Coupling tip and use thereof

Abstract

Provided are a coupling tip and a use thereof. The coupling tip is filled with a ligase that is used for catalyzing the formation of a complex between a targeting molecule and a linker or a linker-payload. A reaction is carried out within the provided coupling tip to generate a conjugated drug XDC. The coupling tip involves simple operation and high efficiency, can lower the technical barrier for experimenters, and allows for on-demand use, thereby saving reagents and minimizing waste.

Description

Coupling gun head and its application TECHNICAL FIELD

[0001] The present application relates to a coupling gun head and its application, in particular to a coupling gun head for preparing a conjugated drug and a coupling box matched therewith, and the application of the coupling gun head and the coupling box in preparing a conjugated drug (especially in early screening of a conjugated drug). BACKGROUND

[0002] Antibody conjugated drugs (ADC) are covalently connected by a linker between a monoclonal antibody targeting specific antigens and a small molecule cytotoxic drug, which has both the strong killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs; ADC can efficiently kill tumor cells, has lower side effects than chemotherapy drugs, and has better efficacy than traditional antibody tumor drugs.

[0003] In order to specifically deliver immune agonists to the tumor microenvironment and trigger local immune activation cascades, antibody-immune agonist conjugated drugs (AIAC, i.e. covalently connecting immune agonists to tumor-targeting antibodies) have become a promising therapy to overcome current cancer immunotherapy resistance mechanisms and immune agonist side effects. The products currently in clinical trials are NJH395 of Novartis, BDC-1001 of Bolt Biotherapeutics and SBT6050 of Silverback Therapeutics, all of which target HER2 and the small molecule compound is a TLR7 / 8 agonist. The above-mentioned AIAC action mechanisms include the following aspects: first, the antibody part inhibits HER2 receptor dimerization and HER2 ectodomain shedding, promotes receptor internalization and / or degradation, inhibits downstream signaling pathways and stimulates antibody-dependent cellular cytotoxicity (ADCC) to kill tumor cells overexpressing HER2; second, antigen-presenting cells (such as macrophages and dendritic cells) directly phagocytose and eliminate tumor cells through antibody-dependent cellular phagocytosis (ADCP); the release of immune agonists in antigen-presenting cells leads to further activation of antigen-presenting cells and enhances antigen presentation; finally, activated antigen-presenting cells can enhance tumor antigen-specific T cell activation, leading to cytotoxic T cell-mediated immune responses against tumor neoantigens.

[0004] Nuclear medicine plays an increasingly significant role in the diagnosis and treatment of cancer due to its potential and advantages of integration of diagnosis and treatment. According to different functions, nuclear medicine can be divided into diagnostic and therapeutic nuclear medicine. The former mainly includes single photon (gamma ray) drugs and positron (beta +Nuclear medicine is a branch of medical science that uses radioactive substances (radiopharmaceuticals) to diagnose and treat diseases. Radiopharmaceuticals are drugs that contain radioactive isotopes (radionuclides) that emit radiation (mainly alpha rays, beta rays, and Auger electrons). They are used in single photon emission computed tomography (SPECT / CT) and positron emission tomography (PET / CT) imaging. At the molecular level, nuclear medicine can study the function and metabolism of radionuclides in the human body to achieve rapid and real-time imaging. Therapeutic nuclear medicine is a type of drug that uses the energy released by radionuclides (mainly alpha rays, beta rays, and Auger electrons) for treatment. Among them, radionuclide drug conjugates (RDC) combine precise targeting and real-time sensitive imaging or powerful killing, bringing more obvious clinical benefits to patients and better achieving precise treatment. Radionuclide drug conjugates (RDC) are usually composed of four parts: targeting ligand, linker, radionuclide, and chelator.

[0005] Antibody-oligonucleotide conjugates (AOC) are a new type of chimeric biomolecule composed of antibodies and oligonucleotides. As a new type of drug, AOC can achieve effective targeted therapy by coupling monoclonal antibodies and oligonucleotides, and has better targeting and pharmacokinetic properties than oligonucleotides.

[0006] XDC is a general term for various conjugate drugs, not limited to ADC, AOC, AIAC, and RDC. It is mainly composed of a targeting molecule, a linker, and a payload. There are many problems in the early screening of XDC: 1) manual operation is cumbersome and the process is complex; 2) there are many impurities in the XDC product, which brings separation problems; 3) it is difficult to efficiently screen suitable XDC molecules. SUMMARY

[0007] In one aspect, the present application provides a conjugate gun head, which is filled with a ligase that catalyzes the formation of a targeting molecule-linker complex or a conjugate drug XDC from a targeting molecule and a linker-payload. The targeting molecule-linker complex can be chemically linked to the payload to form the conjugate drug XDC.

[0008] In some embodiments, the ligase catalyzes the formation of a conjugate drug XDC from a targeting molecule and a linker-payload.

[0009] In some embodiments, the targeting molecule-linker complex can be chemically linked to the payload to form the conjugate drug XDC.

[0010] In some embodiments, the targeting molecule is selected from one or more combinations of the following group: a ligand, a polypeptide, an antibody or an antigen-binding fragment thereof, or an antibody mimetic.

[0011] In some embodiments, the targeting molecule is a specific binding partner for one or more targets in the group of PDL1, FRa, 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, EphA2, 5T4, FOLR1, LAMP1, Cadherin6, FGFR2, FGFR3, CA6, CanAg, integrin aV, TDGF1, EphrinA4, TROP2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, Tissue Factor, or ROR1 / 2.

[0012] In some embodiments, the targeting molecule is an antibody. In some embodiments, the targeting molecule is a mono-, bi- or tri-antibody. In some embodiments, the targeting molecule is selected from the group consisting of one or more of an anti-PDL1 antibody, an anti-FRα antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 / TNFRSF8 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD44v6 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD117 / KITk antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD142 antibody, an anti-CD174 antibody, an anti-CD227 / MUC1 antibody, an anti-CD352 antibody, an anti-CLDN18.2 antibody, an anti-DLL3 antibody, an anti-ErbB2 / HER2 antibody, an anti-CN33 antibody, an anti-GPNMB antibody, an anti-ENPP3 antibody, an anti-Nectin-4 antibody, an anti-EGFRvIII antibody, an anti-SLC44A4 / AGS-5 antibody, an anti-CEACAM5 antibody, an anti-PSMA antibody, an anti-TIM1 antibody, an anti-LY6E antibody, an anti-LIV1 antibody, an anti-Nectin4 antibody, an anti-SLITRK6 antibody, an anti-HGFR / cMet antibody, an anti-SLAMF7 / CS1 antibody, an anti-EGFR antibody, an anti-BCMA antibody, an anti-AXL antibody, an anti-NaPi2B antibody, an anti-GCC antibody, an anti-STEAP1 antibody, an anti-MUC16 antibody, an anti-Mesothelin antibody, an anti-ETBR antibody, an anti-EphA2 antibody, an anti-5T4 antibody, an anti-FOLR1 antibody, an anti-LAMP1 antibody, an anti-Cadherin6 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-CA6 antibody, an anti-CanAg antibody, an anti-integrin αV antibody, an anti-TDGF1 antibody, an anti-EphrinA4 antibody, an anti-TROP2 antibody, an anti-PTK7 antibody, an anti-NOTCH3 antibody, an anti-C4.4A antibody, an anti-FLT3 antibody, an anti-B7H3 / 4 antibody, an anti-Tissue Factor antibody, or an anti-ROR1 / 2 antibody.

[0013] In some embodiments, the payload is selected from the group consisting of one or more of the following: small molecule compounds (e.g., inhibitors, toxins (such as cytotoxins) and polypeptides (e.g., protein tags, bioactive peptides)), glycans, PEG moieties, radionuclides, cytokines, immunomodulators, nucleic acids and analogs thereof (e.g., siRNA, miRNA, ASO and hnRNA), tracer molecules (e.g., fluorophores and fluorescent molecules), macromolecules (e.g., protein toxins and enzymes). In some embodiments, the payload is selected from the group consisting of small molecule compounds, immunomodulators, nucleic acids and analogs thereof, tracer molecules, radionuclides, peptidomimetics, glycans and PEG moieties. In some embodiments, the payload is selected from the group consisting of bioactive peptides, cytokines, antibodies, antibody fragments and protein receptors. In some embodiments, the payload is selected from the group consisting of small molecule compounds, nucleic acid molecules and tracer molecules. In some preferred embodiments, the payload is selected from small molecule compounds. In some embodiments, the payload is selected from the group consisting of cytotoxins and fragments thereof. In some embodiments, the payload is one or more radionuclides. In some embodiments, the payload is one or more cytokines. In some embodiments, the payload is one or more immunomodulators. In some embodiments, the cytotoxin is selected from drugs that target the microtubular cytoskeleton.

