An antibody conjugate and uses thereof

Abstract

The application discloses an antibody conjugate and application thereof, and relates to the technical field of antibody coupling. The application provides a technology for directional modification of disulfide bonds of an antibody, and the antibody is coupled with a carrier and a conjugate partner by using a linker, and the antibody conjugate prepared by the method has good uniformity, and has obvious advantages of high detection sensitivity and good stability compared with an antibody conjugate prepared by a conventional process, so that the application provides a path for development and application of a diagnostic reagent.

Description

Technical Field

[0001] This invention relates to the field of antibody conjugation technology, and more specifically, to an antibody conjugate and its applications. Background Technology

[0002] In the diagnostic field, EDC, NHS, or maleimide are commonly used to conjugate the carboxyl, amino, or thiol groups of proteins. This protein conjugation technique has two problems that affect the sensitivity and precision of the reagents. First, the randomness of the conjugation site: Taking IgG1 antibodies as an example, they contain an average of 80 lysine residues, 20 of which are located in solvent-accessible sites. Some solvent-accessible lysine residues are located in the antigen recognition region of the Fab. Therefore, when using the amino group of the antibody as the conjugation site, the random distribution of the amino group within the antibody leads to heterogeneity of the antibody conjugate and antibody inactivation. Second, low conjugation efficiency: The second-order reaction kinetics of NHS with the amino group is 10... -1 ~10 2 M -1 S -1 When equilibrium is reached, the yield of the conjugate is 40%, which results in a low product yield, waste of raw materials, and affects the sensitivity of the reagent.

[0003] The second-order reaction kinetics of maleimide and thiol group reach 10. 2 ~10 3 M -1 S -1 The conjugate yield was 80%, but the antibody's cysteine ​​residues existed in the form of disulfide bonds, resulting in very few free thiol groups accessible to the solvent. Another method was to convert the antibody's amino group to a thiol group using Traut's reagent (2-iminothiacyclopentane), but this method still suffered from low conversion efficiency and randomness in the coupling site. Summary of the Invention

[0004] The purpose of this invention is to provide an antibody conjugate and its application.

[0005] This invention is implemented as follows:

[0006] In a first aspect, embodiments of the present invention provide an antibody conjugate suitable for immunodiagnostics, the structure of which comprises, in sequence, an antibody-linker-vector-conjugation partner.

[0007] The linker is directionally coupled to the antibody via interchain disulfide bonds. Preferably, the linker and the antibody are connected via a sulfur-carbon bridge.

[0008] Secondly, embodiments of the present invention provide an antibody conjugate suitable for immunodiagnostics, comprising the following structure:

[0009]

[0010] From left to right, they are the conjugation partner, vector, linker, and antibody; the linker forms a sulfur-carbon bridge (SCCCS) linker with the antibody chain, where the two sulfur atoms in the SCCS come from two cysteine ​​residues of the antibody, and the three carbon atoms in the SCCS come from the linker compound. n and m are both integers and independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0011] The linker and carrier can be connected via a suitable coupling technique. Commonly used techniques in the field include connecting the linker via the reaction of the NHS ester on the linker with an amino group on the carrier, connecting the linker via the reaction of the maleimide on the linker with a thiol group on the carrier, or connecting the linker via a first click chemical group on the linker and a second click chemical group on the carrier through a click chemical reaction. Click chemistry is widely used in bioconjugation, biolabeling, and materials science in the pharmaceutical and biotechnology industries due to its mild conditions and high selectivity. For demonstration purposes, the preparation of the antibody conjugates, antibody-linker-carrier products of this invention will primarily be achieved through click chemical reactions, but this does not necessarily mean that click chemical group connection is required. Regardless of the coupling method used, the goal is the same: connection. Other coupling methods (including but not limited to connecting NHS ester with an amino group, and maleimide with a thiol group) can also achieve the connection goal, although the reaction rate is slower and more byproducts may be generated. However, those skilled in the art can subsequently adjust the process through purification and separation to obtain the same final product. In some embodiments, the linker and carrier in the antibody conjugate are connected via orthogonal click chemical groups, having the structure shown below:

[0012]

[0013] Wherein, R1 is the first click chemical group, R2 is the second click chemical group, and n and m are both integers and are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0014] Thirdly, embodiments of the present invention provide an antibody-linker-vector suitable for immunodiagnostics, comprising the following structure:

[0015]

[0016] From left to right: vector, linker, and antibody;

[0017] Wherein, R1 is the first click chemical group, R2 is the second click chemical group, and n and m are both integers and are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0018] This type of antibody-linker-vector product is suitable for free labeling in downstream processes, and subsequent conjugation of selectable coupling partners can be performed on the vector as needed.

[0019] The antibody conjugate or antibody-linker-vector described in this invention is not limited in the number of linkers in the structure and can have one or more linkers. That is, the antibody can have multiple disulfide bonds, each of which can be independently reduced to a thiol group and then recombine with a linker to form a sulfur-carbon bridge bond. In this way, one antibody can link multiple linkers, and each linker can independently link a conjugation partner.

[0020] In the carrier described in this invention, the conjugation partner and the linker are indirectly connected through the carrier, which is selected from at least one of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethyleneimine, and polypropyleneimine. Specifically, the polysaccharides include cross-linked sucrose and dextran; the protein carriers include at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin.

[0021] For the “conjugation partner-carrier-R2 (also written as R2-carrier-conjugation partner)” unit contained in the antibody conjugate structure of the present invention, the hyphen “-” between R2 and the carrier, and between the carrier and the conjugation partner, does not limit whether R2 is directly bonded to the carrier or whether the carrier is directly bonded to the conjugation partner. R2 can be directly bonded to the carrier, R2 can be indirectly bonded to the carrier through small molecule chemical structural units, or it can be indirectly bonded to the carrier through macromolecular biological materials. The carrier can be directly bonded to the conjugation partner, the carrier can be indirectly bonded to the conjugation partner through small molecule chemical structural units, or it can be indirectly bonded to the conjugation partner through macromolecular biological materials. Experimental data provided later demonstrate that these factors do not affect the detection sensitivity of the final prepared antibody conjugate.

