the temperature of the reaction system is 10–30°C; and / or The pH of the reaction system is 6.5–8.5; preferably, the pH of the reaction system is 7.0–8.0 or 7.1–7.8; and / or The reaction system further includes an amino acid-based co-solvent; preferably, the amino acid-based co-solvent is arginine or histidine; preferably, the concentration of arginine or histidine in the reaction system is 10–70 mM. The application of the reaction system according to any one of claims 1 to 9 in the preparation of bioconjugates, wherein, The coupling efficiency of the bioconjugate is at least 75%; preferably, the coupling efficiency of the bioconjugate is at least 82.5%, 85% or 90%. A method for preparing a bioconjugate, comprising: Ligase in Ni 2+ Under the action of [a catalyst], a target molecule is catalyzed to form a target molecule-linker complex or bioconjugate with a linker or linker-loaded material. The target molecule-linker complex can be linked with the load to form a bioconjugate. The ligase contains a sortase sequence. The target molecule contains a ligase donor substrate recognition sequence or a ligase acceptor substrate recognition sequence. The linker or linker-loaded material contains a corresponding ligase acceptor substrate recognition sequence or a ligase donor substrate recognition sequence. The C-terminus of the ligase donor substrate recognition sequence contains the amino acid sequence J1HJ2, where J1 is any one amino acid, preferably G or A, and J2 is 0 to 10 amino acids, preferably 0 to 10 H. The Ni in the reaction system [is present in the reaction system]. 2+ The concentration of Ni is 0.06–30 mM; preferably, the Ni in the reaction system 2+ The concentration of Ni is 0.06–15 mM; more preferably, the Ni in the reaction system 2+ The concentration is 0.06–10 mM or 0.1–5 mM. The preparation method according to claim 11, wherein, The coupling efficiency of the bioconjugate is at least 75%; preferably, the coupling efficiency of the bioconjugate is at least 82.5%, 85%, or 90%. The preparation method according to claim 11, wherein, The molar ratio of the ligase donor substrate recognition sequence to the linker or the linker-load in the target molecule is (1:1.5) to (1:14); preferably, the molar ratio of the ligase donor substrate recognition sequence to the linker or the linker-load in the target molecule is (1:2) to (1:6) or (1:7.5) to (1:14). Preferably, when each of the target molecules contains one ligase donor substrate recognition sequence, the molar ratio of the target molecule to the linker or the linker-load is (1:1.5) to (1:14); preferably, the molar ratio of the target molecule to the linker or the linker-load is (1:2) to (1:6). Preferably, when each target molecule contains two ligase donor substrate recognition sequences, the molar ratio of the target molecule to the linker or the linker-load is (1:3) to (1:28); preferably, the molar ratio of the target molecule to the linker or the linker-load is (1:4) to (1:12). The preparation method according to any one of claims 11 to 13, wherein, The target molecule is selected from one or more of the following group: ligand, aptamer, peptide, antibody or antigen-binding fragment thereof or antibody mimic; and / or The loading material is selected from one or more of the group consisting of: small molecule compounds, peptides, polysaccharides, PEG, radionuclides, cytokines, immunomodulators, nucleic acids or analogues thereof, and tracer molecules; and / or The ligase is a sortase or a sortase-dehalogenase fusion protein (sortase-Halo); and / or; Ni in the reaction system 2+ Derived from soluble nickel salts; preferably, the nickel salts are nickel sulfate and / or nickel acetate. The preparation method according to claim 14, wherein, The sortase enzyme is immobilized on a support; preferably, the sortase-Halo is immobilized on a support containing a haloalkyl linker; more preferably, the support contains a chloroalkyl group, such that the sortase enzyme is immobilized on the support through a covalent interaction between the chloroalkyl group and the dehalogenase (Halo). The preparation method according to claim 14, wherein, The targeting molecule contains a ligase donor substrate recognition sequence at its N-terminus or C-terminus, and the linker or linker-load contains a ligase acceptor substrate recognition sequence; preferably, the ligase donor substrate recognition sequence is LPXTGJ1HJ2, where X is a natural or non-natural amino acid, J1 is any one amino acid, preferably G or A, and J2 consists of 0 to 10 amino acids, preferably 0 to 10 H atoms; preferably, the ligase donor substrate recognition sequence is LPXTGGH, LPXTGAH, or LPXTGGHHHHHH; the ligase acceptor substrate recognition sequence is (Gly). n n is an integer from 2 to 20. The preparation method according to claim 14, wherein, The concentration of the target molecule in the reaction system is 1–60 mg / mL or 0.01–1 mM; preferably, the concentration of the target molecule in the reaction system is 3–40 mg / mL or 0.03–0.6 mM; more preferably, the concentration of the target molecule in the reaction system is 5–25 mg / mL or 0.034–0.17 mM. The preparation method according to claim 14, wherein, The molar ratio of ligase to target molecule in the reaction system is (0.3–20):1; preferably, the molar ratio of ligase to target molecule in the reaction system is (0.5–14):1 or (0.58–12):1; more preferably, the molar ratio of ligase to target molecule in the reaction system is (1–6):