[0014] In some preferred embodiments, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and derivatives thereof, indol-sulfonamides, vinca alkaloids (such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine), dolastatin 10 and analogs, halichondrin B and eribulin, indol-3-oxamides, podophyllotoxins, 7-diethylamino-3(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide. In other embodiments, the cytotoxin is a DNA topoisomerase inhibitor, such as camptothecins and derivatives thereof, mitoxantrone, mitoguazone. In some preferred embodiments, the cytotoxin is selected from the group consisting of nitrogen mustards (such as chlorambucil, chlornaphazine, mechlorethamine oxide hydrochloride, novembichin, phenamet, phenesterine), cholophosphamide, estramustine, ifosfamide, melphalan, prednimustine, trofosfamide, uracil mustard. In some preferred embodiments, the cytotoxin is selected from the group consisting of nitrosoureas (such as carmustine, lomustine, nimustine, ranimustine), chloroambucil, formoterol. In some embodiments, the cytotoxin is an aziridine.In some preferred embodiments, the cytotoxin is selected from the group consisting of benzodopa, carboquone, meturedepa, and uredepa.

[0015] In some embodiments, the cytotoxin is an antitumor antibiotic. In some preferred embodiments, the cytotoxin is an enediyne antibiotic. In some selected embodiments, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, and aclacinomycin.

[0016] In some preferred embodiments, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, and cardinophyllin, dactinomycin, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric adriamycin, rodo rubicin, rufocromomycin, streptozocin, zinostatin, zorubicin. In some embodiments, the cytotoxin is a trichothecene. In some embodiments, the cytotoxin is selected from the group consisting of T-2 toxin, verrucarin A, bacillocporin A, and anguidine.

[0017] In some embodiments, the cytotoxin is an antitumor amino acid derivative. In some preferred embodiments, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In some embodiments, the cytotoxin is a folic acid analog. In some preferred embodiments, the cytotoxin is selected from the group consisting of dimethyl fumarate, methotrexate, pteropterin, trimetrexate, and edatrexate. In some embodiments, the cytotoxin is a purine analog. In some preferred embodiments, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, thioguanine. In yet another embodiment, the cytotoxin is a pyrimidine analog. In some preferred embodiments, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine. In some embodiments, the cytotoxin is an androgen. In some preferred embodiments, the cytotoxin is selected from the group consisting of calusterone, dromostanolone propionate, epitiostanol, 5α- reductane, testolactone. In other embodiments, the cytotoxin is an antiadrenaline. In some embodiments, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In some embodiments, the cytotoxin is an antiandrogen. In some embodiments, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin acetate, and goserelin. In some embodiments, the cytotoxin is selected from a protein kinase inhibitor and / or a proteasome inhibitor. In some particular embodiments, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicine, taxoids, auristatins, and maytansinoids.In some embodiments, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D).

[0018] In some embodiments, the radionuclide comprises: 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.

[0019] In some embodiments, the ligase is selected from the group consisting of one or more of a sortase enzyme, a transglutaminase, a formylglycine-generating enzyme, a tyrosinase, an asparagine ligase, and an endoglycosidase.

[0020] In some embodiments, under the action of a ligase, the targeting moiety (Target) forms a complex Target-La or Target-La-P with the linker (La') or the linker-load (La'-P); the targeting moiety contains a ligase recognition substrate, and La' comprises a ligase recognition substrate. In some embodiments, the targeting moiety comprises a ligase donor recognition substrate, and La' comprises a ligase acceptor recognition substrate. In some embodiments, La' comprises a ligase donor recognition substrate, and the targeting moiety comprises a ligase acceptor recognition substrate.

[0021] In some embodiments, the sortase enzyme is sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), or sortase F (SrtF), but not limited to. In some embodiments, the ligase is sortase A, the ligase donor recognition substrate is the sequence LPX1TGX2(SEQ ID NO: 1), wherein X1is any natural or unnatural amino acid, and X2is absent or an amino acid fragment comprising 1-10 amino acids; and the ligase acceptor recognition substrate is the sequence (Gly) m , wherein m is an integer from 2 to 10, such as 2, 3, 4, 5, or 10.

[0022] In some embodiments, the ligase is transglutaminase (TGase), which catalyzes the reaction of glutamine with lysine and its derivatives, and through which the site-specific coupling of the targeting moiety and the linker or linker-load (La' or La'-P) of the present application can be achieved. In some embodiments, the targeting moiety and La' comprise a transglutaminase donor recognition substrate and a transglutaminase acceptor recognition substrate, respectively. In some embodiments, the transglutaminase donor recognition substrate comprises glutamine. In some embodiments, the targeting moiety contains glutamine. In some embodiments, the targeting moiety contains the peptide LLQG (SEQ ID NO: 2). In some embodiments, the transglutaminase acceptor recognition substrate comprises -NH2. In some embodiments, La' comprises -NH2, for example, La' comprises -C 1-10 alkylene-NH2or lysine. In some embodiments, the targeting moiety comprises the LLQG peptide segment, and TGase can specifically recognize the glutamine in the sequence of the LLQG peptide segment, so that the targeting moiety and the linker or linker-load (La' or La'-P) are coupled.

[0023] In some embodiments, the ligase is formylglycine-generating enzyme (FGE), which can specifically recognize a CX3PX4R pentapeptide sequence, where X3and X4are any natural or unnatural amino acid. The FGE replaces the side chain of cysteine with an aldehyde group, which can further react with an FGE acceptor substrate recognition structure to form a stable structure. For example, the aldehyde group reacts with dimethylated 2-(hydrazinomethyl)-3-indole to form a stable chemical bond via a HIPS reaction at near neutral pH. In some embodiments, the targeting moiety comprises the recognition sequence CX3PX4R, L a’ comprises where the wavy line indicates the site of attachment to the rest of La’.

[0024] In some embodiments, the ligase is tyrosinase, which oxidizes tyrosine to 1,2-quinone, which can undergo a cycloaddition reaction with a variety of structures; for example, with various bicyclo[6.1.0]nonyne (BCN) derivatives. In some embodiments, the targeting moiety and L a’ comprise a tyrosinase donor recognition substrate and a tyrosinase acceptor recognition substrate, respectively. In some embodiments, the tyrosinase donor recognition substrate comprises tyrosine. In some embodiments, the targeting moiety comprises tyrosine. In some embodiments, the modified targeting moiety comprises tyrosine. In some embodiments, the tyrosinase acceptor recognition substrate comprises any structure that can undergo a cycloaddition reaction with 1,2-quinone to form a stable product. In some embodiments, L a’ comprises a bicyclo[6.1.0]nonyne.

[0025] In some embodiments, the ligase is an asparagine ligase, such as Singzyme and butelase. Singzyme specifically recognizes a ligase donor recognition substrate NX5L, where X5is any natural or unnatural amino acid, which allows it to undergo ligation with a ligase acceptor recognition sequence GI, mediated by the ligase.

[0026] Butelase is an asparagine ligase derived from Lotus japonicus, which specifically recognizes the Asn-His-Val (NHV) amino acid sequence at the carboxy terminus of a polypeptide and catalyzes the ligation of the Asn residue with any kind of amino acid residue at the amino terminus of the same or another polypeptide to form a peptide bond.

[0027] In some embodiments, the ligase is an endoglycosidase having glycosyltransfer activity. In some embodiments, the endoglycosidase is an N-acetylglucosamine endohydrolase. In some embodiments, the N-acetylglucosamine endohydrolase is at least one selected from the group consisting of Endo S (Endoglycosidase-S, Streptococcus pyogenes endoglycosidase-S), Endo F3 (Endoglycosidase-F3, Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); or the N-acetylglucosamine endohydrolase is at least one selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

[0028] In some embodiments, the method for preparing the conjugated drug is based on site-directed conjugation at the N-glycosylation site of the Fc region, and the method comprises the following steps:

[0029] (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an endoglycosidase, the Fc containing a GlcNAc motif;

[0030] (2) covalently linking the donor containing the oxazoline oligosaccharide to the protein containing the Fc motif by catalysis of the endoglycosidase.

[0031] In some embodiments, the protein containing the Fc region is an antibody. In some embodiments, the protein containing the Fc region is a fusion protein of an antibody fragment and Fc, such as a fusion protein of scFv or VHH and Fc.

[0032] In some embodiments, the donor of the oxazoline oligosaccharide contains or does not contain a payload. In some embodiments, the donor of the oxazoline oligosaccharide without the payload is covalently linked to the protein containing the Fc region, and then covalently linked to the payload.

[0033] In some embodiments, the barrel of the coupling gun head is hollow and has an open upper end and a lower end, the upper end is used to connect to a pipette, and the lower end is provided with a liquid inlet and outlet; the barrel is filled with a ligase, and the barrel is further provided with an upper filter element and a lower filter element, and the ligase is filled in the barrel and located between the upper filter element and the lower filter element.

[0034] In some embodiments, the gun body is tapered from bottom to top, and the upper end of the gun body has an interface for connecting with a pipette. In some embodiments, the upper end of the gun body is snap-connected with a pipette. In some embodiments, the pipette is a manual pipette, an electric pipette, or an automatic pipette; the automatic pipette comprises a servo motor, a seal ring, a piston structure, a lead screw, a Tip head device, a sensor, a driver, and / or a communication module.