[0022] For the antibody conjugates described in this invention, the number of conjugation partners linked to the same linker in the structure is not limited; the same linker can link one or more conjugation partners. That is, the conjugation partners are indirectly connected to the linker through the carrier, forming a conjugation partner-carrier-linker structure. One carrier can link multiple conjugation partners, so the same linker can indirectly link multiple conjugation partners. Each conjugation partner can independently generate a detection signal, which amplifies the signal when detecting the sample.

[0023] Fourthly, embodiments of the present invention provide a method for preparing antibody conjugates as described in the foregoing embodiments, comprising: performing an addition reaction between an antibody-linker and an R2-carrier-conjugation partner to obtain an antibody conjugate; wherein the antibody-linker has the following structure:

[0024]

[0025] n and m are both integers and are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

[0026] Fifthly, embodiments of the present invention provide the application of antibody conjugates or antibody-linker-carriers as described in the foregoing embodiments in the preparation of diagnostic reagents or kits.

[0027] In a sixth aspect, embodiments of the present invention provide a kit comprising an antibody conjugate or antibody-linker-vector as described in the foregoing embodiments.

[0028] The present invention has the following beneficial effects:

[0029] This invention provides a technique for targeted modification of the disulfide bonds of antibodies for coupling, comprising the use of 2-(toluenesulfonyl)methylacrylamide-(PEG). m -amide-(PEG) n -R1, as shown below Used as a linker for efficient conjugation of antibodies and conjugation partners-carriers, the antibody conjugates prepared by this method exhibit good uniformity and have significant advantages over antibody conjugates prepared by conventional processes, such as high detection sensitivity and good stability, thus providing a pathway for the development and application of diagnostic reagents. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram showing the reconstruction of antibody disulfide bonds after reduction by a reducing agent.

[0032] Figure 2The results are based on the number of disulfide bond reductions of antibodies as shown by gel electrophoresis (lanes 1-3) and the number of disulfide bond reductions after being re-linked by TSMA (lanes 4-6).

[0033] Figure 3 The effect of TCEP concentration on the number of antibody disulfide bonds reduced, as shown by gel electrophoresis;

[0034] Figure 4 The chromatogram of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine;

[0035] Figure 5 The mass spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine is shown.

[0036] Figure 6 The mass spectrometry peak analysis chromatogram of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine;

[0037] Figure 7 The carbon NMR spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine;

[0038] Figure 8 The NMR spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine is shown in the figure. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] This invention provides an antibody conjugate comprising a conjugation partner, a vector, a linker, and an antibody, having the following structure:

[0041]

[0042] Wherein, n and m are both integers and are independently selected from 0 to 24. Preferably, m+n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. Preferably, m is not zero. Preferably, n is not zero. Preferably, neither m nor n is zero. Preferably, m+n≥4.

[0043] In some embodiments, the carrier and the linker are connected by an amide bond, which is, for example, a bond formed by the reaction of an NHS ester and an amino group.

[0044] In some embodiments, the carrier and the linker are connected by a thioether bond, which is, for example, a bond formed by the reaction of maleimide and a thiol group.

[0045] In some embodiments, the support and linker are linked by click chemical groups; for example, the support is modified with R2, and the linker is modified with R1, with the structure being 2-(toluenesulfonyl)methylacrylamide-(PEG). m -amide-(PEG) n -R1, as shown below:

[0046] R1 and R2 are orthogonal pairs of click chemical groups that can be linked together based on click chemical reactions.

[0047] The 2-(toluenesulfonyl)methacrylamide structure in the linker of this invention acts as a reconstructive crosslinking agent, enabling the two free thiol groups on the antibody to be re-bridged together, forming a carbon-sulfur bond in the SCS. (See reference...) Figure 1 The optimal number of PEG molecules in the linker, i.e., the value of m+n, is 4 to 24. The number of PEG molecules in the linker can adjust the spacing between the conjugate partner and the antibody. If the spacing is too close, it will affect the spatial structure of the antibody; if the spacing is too far, it will affect the stability of the antibody conjugate.

[0048] In some embodiments, n and m are both integers and independently selected from 0 to 24, specifically any one or any two of the following: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. Commonly used reconstructing crosslinking agents and click chemical groups are highly hydrophobic organic compounds, making it difficult for them to enter the disulfide bond positions of water-soluble antibodies. For example, bis(ethylene sulfonamide) disclosed in CN107400072B as a reconstructing crosslinking agent has poor water solubility, affecting the efficiency of disulfide bond reconstruction. This invention improves water solubility and increases the efficiency of disulfide bond reconstruction by designing and introducing PEG molecules into the reconstructing crosslinking agent, thus significantly improving the detection signal intensity of the final conjugated product.

[0049] In some embodiments, the sulfur-carbon bridges in the structural formula of the antibody conjugate are located between antibody hinge regions, between antibody light and heavy chains, or between antibody heavy chains. The locations between antibody heavy chains include between CH1 regions and between Fc regions.

[0050] The antibodies described herein are interpreted in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, as well as chimeric antibodies and antigen-binding fragments of antibodies, provided that they exhibit the desired biological activity and have disulfide bonds that can crosslink with the 2-(toluenesulfonyl)methylacrylamide structure to form sulfur-carbon bridges upon reduction.