1. The preparation method according to claim 14, wherein, When the ligase is Ca 2+ In the case of a calcium-dependent reaction, a soluble calcium salt needs to be added to the reaction system, and the Ca in the reaction system... 2+ The concentration of Ca is 0.05–20 mM; preferably, the concentration of Ca in the reaction system is... 2+ The concentration is 1–10 mM or 3–8 mM; and / or The temperature of the reaction system is 4–40°C; preferably, the temperature of the reaction system is 10–30°C; and / or; The pH of the reaction system is 6.5–8.5; preferably, the pH of the reaction system is 7.0–8.0 or 7.1–7.8; and / or The reaction system further includes an amino acid-based co-solvent; preferably, the amino acid-based co-solvent is arginine or histidine; preferably, the concentration of arginine or histidine in the reaction system is 10–70 mM; and / or The reaction time of the coupling reaction is 10-150 min, preferably 15-90 min, more preferably 20-60 min, and even more preferably 20-40 min. The preparation method according to claim 16, wherein, The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 85%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 4–40℃; e) The pH value of the reaction system is 7.0–8.5; f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM; or The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:5) to (1:12) to form a bioconjugate with a coupling efficiency of at least 87.5%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system (0.58–12):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 4–40℃; e) The pH value of the reaction system is 7.3–8.5; f) The Ni in the reaction system 2+ The concentration is 0.25–3 mM; or The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains two ligase donor substrate recognition sequences. The target molecule and the linker-load material are coupled in a molar ratio of (1:7) to (1:12) to form a bioconjugate with a coupling efficiency of at least 90%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 10–30 mg / mL or 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system is (0.58–11.64):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 10–40℃; e) The pH value of the reaction system is 7.3–7.8; f) The Ni in the reaction system 2+ The concentration ranges from 0.25 to 1.9 mM. The preparation method according to claim 16, wherein, The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 82.5%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 4–40℃; e) The pH value of the reaction system is 7.0–8.5; f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM; or The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:4) to (1:12) to form a bioconjugate with a coupling efficiency of at least 85%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 4–40℃; e) The pH value of the reaction system is 7.0–8.5; f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM; g) The reaction system contains 20–70 mM arginine; or The target molecule is an antibody or antigen-binding fragment, and the C-terminus or N-terminus of the target molecule is linked with a Sortase donor substrate recognition sequence LPXTGJ1HJ2. Each target molecule contains one ligase donor substrate recognition sequence. The target molecule and the linker-load material are coupled in a molar ratio of (1:8) to (1:12) to form a bioconjugate with a coupling efficiency of at least 89.5%. The coupling reaction must be carried out under the following conditions: a) The concentration of the target molecule in the reaction system is 0.068–0.4 mM; b) The molar ratio of ligase to target molecule in the reaction system is (0.58–12):1; c) When the ligase is Ca 2+ When using sortase-dependent enzymes, a soluble calcium salt needs to be added to the reaction system. The amount of Ca in the reaction system... 2+ The concentration is 1–10 mM; or when the ligase is Ca 2+ When using sortase-independent enzymes, soluble calcium salts do not need to be added to the reaction system; d) The temperature of the reaction system is 4–40℃; e) The pH value of the reaction system is 7.0–8.5; f) The Ni in the reaction system 2+ The concentration is 0.15–5 mM; g) The reaction system contains 20-70 mM arginine.

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