[0035] In a more preferred embodiment, the upper end of the gun body has an interface for adapting connection with a pipette.

[0036] In some embodiments, the ligase is selected from the group consisting of one or more combinations of sortase enzyme, transglutaminase, formylglycine-generating enzyme, tyrosinase, asparagine ligase, and endoglycosidase.

[0037] In some embodiments, the ligase is immobilized on a coupling medium. In some embodiments, the coupling medium is selected from the group consisting of one or more combinations of resin (e.g., agarose resin, silicone resin, polymethyl methacrylate resin, epoxy resin, or cellulose resin), gel (e.g., alginate hydrogel), bead / microsphere / particle (e.g., polystyrene bead, magnetic particle), thin film, and matrix. In some embodiments, the coupling medium is selected from the group consisting of one or more combinations of agarose resin, silicone resin, polymethyl methacrylate resin, and cellulose resin. In a specific embodiment, the coupling medium is a highly cross-linked agarose resin.

[0038] In some embodiments, the ligase is immobilized on the coupling medium by adsorption, covalent or non-covalent binding, entrapment, encapsulation, or cross-linking. In some embodiments, the surface of the coupling medium is modified to contain one or more functional groups, so that the ligase can be covalently immobilized on the coupling medium. In some embodiments, the coupling medium contains one or more chemically active functional groups that can form covalent bonds with reactive groups (e.g., amine, thiol, and carboxylate) of the ligase or its fusion protein, or with reactive groups in a haloalkyl substrate, or the coupling medium contains one or more binding partners of corresponding binding tags / affinity labels contained in the ligase fusion protein.

[0039] In some embodiments, the ligase is filled in the gun body in the form of being immobilized in a coupling medium selected from the group consisting of one or more combinations of resin, gel, and matrix; preferably, the coupling medium is selected from the group consisting of one or more combinations of agarose resin, silicone resin, polymethyl methacrylate resin, and cellulose resin.

[0040] In some embodiments, the coupling medium comprises a chemically active functional group that can form a covalent bond with a reactive group on the ligase or fusion protein thereof, such as an amine, thiol, and carboxylate, or with a reactive group in the haloalkyl substrate. In some embodiments, the coupling medium comprises a functional group selected from the group consisting of a cyanate ester, isothiocyanate, isocyanate, carbodiimide, N-hydroxysuccinimide (NHS) ester, amine, carbonate, epoxide, maleimide, haloacetyl, aziridine, chloroformate, and aliphatic aldehyde.

[0041] In some embodiments, the coupling medium is an epoxy-activated resin, a CNBr (cyanogen bromide)-activated resin, or an NHS-activated resin, preferably an epoxy-activated resin. In some embodiments, the coupling medium is an epoxy-activated Sepharose resin, preferably an epoxy-activated highly cross-linked Sepharose resin. In some embodiments, the epoxy-activated resin is pre-treated to introduce an amino group prior to reaction with the haloalkyl substrate.

[0042] In some preferred embodiments, the pre-treatment of the epoxy-activated resin is performed using ammonia. In some preferred embodiments, the pre-treatment of the epoxy-activated resin results in the introduction of an amino group on the oxirane ring and the ring opening of the oxirane ring provides a hydroxyl group. In a particular embodiment, the pre-treatment of the epoxy-activated resin results in the introduction of an amino group on the oxirane ring and the ring opening of the oxirane ring provides a hydroxyl group, which is optionally esterified with an esterifying reagent (e.g., an acetylating reagent, such as Ac2O) in a subsequent procedure for the preparation of the support. Such pre-treated epoxy-activated resin is within the scope of "epoxy-activated resin" as defined above. In some preferred embodiments, the resin is a Sepharose resin (such as a highly cross-linked Sepharose resin) or a polymethyl methacrylate resin.

[0043] In some other embodiments, the coupling medium comprises one or more binding partners of the respective binding tag / affinity tag, which is comprised in the ligase or fusion protein thereof, such as an additional tag or affinity tag. Examples of binding tag / affinity tag and corresponding binding partners can include, but are not limited to, His tag and Ni 2+ , biotin / SPB tag / Strep tag / Strep tag II and streptavidin / avidin / neutravidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca 2+ , MBP and amylose, S tag and RNase S-protein, SNAP tag and benzylguanine (BG) derivative, and CLIP tag and benzylcytosine (BC) derivative.

[0044] In some embodiments, the ligase is a fusion protein, the ligase is covalently linked to a dehalogenase (Halo) or a variant thereof, and is immobilized on a coupling medium containing a haloalkyl linker via the dehalogenase.

[0045] In some embodiments, wherein the Halo is derived from a bacterial haloalkane dehalogenase that catalyzes the dissociation of a halogen from a haloalkyl moiety and forms a covalent bond with the dehalogenated alkyl moiety, and is mutated to prevent hydrolysis of the formed covalent bond. In some embodiments, the dehalogenase is derived from a haloalkane dehalogenase of Xanthobacter autotrophicus or Rhodococcus rhodochrous, wherein a residue involved in hydrolysis is mutated, for example, wherein a histidine residue at position 272 corresponding to the amino acid residue of the Rhodococcus rhodochrous dehalogenase is mutated to form a stable ester intermediate. In some embodiments, the amino acid sequence of the dehalogenase is the sequence set forth in SEQ ID NO: 4 from position 1 to position 297 or has 90% identity compared to the sequence thereof.

[0046] In some embodiments, the ligase is covalently linked to a dehalogenase, i.e., Halo-ligase. In some embodiments, one end of the ligase is covalently linked to a dehalogenase, and the other end is covalently linked to a His tag. In some embodiments, the amino terminus of the ligase is covalently linked to a dehalogenase, and the carboxyl terminus of the ligase is covalently linked to a His tag, i.e., Halo-ligase-His. In some embodiments, there is a linker sequence between the ligase and the dehalogenase, such as GA, GGGGS (SEQ ID NO: 3).

[0047] In some embodiments, the ligase fusion protein is Halo-sortase, Halo-endoglycosidase, Halo-transglutaminase, Halo-formylglycine-generating enzyme, Halo-tyrosinase, Halo-asparagine ligase.

[0048] In some embodiments, the coupling medium comprises a chloroalkyl linker such that the ligase is immobilized on the coupling medium via a covalent interaction between the chloroalkyl linker and the dehalogenase. In some embodiments, the chloroalkyl linker is generated from a chloroalkyl substrate having the structure of Formula (I):

[0049] wherein 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.

[0050] In some embodiments, the coupling medium has the structure of Formula (II):

[0051] wherein 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;

[0052] are resins, gels, films, microspheres, and matrices. In some embodiments, are resin microspheres. In some embodiments, are agarose resins, such as highly cross-linked agarose resin microspheres or polymethylmethacrylate microspheres.

[0053] In some embodiments, the gun body is also filled with a storage solution for maintaining the activity of ligase, which is located between the coupling medium and the upper filter core. The storage solution prevents the coupling medium from drying and the ligase from losing activity. In some embodiments, the storage solution comprises Tris-HCl, NaCl, and the pH value of the storage solution is 6-8. In some embodiments, the storage solution further comprises ethanol or Tween (such as Tween 20 or Tween 80). In some embodiments, the storage solution comprises 10-30 mM Tris-HCl, 110-180 mM NaCl, and the pH value of the storage solution is 6-8. In some embodiments, the concentration of Tris-HCl in the storage solution is 10, 11, 18, 19, 20, 22.5, 24, 25, 27, or 30 mM. In some embodiments, the concentration of NaCl in the storage solution is 110, 111, 118, 120, 121, 122, 122.5, 134, 138, 147, 150, 162, 174, 177, or 180 mM. In some embodiments, the concentration of ethanol in the storage solution is 10%-30% (V / V). In some embodiments, the concentration of ethanol in the storage solution is 10%, 11.4%, 14%, 16%, 18%, 20%, 21.2%, 24%, 28%, or 30%. In some embodiments, the concentration of Tween in the storage solution is 0.001%-1% (W / V). In some embodiments, the concentration of Tween in the storage solution is 0.001%, 0.004%, 0.009%, 0.01%, 0.02%, 0.03%, 0.06%, 0.1%, 0.19%, 0.2%, 0.3%, 0.4%, 0.7%, 0.9%, or 1%.

[0054] In some embodiments, the ligase is a sortase enzyme, and the storage solution comprises: 10-30 mM Tris-HCl, 110-180 mM NaCl, 10-30% ethanol; and the pH of the storage solution is 6-8. In some embodiments, when the ligase is a sortase enzyme, the concentration of Tris-HCl in the storage solution is 10, 11, 18, 19, 20, 22.5, 24, 25, 27, or 30 mM; the concentration of NaCl is 110, 111, 118, 120, 121, 122, 122.5, 134, 138, 147, 150, 162, 174, 177, or 180 mM; the concentration of ethanol is 10%, 11.4%, 14%, 16%, 18%, 20%, 21.2%, 24%, 28%, or 30%; and the pH of the storage solution is 6.1, 6.2, 6.4, 6.6, 6.8, 6.9, 7, 7.1, 7.2, 7,4, 7.6, or 8.