[0051] In some embodiments, R1 and R2 are click chemistry groups capable of orthogonal pairing. Click chemistry is an orthogonal linking reaction, commonly of two types: the first is the copper-catalyzed azido-alkynyl cycloaddition reaction (CuAAC) or the copper-free azido-dibenzocyclooctylene strain-promoted cycloaddition reaction (SPAAC); the second is the tetrazine-trans-cyclooctene electron-reversing demand addition reaction, which is currently the fastest orthogonal reaction discovered, with second-order reaction kinetics reaching 30,000 M. -1 S -1 Tetraazines with strong electron-withdrawing groups are less stable than hydrogen-substituted tetraazines and less stable than methyl-substituted tetraazines. Hydrogen-substituted tetraazines exhibit exceptionally fast kinetics (10000 M). -1 S -1 Methyltetrazine (MTz) is typically at least 10 times faster than methyl-substituted tetrazines. Therefore, a balance between reaction rate and stability needs to be struck in the selection of the tetrazine group. The inventors discovered that 1 mg / mL of methyltetrazine-modified IgG antibody and TCO-modified AP (alkaline phosphatase) can be completely converted into an IgG-AP conjugate within 90 min, and the activity of the methyltetrazine-modified IgG antibody only decreases by 10-20% after being placed at 4°C for one month. Based on these results and considering the application scenarios in the IVD field, methyltetrazine (MTz) and trans-cyclooctene (TCO) are preferred as click chemistry orthogonal pairs in this invention; other click chemistry orthogonal pairs can achieve similar results.

[0052] In some embodiments, the first click chemical group and / or the second click chemical group are selected from any one of: methyltetrazine (MTz), trans-cyclooctene (TCO), azide (N3), dibenzocyclooctene (DBCO), tetrazine (Tz), alkynes, cyclopropanecyclooctene (BCN), and cyclopropene.

[0053] Preferably, both the first click chemical group and the second click chemical group are selected from either methyltetraazine (MTz) or trans-cyclooctene (TCO).

[0054] The antibody conjugates prepared by this invention are superior to those prepared by conventional processes, mainly in terms of higher yield, clearer conjugation sites, and better uniformity. Furthermore, detection kits formulated using the antibody conjugates of this invention exhibit better sensitivity and correlation than those prepared using conventional processes.

[0055] In some embodiments, the conjugation partner is a marker selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers; preferably, the fluorescent dye is selected from at least one of fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives; preferably, the enzyme is selected from any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase; preferably, the radioisotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, and 110mI. At least one of n, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F; preferably, the chemiluminescent reagent is selected from at least one of luminol, isoluminol, N-(4-aminobutyl)-N-ethyl isoluminol, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenol and its derivatives, and peroxazone and its derivatives; preferably, the nanoparticle label is selected from any one of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles; preferably, the colloid is selected from at least one of colloidal metals, dispersed dyes, dye-labeled microspheres, and latex; preferably, the colloidal metal is selected from at least one of colloidal gold, colloidal silver, and colloidal selenium.

[0056] In some embodiments, the conjugation partner is a solid support selected from at least one of microspheres, plates, and membranes; preferably, the conjugation partner is selected from at least one of magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0057] In some embodiments of the present invention, a method for preparing antibody conjugates based on click chemistry linkage is provided, comprising: performing an addition reaction between antibody-linker-R1 and R2-carrier-conjugation partner to obtain an antibody conjugate; wherein the antibody-linker-R1 has the following structure:

[0058]

[0059] In some embodiments, the molar ratio of antibody-linker-R1 to R2-vector-conjugation partner is (0.5-4):(0.5-4), specifically, it can be any one or any two of the following: 0.5:4, 0.5:3.5, 0.5:3, 0.5:2.5, 0.5:2, 0.5:1.5, 0.5:1, 1:1, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 2:4, 2:3.5, 2:3, 2:2.5, 2:1.5, 2:1, 2:1, 3:4, 3:3.5, 3:2.5, 3:2, 3:1.5, 3:1, 4:3.5, 4:3, 4:2.5, 4:2, 4:1.5, 4:1.

[0060] In some embodiments, the reaction conditions for the addition reaction include: 4–37°C, 0.5–16 h; optionally, the reaction temperature of the addition reaction can be any one or a range between any two of 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, and 37°C; the reaction time of the addition reaction can be 0. The range between any one or any two of the following: 5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, and 16h.

[0061] In some embodiments, the preparation method further includes the preparation of an antibody-linker: mixing and reacting the linker with an antibody whose disulfide bonds have been reduced and opened to obtain an antibody-linker; the linker has the following structure:

[0062]

[0063] In some embodiments, the molar ratio of the linker and the antibody whose disulfide bond is opened by reduction is 1:(10-30). Specifically, this molar ratio can be any one or any two of 1:10, 1:15, 1:20, 1:25, and 1:30.

[0064] In some embodiments, the reaction conditions for antibodies whose linkers and disulfide bonds are opened by reduction include: 0–10°C, 11–21 h. Specifically, the reaction temperature can be any one or any two of 0°C, 2°C, 4°C, 6°C, 8°C, and 10°C; the reaction time can be any one or any two of 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, and 21 h.

[0065] In some embodiments, the preparation method further includes the preparation of antibodies whose disulfide bonds are opened by reduction: reducing the antibody under the action of a reducing agent to obtain antibodies whose disulfide bonds are opened by reduction.

[0066] In some embodiments, the reducing agent includes at least one of 2-MEA and TCEP.

[0067] 2-Mercaptoethylamine hydrochloride (2-MEA) is a mild reducing agent that specifically reduces disulfide bonds in the hinge region of antibodies without reducing disulfide bonds at other sites. TCEP is another mild disulfide bond reducing agent. At a certain TCEP concentration, taking IgG as an example, the reduction order of antibody disulfide bonds is: interchain disulfide bonds > upper hinge region disulfide bonds > lower hinge region disulfide bonds > intrachain disulfide bonds.

[0068] In some embodiments, the concentration of 2-MEA used includes at least one of 3eq, 5eq, and 10eq.

[0069] In some embodiments, the molar ratio of the reducing agent to the antibody is (5–15):1. Specifically, this molar ratio can be any one or any two of the following: 5:1, 7:1, 9:1, 11:1, 13:1, and 15:1.

[0070] For ease of demonstration, the term "antibody-specific click chemical group" as used below refers to the antibody-linker structure of the present invention with a specific click chemical group.