[0055] In some embodiments, the ligase is an endoglycosidase, and the storage solution comprises: 10-30 mM Tris-HCl, 110-180 mM NaCl, 0.001-1% Tween (Tween 20 or Tween 80); and the pH of the storage solution is 6-8. In some embodiments, when the ligase is an endoglycosidase, the concentration of Tris-HCl in the storage solution is 10, 11, 18, 19, 20, 22.5, 24, 25, 27, or 30 mM; the concentration of NaCl is 110, 111, 118, 120, 121, 122, 122.5, 134, 138, 147, 150, 162, 174, 177, or 180 mM; the concentration of Tween is 0.001%, 0.004%, 0.009%, 0.01%, 0.02%, 0.03%, 0.06%, 0.1%, 0.19%, 0.2%, 0.3%, 0.4%, 0.7%, 0.9%, or 1%; and the pH of the storage solution is 6.1, 6.2, 6.4, 6.6, 6.8, 6.9, 7, 7.1, 7.2, 7,4, 7.6, or 8.

[0056] In some embodiments, the maximum range of the coupling gun head is 90 μL-5 mL. In some embodiments, the maximum range of the coupling gun head is 100 μL, 200 μL, and 1 mL.

[0057] In some embodiments, when the maximum range of the coupling gun head is 1000 μL, the volume of the coupling medium (with immobilized ligase) filled in the gun body is 100-180 μL, and the volume of the storage solution filled in the gun body is 550-730 μL. In some embodiments, the volume of the coupling medium filled in the gun body is 100, 120, 122.5, 130, 144, 148, 150, 151, 162, 170.8, or 180 μL. In some embodiments, the volume of the storage solution filled in the gun body is 550, 559, 560, 580, 590, 600, 614, 650, 672, 690, 700, or 730 μL. In some embodiments, when the maximum range of the coupling gun head is 1000 μL, the volume of the coupling medium filled in the gun body is 150 μL, and the volume of the storage solution filled in the gun body is 600 μL. In some embodiments, when the maximum range of the coupling gun head is 1000 μL, the volume of the coupling medium filled in the gun body is 145 μL, and the volume of the storage solution filled in the gun body is 575 μL. In some embodiments, when the maximum range of the coupling gun head is 1000 μL, the volume of the coupling medium filled in the gun body is 165 μL, and the volume of the storage solution filled in the gun body is 605 μL.

[0058] In some embodiments, when the maximum range of the coupling gun head is 200 μL, the volume of the coupling medium filled in the gun body is 15-45 μL, and the volume of the storage solution filled in the gun body is 100-135 μL. In some embodiments, the volume of the coupling medium filled in the gun body is 15, 20, 22.5, 24, 26, 28, 30, 31, 32, 35.8, 38, 40, or 45 μL. In some embodiments, the volume of the storage solution filled in the gun body is 100, 104, 106, 110.2, 115, 119, 120, 121, 122, 126, 130, or 135 μL. In some embodiments, when the maximum range of the coupling gun head is 200 μL, the volume of the coupling medium filled in the gun body is 30 μL, and the volume of the storage solution filled in the gun body is 120 μL. In some embodiments, when the maximum range of the coupling gun head is 200 μL, the volume of the coupling medium filled in the gun body is 35 μL, and the volume of the storage solution filled in the gun body is 115 μL.

[0059] The present application can simultaneously consider the efficient catalysis of the coupling medium immobilized ligase and the preparation of more complex (XDC) under the premise of low amount of ligase by adjusting the volume of the coupling medium and the storage solution through experiments.

[0060] In a preferred embodiment, the upper filter core is fixed in the gun body, the thickness of the upper filter core is 1-5 mm, and the pore size of the filter hole of the upper filter core is 10-100 μm. In some embodiments, the thickness of the upper filter core is 1, 2, 2.4, 3, 3.4, or 5 mm. In some embodiments, the pore size of the filter hole of the upper filter core is 10, 30, 40, 45, 48, 50, 51, 54, 60, 70, 80, or 100 μm. More preferably, the pore size of the filter hole of the upper filter core is 20-80 μm; further 40-60 μm.

[0061] In a preferred embodiment, the lower filter core is fixed in the gun body, the thickness of the lower filter core is 1-5 mm, and the pore size (diameter) of the filter hole of the lower filter core is 10-100 μm. In some embodiments, the thickness of the upper filter core is 1, 2, 2.4, 3, 3.4, or 5 mm. In some embodiments, the pore size of the filter hole of the upper filter core is 10, 30, 40, 45, 48, 50, 51, 54, 60, 70, 80, or 100 μm. More preferably, the pore size of the filter hole of the lower filter core is 30-90 μm; further 50-80 μm.

[0062] In some embodiments, the material of the upper filter core and the lower filter core is hydrophobic. In some embodiments, the material of the upper filter core and the lower filter core is hydrophobic resin. In some embodiments, the material of the upper filter core and the lower filter core is hydrophobic polyethylene resin, in particular ultra-high molecular weight polyethylene.

[0063] In some embodiments, the material of the upper filter core and the lower filter core is hydrophobic polyethylene resin, and the pore size of the upper filter core and the lower filter core is 35-60 μm. By adjusting the material and pore size of the upper filter core and the lower filter core through experiments, the sealing of the storage liquid, the flexible suction and discharge of the liquid by the gun head can be simultaneously considered.

[0064] In a more preferred embodiment, the upper filter core and the lower filter core are fixed on the gun body by ultrasonic welding or interference fit. In some embodiments, a clamping groove is provided inside the gun body, and the upper filter core and the lower filter core are fixed in the clamping groove.

[0065] In a second aspect, the present application also provides a coupling box comprising a plurality of the coupling gun heads.

[0066] In some embodiments, the coupling box comprises a box body and a box cover, the coupling gun heads are arranged in the box body, the upper end and the lower end of the coupling gun head are respectively provided with an upper sealing gasket and a lower sealing gasket, and the upper sealing gasket and the lower sealing gasket seal the coupling gun head to form a sealed space, thereby avoiding or reducing the leakage of the storage liquid or the reaction liquid of the coupling gun head.

[0067] In some embodiments, the upper and lower sealing pads are made of elastic material. In some embodiments, the material of the upper and lower sealing pads is silica gel. In some embodiments, the upper sealing pad is arranged on the inner surface of the box cover, such as fixed on the inner surface of the box cover. In some embodiments, the upper sealing pad is fixed on the inner surface of the box cover by secondary injection molding. In some embodiments, the upper sealing pad is an upper silica gel pad. In some embodiments, the lower surface of the upper silica gel pad has a plurality of downwardly extending protrusions, which are inserted into the upper end of the coupling gun head after the box cover is covered on the box body, and are sealed after being squeezed and deformed.

[0068] In some embodiments, the lower sealing pad is arranged on the bottom of the box body, such as fixed on the inner surface of the bottom of the box body. In some embodiments, the lower sealing pad is a lower silica gel pad. In some embodiments, the upper surface of the lower silica gel pad is formed with a plurality of grooves, and the lower end of the coupling gun head is inserted into the grooves to be wrapped by the groove walls. In some embodiments, the grooves are cylindrical.

[0069] In some embodiments, the lower sealing pad comprises a plurality of integrally formed sealing plugs, each of which is provided with a groove, and each of the sealing plugs corresponds to a coupling gun head, and the lower end of the coupling gun head is inserted into the groove of the corresponding sealing plug to be wrapped by the sealing plug. In some embodiments, the lower sealing pad is composed of a plurality of arrayed sealing plugs. In some embodiments, the grooves are cylindrical.

[0070] In some embodiments, the lower sealing pad is arranged on the bottom of the box body and fixed on the inner surface of the bottom of the box body by secondary injection molding.

[0071] The present application can avoid the leakage of the storage liquid during the transportation of the coupling gun head and the leakage of the reaction liquid in the coupling gun head during the mixing in the mixing instrument by the upper and lower silica gel pads.

[0072] In some embodiments, the box body is provided with a support for accommodating the coupling gun head, and the support is located between the upper and lower sealing pads. In some embodiments, the support is fixedly installed on the box body (such as integrally formed), and the support is provided with a fixing hole slot for accommodating the coupling gun head, and the coupling gun head is sealed by the lower sealing pad after passing through the fixing hole slot. In some embodiments, the support comprises a placement plate fixed in the box body (such as integrally formed), and the placement plate is provided with a fixing hole slot, and the coupling gun head is sealed by the lower sealing pad after passing through the fixing hole slot.

[0073] In some embodiments, the box is provided with a support for placing the coupling gun head, which is located between the upper and lower sealing pads. In some embodiments, the support comprises a base fixedly installed on the box body, and the base is provided with a placement plate, and the base and the placement plate are both provided with fixed holes and grooves, and the coupling gun head is sealed by the lower sealing pad after sequentially passing through the fixed holes and grooves of the placement plate and the base.

[0074] In a preferred embodiment, the box cover is detachably connected with the box body by buckling. In a preferred embodiment, the box cover and the box body are spring-buckled.

[0075] In a preferred embodiment, one or more positioning structures extending upward and downward are arranged on the outer surface of the box body, and the positioning structures comprise positioning grooves or positioning protrusions; and the coupling box is fixed on the mixing instrument through the positioning structures.

[0076] In some embodiments, the coupling box contains a plurality of coupling gun heads, such as 48, 96, 192, or 384.

[0077] In a third aspect, the application also provides a preparation method of a conjugated drug (XDC) using the coupling gun head. The preparation method comprises the following steps: S1, after discharging the storage liquid in the coupling gun head, eluting with an eluent; S2, using the coupling gun head to suck the mixed reaction liquid and contact with the ligase therein, or using the coupling gun head to sequentially suck a plurality of reaction liquids and contact with the ligase therein; S3, continuously mixing, and the ligase catalyzing to form the conjugated drug XDC or the targeting molecule-ligand complex. The targeting molecule-ligand complex can be further chemically connected with the payload to form the XDC.

[0078] In some embodiments, the XDC comprises an ADC, an AOC, an AIAC, and an RDC.

[0079] In some embodiments, the targeting molecule is selected from one or more combinations of the following group: a ligand, a polypeptide, an antibody or an antigen-binding fragment thereof, or an antibody mimetic.

[0080] In some embodiments, the reaction liquid comprises a targeting molecule, a payload, a ligand, a ligand-payload, and / or a combination thereof.

[0081] In some embodiments, the ligase is selected from one or more combinations of the following group: a sortase enzyme, a transglutaminase, a formylglycine-generating enzyme, a tyrosinase, an asparagine ligase, and an endoglycosidase.

[0082] In some embodiments, in step S3, the coupling gun head is loaded into the coupling box and placed in the mixing instrument for mixing.

[0083] In another aspect, the present application also provides the coupling gun head and / or the coupling box for early screening of conjugated drugs (XDC).

[0084] In some embodiments, the coupling gun head sucks and / or discharges reagents by an automatic pipette, and completes the coupling reaction.

[0085] In some embodiments, the automatic pipette comprises a servo motor, a sealing ring, a piston structure, a lead screw, a Tip head device (connecting multiple coupling gun heads at a time), a sensor, a driver and / or a communication module.

[0086] In some embodiments, the coupling box is opened by a mechanical arm, and the automatic pipette sucks and / or discharges reagents, and completes the coupling reaction.

[0087] In some embodiments, the coupling box is opened by a mechanical arm, and the automatic pipette sucks and / or discharges reagents, and completes the coupling reaction.

[0088] In some embodiments, the device further comprises an automatic pipette for sucking and / or discharging reagents, a mechanical arm for opening the box cover in the coupling box, and a vortex mixer. In some embodiments, the automatic pipette comprises a servo motor, a sealing ring, a piston structure, a lead screw, a Tip head device, a sensor, a driver and / or a communication module.

[0089] The present application has the following advantages compared with the prior art by adopting the above scheme:

[0090] 1) The coupling gun head of the present application, wherein a ligase is arranged and can be directly loaded onto a pipette, can conveniently suck a reaction solution into the coupling gun head, mix it with the ligase in the coupling gun head, and react in the coupling gun head to generate a conjugated drug XDC; the operation is convenient, efficient, reduces the technical threshold of experimental personnel, and can be taken at any time, saves reagents and avoids waste;

[0091] 2) The ligase is fixed on a resin microsphere by a dehalogenase, which can experimentally recycle the ligase multiple times, and can avoid contamination of the ligase to the XDC product, thereby reducing the difficulty and cost of XDC separation;

[0092] 3) The storage agent filled in the coupling gun head can prevent the ligase from drying, thereby avoiding the loss of activity of the ligase; when needed, the storage agent can be directly discharged from the liquid outlet at the lower end;

[0093] 4) Coupling gun head and coupling box are matched, and the coupling box can provide good sealing effect for the coupling gun head, so as to reduce or even avoid liquid leakage and pollution in the coupling gun head; thus, the coupling gun head can be further fixed in the coupling box after absorbing the reaction liquid, and the coupling box is placed in the mixing instrument to be fully mixed, so that the preparation of the coupling drug is more convenient, and batch preparation can be performed;

[0094] 5) The XDC technology threshold is high, and it is quite challenging to screen out suitable XDC; the matched coupling gun head and coupling box or the automatic coupling equipment provided in the present application provide convenience and efficiency for early screening of XDC. BRIEF DESCRIPTION OF DRAWINGS

[0095] In order to more clearly illustrate the technical solutions of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0096] Fig. 1 is a sectional view of a coupling gun head according to an embodiment of the present application;

[0097] Fig. 2 is a structural view of a coupling box according to an embodiment of the present application;

[0098] Fig. 3 is an exploded view of a coupling box according to an embodiment of the present application, wherein only one coupling gun head is shown;

[0099] Fig. 4 is a perspective view of a coupling box loaded on a mixing instrument according to an embodiment of the present application.

[0100] Fig. 5 is a front view of a coupling box loaded on a mixing instrument according to an embodiment of the present application.

[0101] Fig. 6 is a sectional view of A-A in Fig. 5.

[0102] In the above drawings, 100 is a coupling gun head; 110 is a gun body; 111 is a liquid inlet and outlet; 112 is a joint; 120 is a coupling medium; 130 is an upper filter element; 140 is a lower filter element; 150 is a storage agent; 200 is a coupling box; 210 is a box body; 211 is a positioning groove; 220 is a box cover; 221 is a buckle; 230 is a lower sealing gasket; 231 is a sealing plug; 240 is an upper sealing gasket; 250 is a support; 300 is a mixing instrument; 301 is a mounting frame; 302 is a rotating shaft; 303 is a rotating bin module; 331 is a rotating bin; 305 is a pressing member; 36 is a timer; and 37 is a speed regulator. DETAILED DESCRIPTION

[0103] The preferred embodiments of the present application will be described in detail below with reference to the drawings. It should be noted that the description of these embodiments is intended to help understand the present application and is not intended to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0104] In the present application, the terms "upper", "lower", and "horizontal" are intended to facilitate understanding of the structure of the coupling gun head by those skilled in the art, and are not intended to limit the state of the coupling gun head after it is installed into the coupling box.

[0105] The terms "first", "second", and the like are used to describe various information, but these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other, and do not indicate a particular order or importance. In fact, the expressions "first", "second", and the like can be used interchangeably. For example, the first information can also be referred to as the second information, and similarly, the second information can also be referred to as the first information without departing from the scope of the present disclosure.

[0106] As shown in the specification and claims, the terms "comprise" and "include" only indicate the inclusion of the steps and elements explicitly identified, and these steps and elements do not constitute an exclusive list, and the method or device can also include other steps or elements.

[0107] As used herein, the expression "at least one" or "one or more" or "a" or "an" means one, two, three, four, five, six, seven, eight, nine, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred, or more, and the like. As used herein, unless specifically indicated to the contrary, "one" and "a" should be understood as "at least one" or "at least one".

[0108] When a particular amount, concentration, or other value or parameter is expressed in a range, a preferred range, or a preferred upper or lower limit, it is understood that the disclosure specifically contemplates the inclusion of any range formed by combining any upper limit or preferred upper limit with any lower limit or preferred lower limit, regardless of whether the range is explicitly listed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints, all integers and fractions within the range, and interval values between any two numerical values. For example, the expression "u is an integer from 1 to 20" should be understood as u being any integer from 1 to 20, for example, u can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions should also be understood in a similar manner.

[0109] The terms "about" and "approximately," when used in connection with a numerical variable, such as a concentration, isoelectric point (pi), pH, temperature, or a particular range, generally mean that the numerical value of the variable and all values within the experimental error (e.g., within the 95% confidence interval of the mean) of the variable or within 10% of the particular value, or a broader range.

[0110] As used herein, "sequence identity" has the art-recognized meaning, and the percent sequence identity between two polypeptides can be calculated by aligning the two sequences using publicly available algorithms, such as the Basic Local Alignment Search Tool (BLAST) and Fast Accurate and Sequence ThresholD Algorithm (FASTA) based on local alignment algorithm search tools (see, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994). While there are a variety of methods for measuring identity between two polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0111] The term "targeting molecule" refers to a molecule that has affinity for a particular target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). A targeting molecule can deliver a payload to a specific site in the body through targeted delivery. A targeting molecule can recognize one or more targets. The specific target 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 receptors. Examples of targeting molecules include, but are not limited to, antibodies, antibody fragments, binding proteins for a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0112] An "amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid, either directly or in the form of a precursor. Individual amino acids are encoded by nucleic acids composed of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The encoding of the same amino acid by different codons is referred to as "degeneracy of the genetic code." Amino acids include natural amino acids and non-natural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0113] The term "antibody" as used herein is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The antibodies can be of any subclass (e.g., IgG, IgE, IgM, IgD, and IgA) or class and can be derived from any suitable species. In some embodiments, the antibodies are of human or murine origin. The antibodies can also be fully human antibodies, humanized antibodies, or chimeric antibodies prepared by recombinant methods.