[0071] In some embodiments, the preparation method further includes the preparation of a linker: combining TSMA and R1-(PEG) n -NHS ester mixing reaction, wherein the TSMA is 2-(toluenesulfonyl)methacrylamide-(PEG). m -Amide, with the following structural formula:

[0072]

[0073] In some embodiments, the TSMA and R1-(PEG) nThe molar ratio of -NHS ester is (0.5~1.5):(0.5~1.5). Specifically, this molar ratio can be any one or any two of 0.5:1.5, 0.5:1, 0.5:1:1:0.5, 1.5:1.

[0074] In some embodiments, the TSMA and R1-(PEG) n The reaction conditions for -NHS esters include: 10–30℃, 10–14h. Specifically, the reaction temperature can be any one or any two of the following: 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃; the reaction time can be any one or any two of the following: 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h.

[0075] In some embodiments, the TSMA and R1-(PEG) n -NHS esters react in organic solvents or water.

[0076] In some embodiments, the organic solvent of the organic phase includes dichloromethane.

[0077] In some embodiments, the TSMA and R1-(PEG) n -NHS esters react with DIEA and / or triethylamine.

[0078] In some embodiments, the molar ratio of DIEA to TSMA is 1:(1 to 4). Specifically, this molar ratio can be any one of 1:1, 1:2, 1:3, 1:4, or a range between any two of them.

[0079] In some embodiments, the molar ratio of triethylamine to TSMA is 1:(1 to 4). Specifically, this molar ratio can be any one of 1:1, 1:2, 1:3, 1:4, or a range between any two of them.

[0080] In some embodiments, the preparation method further includes the preparation of TSMA:

[0081] Using compound A and compound B as starting materials, the reaction of compound A and compound B yields compound C;

[0082] The compound C was reacted with compound D via a substitution reaction to obtain TSMA;

[0083] Wherein, compound A is tert-butoxycarbonyl-imino-polyethylene glycol-amine, with the following structural formula: Compound B is methacrylic acid. Methacryloyl halide (where X is a halogen) and methacrylic anhydride At least one of the following; compound C is tert-butyloxycarbonyl-imino-polyethylene glycol-methacrylamide, with the structural formula: The compound D is 4-toluenesulfonyl chloride. Sodium 4-toluenesulfinate At least one of the following; wherein m is 0 to 24. The specific selection of m is the same as described in the corresponding embodiments above, and will not be repeated here.

[0084] In some embodiments, the molar ratio of compound A to compound B is (0.35–0.85):(0.58–0.98); specifically, this molar ratio can be any one or a range between any two of the following: 0.45:0.58, 0.45:0.78, 0.45:0.98, 0.35:0.58, 0.35:0.78, 0.35:0.98, 0.65:0.58, 0.65:0.78, 0.65:0.98, 0.85:0.58, 0.85:0.78, and 0.85:0.98.

[0085] In some embodiments, the reaction conditions for compound A and compound B include: stirring at 20–30°C for 10–14 h; the reaction temperature can specifically be any one or any two of 20°C, 22°C, 24°C, 26°C, 28°C, and 30°C; the reaction time can specifically be any one or any two of 10 h, 10.5 h, 11 h, 11.5 h, 12 h, 12.5 h, 13 h, 13.5 h, and 14 h.

[0086] In some embodiments, the reaction of compound A and compound B includes: dissolving compound A in a first organic solvent, adding compound B, reacting under the action of a dehydrating agent and / or a hydroxyl activator, removing the solvent, filtering to remove the precipitate, and purifying to obtain compound C.

[0087] In some embodiments, the first organic solvent includes at least one of DMF and dichloromethane;

[0088] In some embodiments, the dehydrating agent comprises dicyclohexylcarbodiimide.

[0089] In some embodiments, the hydroxyl activator includes N-hydroxysuccinimide.

[0090] In some embodiments, the molar ratio of compound C to compound D is (0.33–0.73):(0.60–1.00). Specifically, this molar ratio can be any one or a range between any two of the following: 0.33:0.60, 0.33:0.80, 0.33:1.00, 0.53:0.60, 0.53:0.80, 0.53:1.00, 0.73:0.60, 0.73:0.80, and 0.73:1.00.

[0091] In some embodiments, the reaction conditions for the substitution reaction include: stirring at 20–30°C for 20–28 hours. Specifically, the reaction temperature can be any one or any two of 20°C, 22°C, 24°C, 26°C, 28°C, and 30°C; the reaction time can be any one or any two of 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, and 28 hours.

[0092] In some embodiments, the substitution reaction includes: dissolving compound C in a second organic solvent, adding compound D to react; adding triethylamine after the reaction, and stirring for 10 to 14 hours; the stirring time can be any one or any two of 10 hours, 11 hours, 12 hours, 13 hours, and 14 hours.

[0093] The stirred product was washed and dried to remove the solvent, yielding a crude product. The crude product was dissolved in ethyl acetate, triethylamine was added and refluxed, and the solvent was removed by rotary evaporation to obtain compound E.

[0094] In some embodiments, the second organic solvent comprises dichloromethane.

[0095] In some embodiments, the detergent used for washing is at least one of 1M HCl, saturated sodium bicarbonate solution, saturated sodium thiosulfate solution, and saturated sodium chloride solution.

[0096] This invention provides a linker compound with the structure 2-(toluenesulfonyl)methylacrylamide-(PEG). m -amide-(PEG) n- R1;

[0097]

[0098] Wherein, m and n are both integers and are independently selected from 0 to 24, preferably m+n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; preferably m+n = 4, preferably m is not zero, preferably n is not zero, preferably neither m nor n is zero;

[0099] R1 is the first click chemical group, which can be quickly and easily coupled with substances containing the second click chemical group R2; while the 2-(toluenesulfonyl)methylacrylamide structure can react with two free thiol groups to form a sulfur-carbon bridge structure.

[0100] It is understood that the specific selection of the linker compound is the same as described in the corresponding examples above, and will not be repeated here.