[0114] The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that the class of a heavy chain includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within each (e.g., γ1-γ4). The "class" of an antibody is determined by the nature of the constant domains of its heavy chains. The immunoglobulin subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and the functional specificities conferred are known. All immunoglobulin classes are within the scope of the disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. IgG generally comprises two identical light chain polypeptides of molecular weight approximately 23,000 daltons and two identical heavy chain polypeptides of molecular weight approximately 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain beginning at the "Y" mouth and continuing around the heavy chain through the variable region.

[0115] Light chains can be classified as kappa (K) or lambda (l). Each heavy chain can be combined with either K or l light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, its light and heavy chains are bound together by covalent bonds, and the "tail" portions of the two heavy chains are bound together by covalent disulfide bonds or non-covalent bonds. In a heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y shape to the C-terminus at the bottom of each chain. The immunoglobulin K light chain variable region is Vκ; the immunoglobulin l light chain variable region is Vλ.

[0116] An antibody fragment can comprise a portion of an intact antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CHI domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs). A Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin, or a fragment of the same structure produced by, for example, recombinant expression. A Fab fragment comprises a light chain (comprising VL and CL) and another chain, wherein the other chain comprises a variable region of a heavy chain (VH) and one constant region of a heavy chain (CHI). An F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment of the same structure produced by, for example, recombinant expression. An F(ab')2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including a cysteine that forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment comprising one half of an F(ab')2 fragment (one heavy chain and one light chain). The antibody fragment can comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single chain Fv (scFv), Fv, dsFv, diabodies, Fd, and Fd' fragments, as well as other fragments, including modified fragments. An antibody fragment generally comprises at least or about 50 amino acids, typically at least or about 200 amino acids. An antigen binding fragment can include any such antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), can obtain an antibody that immunospecifically binds an antigen.

[0117] A small molecule compound refers to a molecule of a size comparable to an organic molecule typically used in a drug. The term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but does encompass low molecular weight peptides or derivatives thereof, e.g., dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, a small molecule compound can have a molecular weight of, e.g., about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 2090 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, about 200 to about 500 Da.

[0118] Cytotoxins are substances that inhibit or prevent cellular expression activity, cellular function, and / or cause cell destruction. Currently, cytotoxins commonly used in ADCs are more toxic than chemotherapy drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinin-mediated protein transport, and regulation of apoptosis. Drugs targeting the microtubule cytoskeleton can be, for example, microtubule stabilizers or microtubule polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of microtubule polymerization inhibitors include, but are not limited to, maytansinoids, orlistatins, vincristine alkaloids, colchicine alkaloids, and sarsaparilla toxins. Drugs targeting DNA can be, for example, drugs that directly disrupt DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt DNA structure include, but are not limited to, DNA double-strand breaker, DNA alkylating agents, and DNA intercalators. DNA double-strand disruptors can be, for example, enediyne antibiotics, including but not limited to danendomycin, esperamycin, neomycin, and uncialamycin. DNA alkylating agents can be, for example, DNA dialkylators (DNA cross-linkers) or DNA monoalkylators. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepines. PBD-like dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole-benzo[e]benzo[b] ... (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2)), camptothecins, and anthracyclines. Drugs targeting RNA can be, for example, drugs that inhibit splicing, including, but not limited to, pladienolide. Drugs targeting kinin-mediated protein transport can be, for example, mitotic kinin inhibitors, including, but not limited to, spindle kinin (KSP) inhibitors.

[0119] The term "linker" is a chemical group used to connect the target molecule and the load in a drug conjugation.

[0120] The term "alkyl linker" is a chemical group used to connect the coupling medium to the dehalogenase.

[0121] The synthesis and immobilization of Sortase enzymes herein can refer to patent WO2022160156A, the entire contents of which are incorporated herein. The synthesis and immobilization of endoglycosidases herein can refer to patent WO2024002330A, the entire contents of which are incorporated herein.

[0122] Example 1

[0123] The present embodiment provides a coupling gun head which can be directly loaded into a pipette (such as a pipette gun or an automatic pipette); the air pressure driving device of the pipette provides positive pressure or negative pressure to suck and discharge reaction solution, storage solution or eluent, etc. Referring to Figure 1, the coupling gun head 100 includes a gun body 110, which is hollow and has an open upper end and a lower end, the lower end of the gun body gradually increases from bottom to top, the upper end is provided with a connector 112 for connecting the pipette, and the lower end is provided with a liquid inlet and outlet 111. The material of the coupling gun head 100 is transparent low adsorption polypropylene resin.

[0124] The structure or substance in the gun body 110 from top to bottom is: the connector 112, the upper filter core 130, the storage solution 150, the coupling medium 120 (fixed with ligase), the lower filter core 140 and the liquid inlet and outlet 111. The upper filter core 130 and the lower filter core 140 are fixed in the gun body 110 by interference fit, and the material of the upper filter core 130 and the lower filter core 140 is polyethylene resin, the thickness is 2.6-3.2 μm, and the pore size of the filter hole is 45-56 μm.

[0125] The coupling medium 120 is fixed with ligase, and the coupling medium 120 has the structure of formula (II):

[0126] Wherein u is 3, v is 2, and w is 5;

[0127] is a highly cross-linked agarose resin microsphere.

[0128] If the coupling medium 120 is immobilized with a dehalogenase-sortase fusion protein, its amino acid sequence is MAEIGTGFPFDHPHVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRN IIPHVAPTHR CIAPDLIGMG KSDKPDLGYFFDDHVRFMDA FIEALGLEEV VLVIHDWGSA LGFHWAKRNP ERVKGIAFME FIRPIPTWDE WPEFARETFQ AFRTTDVGRKLIIDQNVFIE GTLPMGVVRP LTEVEMDHYR EPFLNPVDRE PLWRFPNELP IAGEPANIVA LVEEYMDWLH QSPVPKLLFWGTPGVLIPPA EAARLAKSLP NCKAVDIGPG LNLLQEDNPD LIGSEIARWL STLEISGGGG GSGGGGSAKP QIPKDKSKVAGYIEIPDADI KEPVYPGPAT SEQLNRGVSF AEENESLDDQ NISIAGHTFI DRPNYQFTNL KAAKKGSMVY FKVGNETRKYKMTSIRNVKP TAVGVLDEQK GKDKQLTLIT CDDYNEKTGV WETRKIFVAT EVK (SEQ ID NO: 4). The storage solution comprises 20 mM Tris-HCl, 150 mM NaCl and 20% ethanol, and the pH value of the storage solution is 7.2.

[0129] This embodiment provides a coupling gun head 100 with a maximum range of 200 μL, the volume of the coupling medium (immobilized with srotase ligase) filled in the gun body 110 is 30 μL, and the volume of the storage solution filled in the gun body is 120 μL. Here, the "maximum range" refers to the maximum volume of the substance allowed to be added to the coupling gun head 100, which should be less than the volume of the cavity 110.

[0130] This embodiment also provides a coupling gun head 100 with a maximum range of 1000 μL, the volume of the coupling medium (immobilized with srotase ligase) filled in the gun body 110 is 150 μL, and the volume of the storage solution filled in the gun body is 600 μL.

[0131]

[0132] Embodiment 2

[0133] Figs. 2-3 show a coupling box 200 according to the present embodiment, which not only serves as a packaging container for the coupling gun head 100, but also can serve as a sealing and fixing device for the coupling gun head 100 after the coupling gun head 100 is filled with the reaction solution, facilitating subsequent mixing operation and the like, and in particular, facilitating synchronous processing of a batch of coupling gun heads 100. Referring to Figs. 2 and 3, the coupling box 200 includes a plurality of the above-described coupling gun heads 100 (e.g., 96 coupling gun heads 100) arranged in the interior thereof.

[0134] The coupling box 200 includes a box body 210 and a box cover 220, the coupling gun head 100 is arranged in the box body 210, and the coupling box 200 further includes a lower sealing gasket 230 arranged between the lower end of the gun body 110 and the bottom of the box body 210, and an upper sealing gasket 240 arranged between the upper end of the gun body 110 and the box cover 220, the gun body 110 is compressed between the upper sealing gasket 240 and the lower sealing gasket 230 to avoid or reduce liquid leakage in the gun body 110. Further, the coupling box 200 further includes a bracket 250 installed in the box body 210, the bracket 250 is located between the upper sealing gasket 240 and the lower sealing gasket 230.