[0101] The present invention provides a method for preparing a linker as described in the foregoing embodiments, which includes the preparation of TSMA as described in any of the foregoing embodiments and / or the preparation of a linker as described in any of the foregoing embodiments.

[0102] The embodiments of the present invention provide the application of antibody conjugates as described in any of the foregoing embodiments in the preparation of diagnostic reagents or kits.

[0103] Furthermore, embodiments of the present invention provide the application of antibody conjugates as described in any of the foregoing embodiments in the preparation of reagents or kits for improving detection sensitivity.

[0104] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0105] The inventors have been dedicated to researching carrier-based signal amplification technology (a carrier can link multiple signal molecules, and antibodies indirectly link multiple signal molecules through the carrier) in order to improve the detection sensitivity of labeled antibodies. Previously, the inventors labeled antibodies using conventional processes (see Comparative Example 1 below). When developing chemiluminescent reagent-labeled antibodies, they found that introducing a carrier between the antibody and the chemiluminescent reagent for coupling amplification, while increasing the detection signal value, also significantly increased the detection signal value for blank samples (possibly due to the hydrophobicity of the chemiluminescent reagent), thus failing to improve the P / N (signal-to-noise ratio). Later, the inventors attempted a directional coupling labeling process based on antibody disulfide bonds and found that introducing a carrier again amplified the signal. The research and development idea of ​​this invention is to anchor the antibody's disulfide bonds, first reducing and opening the disulfide bonds to obtain two free thiol groups, and then using a heavy bridging reagent to connect the thiol groups to form a sulfur-carbon bridge. Commonly used heavy bridging reagents in existing technologies include disulfone compounds, maleimide compounds, pyridazine compounds, and bis(ethylenesulfonamide) compounds. Other, less frequently reported, compounds include allyl sulfones, bromopyridinidine diones, divinylpyridine, and N-substituted 3-bromo-5-methylenepyrrole-2-ones. This invention designs an allyl sulfone linker and discovers that its use in conjugating antibodies to prepare labeled antibodies, followed by amplification via a carrier, results in labeled antibodies with unexpectedly high sensitivity in immunoassays.

[0106] Example 1

[0107] 2-(Toluenesulfonyl)methacrylamide-(PEG) m -Amide and 2-(toluenesulfonyl)methacrylamide-(PEG) m -amide-(PEG) n Preparation of -R1.

[0108] Unless otherwise specified, all chemical reagents used in the synthesis were purchased from Maclean's Reagents website.

[0109] This embodiment uses m=4 as an example to illustrate the preparation method of the title compound. In fact, the title compound with other m values ​​(including positive integers with zero) can be obtained by changing the number of PEG units in the compound A used in the preparation method (compound A with other m values ​​can also be purchased from Maclean's Reagents website).

[0110] The following example describes the preparation process of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine.

[0111]

[0112] (1) Preparation of 2-(toluenesulfonyl)methacrylamide-(PEG)4-amide (TSMA)

[0113] ①

[0114] a. Dissolve tert-butoxycarbonyl-imino-tetraethylene glycol-amine (compound A, 200 mg, 0.65 mmol) in 10 mL of LDM (N,N-dimethylformamide), add methacrylic acid (compound B, 67 mg, 0.78 mmol), DCC (dicyclohexylcarbodiimide, 193 mg, 0.94 mmol) and NHS (N-hydroxysuccinimide, 119 mg, 1.03 mmol), and stir magnetically at 25 °C for 12 hours.

[0115] b. The solvent was removed by rotary evaporation. The crude product was dissolved in 10 mL of deionized water, and the precipitate was removed by filtration. After removing the solvent by rotary evaporation, the product was purified by column chromatography (ethyl acrylate: n-hexane = 1:1) to obtain tert-butoxycarbonyl-imino-tetraethylene glycol-methacrylamide (compound C) with a yield of about 90%.

[0116] In the above steps, methacrylic acid can be replaced by, but is not limited to, methacryloyl halides (such as methacryloyl chloride, methacryloyl bromide, methacryloyl fluoride, etc.) or methacrylic anhydride. When using methacryloyl halides or methacrylic anhydride, the synthesis steps are as follows:

[0117] a. Dissolve tert-butoxycarbonyl-imino-tetraethylene glycol-amine (compound A, 200 mg, 0.65 mmol) in 10 mL of dichloromethane, add triethylamine (78.8 mg, 108 μL, 0.78 mmol) and methacrylamide or methacrylic anhydride (0.78 mmol) at 0 °C, and stir magnetically for 12 hours.

[0118] b. The solvent was removed by rotary evaporation. The crude product was dissolved in 30 mL of dichloromethane and washed with 1 M HCl and saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. Purification was performed by column chromatography (ethyl acrylate: n-hexane = 1:1) to give tert-butoxycarbonyl-imino-tetraethylene glycol-methacrylamide (compound C) in approximately 90% yield.

[0119] ②

[0120] a. Dissolve compound C (200 mg, 0.53 mmol) in 5 mL of dichloromethane, add 4-toluenesulfonyl chloride (compound D, 152 mg, 0.80 mmol), and stir magnetically at 25 °C for 24 hours.

[0121] b. Triethylamine (162 mg, 223 μL, 1.60 mmol) was then added and stirred for 12 hours. The crude product was washed with 1 M HCl, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation.

[0122] c. The crude product was dissolved in 10 mL of ethyl acetate, and refluxed at 0 °C with triethylamine (162 mg, 223 μL, 1.60 mmol) for 12 hours. After removing the solvent by rotary evaporation, the product was purified by column chromatography (ethyl acrylate: n-hexane = 3:1) to give tert-butoxycarbonyl-2-(toluenesulfonyl)methacrylate (compound E) in approximately 70% yield.

[0123] In the above steps, 4-toluenesulfonyl chloride can be replaced by, but is not limited to, sodium 4-toluenesulfinate. When sodium 4-toluenesulfinate is used, steps a and b in the above synthesis process are as follows:

[0124] a. Compound C (200 mg, 0.53 mmol) was dissolved in 5 mL of dichloromethane, and sodium 4-toluenesulfinate (143 mg, 0.80 mmol) and iodine (204 mg, 0.80 mmol) were added. The mixture was then magnetically stirred at 25 °C for 72 hours.

[0125] b. Triethylamine (162 mg, 223 μL, 1.60 mmol) was then added and stirred for 12 hours. The crude product was washed with 1 M HCl, saturated sodium bicarbonate solution, saturated sodium thiosulfate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation.

[0126] Step c is the same.

[0127] ③ Compound E (26.7 mg, 0.05 mmol) was dissolved in 5 mL of dichloromethane, and trifluoroacetic acid (278 mg, 2.50 mmol) was added. The mixture was magnetically stirred at 25 °C for 12 hours, and the solvent was removed by rotary evaporation to obtain TSMA with a yield of approximately 98%.

[0128] (2) Preparation of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine (TSMA-MTz)

[0129] ① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of dichloromethane, add DIEA (N,N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), and stir until homogeneous. Dissolve methyltetraazine-PEG4-NHS ester (compound F, purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 16.7 mg, 0.03 mmol) in 1 mL of dichloromethane, and add it to the above solution of TSMA after homogeneity.

[0130] ② The mixture was magnetically stirred at 25°C for 12 hours, the solvent was removed by rotary evaporation, the crude product was dissolved in 20 mL of dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and purified by column chromatography (dichloromethane:methanol = 20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine (compound G, TSMA-MTz), with a yield of about 50%.

[0131] The product was detected by liquid chromatography-mass spectrometry and nuclear magnetic resonance. The results are attached. Figure 4-8 This proves that the method successfully prepared 2-(toluenesulfonyl)methylacrylamide-PEG4-amide-PEG4-methyltetraazine.

[0132] The above describes the preparation method of the title compound using n=4 as an example. In practice, title compounds with different n values ​​(including positive integers such as zero) can be synthesized using methyltetraazine-PEGn-NHS esters containing different numbers of PEG units. Furthermore, the methyltetraazine here can be replaced by other tetrazine groups, such as tetrazine (unless otherwise specified, all click chemical group-PEGn-NHS compounds used in this article can be customized from Xi'an Kangfuno Biotechnology Co., Ltd.). For example, during preparation, following the previous steps, with other synthetic steps unchanged, only the methyltetraazine-PEG4-NHS ester needs to be replaced with equimolar amounts of methyltetraazine-NHS ester, tetrazine-PEG4-NHS ester, and tetrazine-NHS ester to synthesize 2-(toluenesulfonyl)methacrylamide-PEG4-amide-methyltetraazine, 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-tetraazine, and 2-(toluenesulfonyl)methacrylamide-PEG4-tetraazine, respectively.

[0133] The compound in this embodiment can also be synthesized in an aqueous phase. An exemplary step for aqueous phase synthesis is as follows:

[0134] Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir until homogeneous. Dissolve methyltetraazine-PEG4-NHS ester (compound F, purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 16.7 mg, 0.03 mmol) or an equimolar amount of tetraazine-PEG4-NHS ester in 1 mL of deionized water, and add the homogeneous solution to the above TSMA solution. Stir the mixture magnetically at 25 °C for 12 hours to obtain an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine or 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-tetraazine.

[0135] (3) Preparation of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(4E)-transcyclooctene (TSMA-TCO).

[0136]

[0137] ① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of dichloromethane, add DIEA (N,N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), and stir until homogeneous. Dissolve (4E)-trans-cyclooctene-PEG4-NHS ester (purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 15.4 mg, 0.03 mmol) in 1 mL of dichloromethane, and add it to the above solution of TSMA after homogeneity.

[0138] ② The mixture was magnetically stirred at 25°C for 12 hours, the solvent was removed by rotary evaporation, the crude product was dissolved in 20 mL of dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and then purified by column chromatography (dichloromethane:methanol = 20:1) to obtain 2-(toluenesulfonyl)methylacrylamide-PEG4-(4E)-transcyclooctene (TSMA-TCO).

[0139] The above describes the preparation method of the title compound using n=4 as an example. Different n values ​​can be synthesized using (4E)-trans-cyclooctene-PEGn-NHS esters containing different numbers of PEG units, to produce 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(4E)-trans-cyclooctene. (4E)-trans-cyclooctene can also be replaced with (2E)-trans-cyclooctene. For example, during preparation, following the previous steps, other synthetic steps remain unchanged; only the (4E)-trans-cyclooctene-PEG4-NHS ester needs to be replaced with equimolar amounts of (4E)-trans-cyclooctene-NHS ester and (2E)-trans-cyclooctene-PEG4-NHS ester to synthesize 2-(toluenesulfonyl)methacrylamide-PEG4-amide-(4E)-trans-cyclooctene and 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(2E)-trans-cyclooctene, respectively.

[0140] The compound of this embodiment can also be synthesized in an aqueous phase. Exemplary steps for aqueous phase synthesis are as follows:

[0141] Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir until homogeneous. Dissolve (4E)-trans-cyclooctene-PEG4-NHS ester (purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 15.4 mg, 0.03 mmol) or an equimolar amount of (2E)-trans-cyclooctene-PEG4-NHS ester in 1 mL of deionized water, and add the homogeneous solution to the above TSMA solution. Stir the mixture magnetically at 25 °C for 12 hours to obtain an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(4E)-trans-cyclooctene or 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(2E)-trans-cyclooctene.

[0142] (4) Preparation of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-azide (TSMA-N3)

[0143] ① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of dichloromethane, add DIEA (N,N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), and stir until homogeneous. Dissolve 0.03 mmol of azide-PEG4-NHS ester in 1 mL of dichloromethane, and add it to the above solution of TSMA.