[0135] In the present embodiment, the bracket 250 is provided with a plurality of fixing holes for accommodating the coupling gun heads 100, the gun body 110 is inserted into the fixing holes to be fixed in the box body 210 by the bracket 250, and the lower end thereof is sealed by the lower sealing gasket 230. The upper sealing gasket 240 is a silica gel pad, which can be elastically deformed after being compressed. The upper sealing gasket 240 is fixed in the box cover 220, and in the present embodiment, the upper sealing gasket 240 is fixed on the inner surface of the box cover 220 by two-shot injection molding. The lower sealing gasket 230 includes sealing plugs 231, the sealing plugs 231 are provided with grooves, each sealing plug 231 corresponds to one coupling gun head 100, and the lower end of the coupling gun head 100 is inserted into the groove of the corresponding sealing plug 231 to be wrapped by the sealing plug 231. In the present embodiment, the sealing plug 231 is a silica gel plug, which is provided with a cylindrical groove, and the lower sealing gasket 230 is composed of a plurality of arrayed sealing plugs 231.

[0136] Another structure of the bracket (not shown in the figure) can also be adopted: the bracket 250 includes a base fixed in the box body 210, the base is provided with a placement plate, the base and the placement plate are both provided with fixing holes, and the coupling gun head 100 is sequentially inserted through the fixing holes of the placement plate and the base and then sealed by the lower sealing gasket 230.

[0137] In some other embodiments, the lower sealing gasket 230 is a silica gel gasket, which is fixed to the inner surface of the bottom of the box body 210, for example, by means of secondary injection molding. The contact surface (upper surface) of the lower sealing gasket 230 is concave, having a plurality of grooves, and the lower end of the gun body 110 is inserted into the grooves to achieve sealing.

[0138] The upper sealing gasket 240 is convex, having a plurality of downwardly extending protrusions, and the protrusions of the upper sealing gasket 240 are inserted into the structure 120 of the gun body 110 after the box cover 220 is put on, and are deformed to achieve sealing.

[0139] The box cover 220 is detachably connected to the box body 210 by means of the buckle 221, and can be opened by a robot hand.

[0140] The outer surface of the box body 210 is provided with one or more positioning structures extending upward and downward. In this embodiment, the positioning structure includes a positioning groove 211. In some other embodiments, the positioning structure can be a positioning protrusion.

[0141] Embodiment 3

[0142] The embodiment provides a mixing instrument (not limited thereto) shown in FIGS. 4 to 6, which includes a mounting frame 301, a rotating shaft 302, a rotating bin module 303, and a fastening structure. The rotating shaft 302 is rotatably arranged on the mounting frame 301, the rotating bin module 303 is arranged on the rotating shaft, the rotating bin module 303 includes a plurality of rotating bins 331, the fastening structure is arranged on the side wall of the rotating bin 331, the rotating bin 331 and the coupling box 200 are relatively fixed by means of the fastening structure and can be quickly disassembled. The mounting frame 301 is provided with a power supply and a motor, the power supply serves as a power source for the rotation of the motor, and the motor (a rotary motor) drives the rotating shaft to rotate, thereby driving the rotating bin module 303 and the plurality of coupling boxes 200 to rotate.

[0143] The rotating shaft 302 is horizontally arranged and can be installed with multiple groups of rotating bin modules 303 and coupling boxes 200. The rotating bins 331 are arranged at intervals along the circumferential direction of the rotating shaft 302, and the rotating bin module 303 composed of a plurality of rotating bins 331 is arranged on the rotating shaft 302. In this embodiment, four rotating bins 331 form a group of rotating bin modules 303 (two rotating bin modules 303 are shown in FIGS. 4 and 5). This arrangement allows the capacity of the mixing instrument 300 to be flexibly configured according to the number of coupling boxes 200 to be processed or the scale of the reaction, and the rotating bin module 303 can be flexibly expanded if a large amount of processing is required.

[0144] The rotating bin 331 has an open end, which is located on the side of the rotating bin 331 far from the rotating shaft 302. The rotating bin 331 also has a cavity for accommodating the coupling box 200. When the coupling box 200 is mounted on the mixing instrument 300, the coupling gun head 100 is arranged horizontally. This design increases the capacity of the mixing instrument 300, and a larger number of coupling boxes 200 can be loaded at one time. The reagents in multiple coupling boxes 200 can be mixed synchronously, thereby improving the efficiency.

[0145] The fastening structure is arranged on at least two opposite side walls of the rotating bin 331. This arrangement can further effectively clamp the coupling box 200 and fix the coupling box 200 relative to the rotating bin 331. After the mixing is completed, the coupling box 200 can be quickly and conveniently separated from the rotating bin 331. The fastening structure includes a plurality of fastening units, which are arranged at intervals along the diameter direction or the circumferential direction of the rotating shaft 302. The fastening unit includes a pressing member 305, which includes a pressing part arranged on the rotating bin 331 and capable of moving. The pressing part has a retracted position and an extended position protruding inward into the cavity. The fastening unit also includes a resilient member arranged between the pressing part and the surface (for example, the inner surface) of the rotating bin 331, for example, a compression spring. By arranging the elastic fastening structure, the coupling box 200 can be conveniently put in or taken out, and at the same time, it can be ensured that the coupling box 200 will not be separated from the rotating bin 331 during the rotation and oscillation. The pressing part of the fastening unit can be inserted into the positioning groove 211 of the coupling box 200, so that the rotating bin 331 is fixedly connected with the coupling box 200, and the coupling box 200 can be quickly separated from the rotating bin 331.

[0146] It should be further noted that the length of the positioning groove 211 is less than the height of the coupling box 200, and the above-mentioned fastening structure is offset from the bottom of the rotating bin 331 by a distance. The coupling box 200 is inserted into the rotating bin 331 in a side-by-side manner. In the first stage of insertion of the coupling box 200, the pressing member 305 is subjected to extrusion, and the resilient member is in a deformed state to allow the wider part of the coupling box 200. When the pressing member 305 enters the positioning groove 211, the resilient member resets, and the pressing part of the pressing member 305 is in its extended position, clamping the coupling box 200. The insertion is convenient and fast, and the elasticity of the resilient member allows the coupling box 200 to be quickly taken out.

[0147] The mixing instrument 300 also includes a timer 36 and a speed regulator 37. The timer 36 is used to adjust the rotation time of the rotating shaft 302, and the speed regulator 37 is used to adjust the rotation speed of the rotating shaft 302 and thereby adjust the vibration effect. The greater the rotation speed, the more obvious the vibration. The speed regulator 37 is configured to control the rotation speed of the rotating shaft 302 to be 20-150 revolutions per minute. By controlling the rotation time and the rotation speed, the mixing rate of the reaction solution and the connecting enzyme can be adjusted.

[0148] In the preparation method, the coupling gun head 100 containing the reaction solution is placed into the coupling box 200, the coupling box 200 is placed into the rotating bin 331 of the rotating bin module 303, and the positioning groove 211 of the coupling box 200 is matched with the fastening structure of the sidewall of the rotating bin 331 to fix the coupling box 200. The power supply is turned on, the motor is started, the motor drives the rotating shaft 302 to rotate, and the reaction solution and the ligase are uniformly mixed by rotation, thereby improving the mixing effect of the reaction solution and the ligase.

[0149] The automatic pipette automatically installs and takes out the coupling gun head 100 from the coupling box 200, then conveniently sucks the reaction solution into the coupling gun head 100 to mix with the ligase, and repositions the coupling gun head 100 in the coupling box 200; the coupling gun head 100 is fixed in the coupling box 200, and under the action of the mixing instrument 300, the antibody conjugated drug screening substance is fully generated in the coupling gun head 100, the operation is convenient, and the antibody conjugated drug can be conveniently and efficiently prepared.

[0150] Embodiment 4

[0151] The embodiment also provides a preparation method of an antibody conjugated drug (XDC), which comprises:

[0152] S1, the switch cover device automatically opens the coupling box 200, the automatic pipette automatically installs the coupling gun head 100, then discharges the storage liquid 150 in the coupling gun head 100, the automatic pipette controls the coupling gun head 100 to suck the eluent to perform elution, and the elution step is repeatedly performed multiple times; in the embodiment, the coupling box is 96 wells, and the automatic pipette can automatically install 96 coupling gun heads 100 at a time;

[0153] S2, after the reaction solution is uniformly mixed, the automatic pipette controls the coupling gun head 100 to suck the reaction solution mixture to contact with the ligase (such as Sortase enzyme or glycoside endo enzyme);

[0154] S3, the automatic pipette automatically repositions the coupling gun head 100 in the coupling box 200; the mechanical arm takes out the coupling box 200 and places it in the mixing instrument 300 to be continuously mixed to form the XDC; a large amount of ADC product can be used for drug screening.

[0155] The above embodiment is only a preferred embodiment for describing the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application.

Claims

1. A coupling gun head, characterized by, The coupling gun head is filled with a ligase for catalyzing the target molecule to form a target molecule-ligand complex with the ligand or catalyzing the target molecule to form a conjugated drug XDC with the ligand-load; the target molecule-ligand complex is chemically connected with the load to form the conjugated drug XDC. Preferably, the ligase is used for catalyzing the target molecule to form a conjugated drug XDC with the ligand-load.

2. The coupling gun head of claim 1, wherein, The target molecule is selected from one or more combinations of the following group: a ligand, a polypeptide, an antibody or an antigen-binding fragment thereof, or an antibody mimetic.

3. The coupling gun head of claim 1 or 2, wherein, The load is selected from one or more combinations of the following group: a small molecule compound, a polypeptide, a glycan, a PEG moiety, a radionuclide, a cytokine, an immunomodulator, a nucleic acid and its analogues, a tracer molecule, and a macromolecule.

4. The coupling gun head of claim 1, wherein, The ligase is selected from one or more combinations of the following group: a sortase enzyme, a transglutaminase, a formylglycine-generating enzyme, a tyrosinase, a asparagine ligase, and an endoglycosidase; Preferably, the ligase is a sortase enzyme; more preferably, the sortase enzyme includes sortase A, sortase B, sortase C, sortase D, sortase E, or sortase F; Preferably, the ligase is an endoglycosidase with glycosyltransferase activity; more preferably, the endoglycosidase is an N-acetylglucosamine endohydrolase selected from at least one of Endo S, Endo F3, Endo S2, Endo Sd, and Endo CC, or the N-acetylglucosamine endohydrolase includes at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

5. The coupling gun head of any one of claims 1 to 4, wherein, The coupling gun head includes a gun body, which is hollow and has an open upper end and a lower end, the upper end is used to connect a pipette, and the lower end is provided with a liquid inlet and outlet; the gun body is further provided with an upper filter core and a lower filter core, and the ligase is filled in the interior of the gun body and located between the upper filter core and the lower filter core.

6. The coupling gun head of claim 5, wherein, The pipette is a manual pipette, an electric pipette, or an automatic pipette; the automatic pipette includes a servo motor, a piston structure, and a Tip head device.

7. The coupling gun head of claim 5, wherein, The thickness of the upper filter core is 1-5 mm, the pore size of the filter hole of the upper filter core is 10-100 μm, preferably 35-60 μm; the thickness of the lower filter core is 1-5 mm, and the pore size of the filter hole of the lower filter core is 10-100 μm, preferably 35-60 μm.

8. The coupling gun head of claim 5, wherein, The materials of the upper filter core and the lower filter core are hydrophobic; preferably, the materials of the upper filter core and the lower filter core are hydrophobic resin; more preferably, the materials of the upper filter core and the lower filter core are hydrophobic polyethylene resin; and / or The upper filter core and the lower filter core are fixed on the gun body by ultrasonic welding or interference fit; or, the gun body is provided with a clamping groove, and the upper filter core and the lower filter core are fixed in the clamping groove.

9. The coupling gun head of claim 5, wherein, The ligase is filled in the gun body in the form of being fixed in a coupling medium selected from one or more combinations of the following group: resin, gel and matrix; preferably, the coupling medium is selected from one or more combinations of the following group: agarose resin, silicone resin, polymethyl methacrylate resin and cellulose resin.

10. The coupling gun head of claim 9, wherein, The ligase is a fusion protein, the ligase is covalently connected with a dehalogenase or a variant thereof, and the ligase is fixed on a coupling medium containing a haloalkyl linker by the dehalogenase; Preferably, the fusion protein is a dehalogenase-sortase enzyme, a dehalogenase-endoglycosidase, a dehalogenase-transglutaminase, a dehalogenase-formylglycine-generating enzyme, a dehalogenase-tyrosinase or a dehalogenase-asparagine ligase; More preferably, the amino terminal of the ligase is covalently connected with a dehalogenase, and there is a linker sequence between the ligase and the dehalogenase.

11. The coupling gun head of claim 10, wherein, The coupling medium comprises a chloroalkyl linker produced from a chloroalkyl substrate having the structure of Formula (I): Wherein, 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; Preferably, the coupling medium has the structure of formula (II): wherein resin, gel or matrix; more preferably, The resin microspheres.

12. The coupling gun head of claim 5, wherein, The gun body is also filled with a storage solution for maintaining the activity of the ligase, the storage solution is located between the coupling medium and the upper filter core, and the storage solution comprises 10-30 mM Tris-HCl, 110-180 mM NaCl, and the pH value of the storage solution is 6-8; preferably, the storage solution further comprises ethanol or Tween; Preferably, when the ligase is a sortase enzyme, the storage solution comprises 10-30 mM Tris-HCl, 110-180 mM NaCl and 10%-30% ethanol (V / V), and the pH value of the storage solution is 6-8; Preferably, when the ligase is an endoglycosidase, the storage solution comprises 10-30 mM Tris-HCl, 110-180 mM NaCl and 0.001%-1% Tween (W / V), and the pH value of the storage solution is 6-8.

13. The coupling gun head of claim 5, wherein, The maximum range of the coupling gun head is 90 μL-5 mL, preferably 100 μL, 200 μL or 1 mL; Preferably, when the maximum range of the coupling gun head is 1 mL, the volume of the coupling medium in the gun body is 100-180 μL, and the volume of the storage solution is 550-730 μL; Preferably, when the maximum range of the coupling gun head is 200 μL, the volume of the coupling medium filled in the gun body is 15-45 μL, and the volume of the storage solution is 100-135 μL.

14. A coupling cassette characterized in that, The coupling box comprises a box body and a box cover, the coupling gun head is arranged in the box body, the upper end and the lower end of the coupling gun head are respectively provided with an upper sealing gasket and a lower sealing gasket, and the upper sealing gasket and the lower sealing gasket seal the coupling gun head to form a sealed space; 15. The coupling cassette of claim 14, wherein, ​ Preferably, the upper and lower sealing gaskets are made of elastic material; more preferably, the material of the upper and lower sealing gaskets is silica gel.

16. The coupling cassette of claim 15, wherein, The upper sealing gasket is arranged on the inner surface of the box cover; preferably, the upper sealing gasket is fixed on the inner surface of the box cover; more preferably, the upper sealing gasket is fixed on the inner surface of the box cover by secondary injection molding. The lower sealing gasket is arranged on the bottom of the box body; preferably, the lower sealing gasket is fixed on the inner surface of the bottom of the box body; more preferably, the lower sealing gasket is fixed on the inner surface of the bottom of the box body by secondary injection molding. The box body is internally provided with a support for accommodating the coupling gun head, and the support is located between the upper and lower sealing gaskets; preferably, the support is fixed in the box body. Preferably, the box cover is detachably connected to the box body by buckling.

17. The coupling cassette of claim 16, wherein, The lower surface of the upper sealing gasket is provided with a plurality of downwardly extending protrusions, and the protrusions are inserted into the upper end of the coupling gun head after the box cover is covered on the box body; and / or The upper surface of the lower sealing gasket is formed with a plurality of grooves, and the lower end of the coupling gun head is inserted into the grooves and wrapped by the groove walls; more preferably, the lower sealing gasket comprises a plurality of integrally formed sealing plugs, each of which is provided with a groove, and the lower end of the coupling gun head is inserted into the groove of the corresponding sealing plug; and / or The support is provided with a fixing hole slot for accommodating the coupling gun head, and the coupling gun head is sealed by the lower sealing gasket after passing through the fixing hole slot; preferably, the support comprises a base fixed in the box body, and the base is provided with a placement plate, and the base and the placement plate are both provided with the fixing hole slot, and the coupling gun head is sequentially passed through the fixing hole slots of the placement plate and the base and then sealed by the lower sealing gasket; preferably, the support comprises a placement plate fixed in the box body, and the placement plate is provided with the fixing hole slot, and the coupling gun head is passed through the fixing hole slot and then sealed by the lower sealing gasket; and / or The outer surface of the box body is provided with one or more positioning structures extending upward and downward, and the positioning structures comprise positioning grooves or positioning protrusions to fix the coupling box on the mixing instrument.

18. An apparatus for early screening of conjugated drugs, characterized in that, The coupling gun head according to any one of claims 1 to 13 and / or the coupling box according to any one of claims 14 to 17.

19. The apparatus of claim 18, wherein, The device further comprises an automatic pipettor for aspirating and / or expelling reagents, a mechanical hand for opening the box cover of the coupling box, and / or a mixing instrument.

20. The apparatus of claim 19, wherein, The automatic pipettor comprises a servo motor, a sealing ring, a piston structure, a lead screw, a Tip head device, a sensor, a driver, and / or a communication module.

21. A preparation method of a conjugated drug (XDC) using the coupling gun head according to any one of claims 1 to 13 and / or the coupling box according to any one of claims 14 to 17; the preparation method comprises the following steps: S1, after expelling the storage liquid in the coupling gun head, using an eluent for elution; S2, using the coupling gun head to aspirate the mixed reaction liquid and contact the ligase therein, or using the coupling gun head to sequentially aspirate a plurality of reaction liquids and contact the ligases therein; S3, constantly mix, ligase catalyzed formation of conjugated drugs XDC.

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