[0144] ② The mixture was magnetically stirred at 25°C for 12 hours, the solvent was removed by rotary evaporation, the crude product was dissolved in 20 mL of dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and then purified by column chromatography (dichloromethane:methanol = 20:1) to obtain 2-(toluenesulfonyl)methylacrylamide-PEG4-azide (TSMA-N3).

[0145] The above describes the preparation method of the title compound of this embodiment using n=4 as an example. In practice, 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEGn-azide with different n values ​​can be synthesized using azide-PEGn-NHS esters containing different numbers of PEG units (the azide-PEGn-NHS esters here can be customized from Shanghai Pengshuo Biotechnology Co., Ltd.). For example, during preparation, referring to the previous steps, other synthesis steps remain unchanged, only needing to replace the azide-PEG4-NHS ester with equimolar amounts of azide-PEG2-NHS ester and azide-PEG8-NHS ester, respectively, to synthesize 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG2-azide and 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG8-azide, respectively.

[0146] The compound of this embodiment can also be synthesized in an aqueous phase. Exemplary steps for aqueous phase synthesis are as follows:

[0147] TSMA (13 mg, 0.03 mmol) was dissolved in 2 mL of deionized water, and the pH was adjusted to 7.0-8.0. The solution was stirred until homogeneous. Azide-PEG4-NHS ester (0.03 mmol) was dissolved in 1 mL of deionized water, and the solution was added to the TSMA solution. The mixture was magnetically stirred at 25 °C for 12 hours to obtain an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-azide.

[0148] (5) Preparation of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-DBCO (TSMA-DBCO)

[0149] ① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of dichloromethane, add DIEA (N,N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), and stir until homogeneous. Dissolve DBCO-PEG4-NHS ester (purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 0.03 mmol) in 1 mL of dichloromethane, and add it to the above solution of TSMA after homogeneity.

[0150] ② The mixture was magnetically stirred at 25°C for 12 hours, the solvent was removed by rotary evaporation, the crude product was dissolved in 20 mL of dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and then purified by column chromatography (dichloromethane:methanol = 20:1) to obtain 2-(toluenesulfonyl)methylacrylamide-PEG4-amide-PEG4-DBCO (TSMA-DBCO).

[0151] The above describes the preparation method of the title compound in this embodiment using n=4 as an example. In practice, DBCO-PEGn-NHS esters containing different numbers of PEG units can be used to synthesize 2-(toluenesulfonyl)methylacrylamide-PEG4-amide-PEGn-DBCO with different n values. For example, during preparation, following the previous steps, other synthesis steps remain unchanged; only the DBCO-PEGn-NHS ester needs to be replaced with equimolar amounts of DBCO-NHS ester and DBCO-PEG2-NHS ester to synthesize 2-(toluenesulfonyl)methylacrylamide-PEG4-DBCO and 2-(toluenesulfonyl)methylacrylamide-PEG4-amide-PEG2-DBCO, respectively. All the above raw materials can be purchased from Xi'an Kangfuno Biotechnology Co., Ltd.

[0152] The compound of this embodiment can also be synthesized in an aqueous phase. Exemplary steps for aqueous phase synthesis are as follows:

[0153] TSMA (13 mg, 0.03 mmol) was dissolved in 2 mL of deionized water, and the pH was adjusted to 7.0-8.0. The solution was stirred until homogeneous. DBCO-PEG4-NHS ester (purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 0.03 mmol) was dissolved in 1 mL of deionized water and added to the TSMA solution. The mixture was magnetically stirred at 25 °C for 12 hours to obtain an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-DBCO.

[0154] Example 2

[0155] Preparation of acridinium ester-support-trans-cyclooctene (AE-support-TCO)

[0156] This embodiment uses acridine ester as an example of a chemiluminescent reagent, but other chemiluminescent reagents can also be used, especially those that do not require enzyme assistance, such as N-(4-aminobutyl)-N-ethylisoluminol. In this embodiment, the carrier can be a protein carrier or a chemical polymer carrier. The protein carrier can be bovine serum albumin (BSA), alkaline phosphatase (AP), horseradish peroxidase, chicken ovalbumin (OVA), hemocyanin (KLH), etc.; the polymer carrier can be cross-linked sucrose, dextran, polylysine, polyethylene glycol, polyethyleneimine, and polypropyleneimine, etc. Protein carriers are preferred because the properties of protein carrier coupling products are generally more stable. In this embodiment, acridine ester and trans-cyclooctene are simultaneously modified and linked on the carrier, and the acridine ester is indirectly linked to trans-cyclooctene through the carrier. In this embodiment, one carrier is modified and linked with multiple acridine esters, which can improve the sensitivity of the reagent. The modification method of acridine ester is described below using BSA as an example; the modification methods of other carriers are similar.

[0157] Bovine serum albumin (5 mg / mL) was dissolved in 100 mM pH 7.4 PBS buffer, and 10 mM TCO-PEG4-NHS (10 eq, dissolved in DMSO) was added. The mixture was reacted at 25°C for 1 hour. Excess TCO-PEG4-NHS was disposed of using Zeba. TM Desalting was performed using a 10 kMWCO desalting column, followed by replacement with 100 mM pH 7.4 PBS buffer. Then, 10 mM acridine ester-NHS (20 eq, dissolved in DMSO) was added, and the reaction was carried out at 25°C for 1 hour. Excess acridine ester-NHS was disposed of using Zeba. TM The desalting column (10K MWCO) was used for desalting, and the solution was replaced with PB (50mM, pH 7.4) buffer. AE-BSA-TCO was prepared, diluted to 4 mg / mL with PB (50mM, pH 7.4) buffer, and stored at 4°C for later use.

[0158] Example 3

[0159] Reduction of antibody disulfide bonds.

[0160] The embodiments of the present invention use IgG1 type CA153 antibody.

[0161] Antibody (5 mg / mL) with Zeba TM The desalting column (10K MWCO) was replaced with PB (50 mM, pH 7.4, 1 mM EDTA) buffer. TCEP (Sigma) was prepared into a 10 mM solution using PB (50 mM, pH 7.4, 1 mM EDTA). 2 eq of TCEP was added to the antibody solution, and the reaction was carried out at 37°C with gentle shaking (400 rpm) for 2 hours. The reduced antibody was stored at 4°C for later use.

[0162] TCEP can be reduced at the antibody disulfide bond to obtain two free thiol groups. Subsequently, a linker molecule with a 2-(toluenesulfonyl)methylacrylamide structure is added. Through the linkage reaction between 2-(toluenesulfonyl)methylacrylamide and the two free thiol groups, they can be reconnected to form a sulfur-carbon bridge bond.

[0163] Example 4

[0164] Reconstruction of antibody sulfur-carbon bridges.

[0165] TSMA-MTz (2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetraazine) was prepared into a 10 mM solution with DMSO under dry conditions. 20 eq of TSMA-MTz was added to the antibody solution obtained in Example 3, and the reaction was carried out at 4°C for 16 hours. Excess chemicals in the antibody solution were removed using Zeba. TM The desalting column (10K MWCO) was replaced with PB (50mM, pH 7.4) buffer. The antibody-linker-methyltetrazine (antibody-MTz) was prepared, diluted to 4 mg / mL with PB (50mM, pH 7.4), and stored at 4°C for later use.

[0166] Example 5

[0167] Preparation of antibody conjugates with directional cross-linking of conjugation partners.

[0168] (1) Preparation of antibody-acridinium ester conjugate: Antibody-linker-methyltetraazine (4 mg / mL) and acridinium ester-trans-cyclooctene (4 mg / mL, dissolved in DMSO) were cross-linked at a molar ratio of 1:10 and reacted at 25°C for one hour to obtain antibody-acridinium ester conjugate 1 of the present invention. After the reaction was complete, it was stored at 4°C for later use.

[0169] (2) Preparation of antibody-BSA-acridone ester conjugate: Antibody-linker-methyltetraazine (4 mg / mL) and acridine ester-BSA-trans-cyclooctene (4 mg / mL) were cross-linked at a molar ratio of 1:1 and reacted at 25°C for one hour to obtain antibody-BSA-acridone ester conjugate 1 of the present invention. After the reaction was complete, it was stored at 4°C for later use.

[0170] Comparative Example 1

[0171] A conventional method for preparing antibody conjugates is provided, as detailed below.

[0172] The conventional principle for preparing antibody conjugates in the IVD field is to use the amino or thiol groups on the side chains of protein amino acids as coupling sites.

[0173] The conventional method for preparing antibody-acrididine ester conjugates is as follows:

[0174] Antibody (5 mg / mL) with Zeba TM The desalting column (10K MWCO) was replaced with PBS (10mM PB, 50mM sodium chloride, pH 7.4, 5mM EDTA) buffer. 10mM NHS-AE (10 eq, dissolved in DMSO) was added to 30µM of the antibody solution, and the reaction was carried out at 25°C for 1 hour. After the reaction, excess reagents were removed by desalting to obtain antibody-acrididium ester conjugate 2, which was stored at 4°C for later use.

[0175] The conventional method for preparing antibody-BSA-acrididine ester conjugates is as follows:

[0176] (1) Thioylation modification of antibodies (IgG-SH):

[0177] Antibody (5 mg / mL) with Zeba TM The desalting column (10K MWCO) was replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0, 5 mM EDTA). Traut's reagent (Pierce) was prepared into a 10 mM solution using PBS (100 mM PB, 50 mM sodium chloride, pH 7.4, 1 mM EDTA). 10 eq of Traut's reagent was added to the antibody solution, and the reaction was carried out with gentle shaking (400 rpm) at 25°C for 2 hours. Excess reagent was removed by desalting, and the thiol-modified antibody was stored at 4°C for later use.

[0178] (2) Maleimide modification of BSA (BSA-MH):

[0179] Bovine serum albumin (5 mg / mL) was administered using Zeba. TM The desalting column (10K MWCO) was replaced with PBS (10mM PB, 50mM sodium chloride, pH 7.4, 5mM EDTA) buffer. 10mM SMCC (10 eq, dissolved in DMSO) was added to 40µM alkaline phosphatase solution, and the reaction was carried out at 25°C for 1 hour. Excess reagents were removed by desalting, and the maleimide-modified AP was stored at 4°C for later use.

[0180] (3) Acridinium ester modification of BSA-MH (AE-BSA-MH):

[0181] BSA-MH (5 mg / mL) with Zeba TMThe desalting column (10K MWCO) was prepared in PBS (10mM PB, 50mM sodium chloride, pH 7.4, 5mM EDTA) buffer. 10mM NHS-AE (10 eq, dissolved in DMSO) was added to 30µM BSA-MH solution, and the reaction was carried out at 25°C for 1 hour. After the reaction, excess reagents were removed by desalting, and the product was stored at 4°C for later use.

[0182] (4) Crosslinking of IgG-SH and AE-BSA-MH:

[0183] The thiol-modified antibody (4 mg / mL) and AE-BSA-MH (4 mg / mL) were reacted in PBS (10 mM PB, 50 mM sodium chloride, pH 7.4) buffer at 25 °C for 1 hour. After the reaction was complete, antibody-BSA-acrididine ester conjugate 2 was obtained and stored at 4 °C for later use.

[0184] Verification Example 1

[0185] Sensitivity assessment of acridine ester-labeled antibody conjugates.

[0186] Sensitivity tests were performed on antibody-acridinium ester label 2 and antibody-BSA-acridinium ester label 2 prepared by conventional processes, and antibody-acridinium ester label 1 and antibody-BSA-acridinium ester label 1 prepared according to the present invention. The sensitivities for low-value samples (1 U / mL) were 4.4, 4.4, 10.5, and 19.2, respectively. Without a BSA carrier, the antibody conjugates prepared using the technique of the present invention showed a 136% increase in sensitivity compared to conventional processes. Furthermore, by adding a BSA carrier to the disulfide bond reconstruction process, the sensitivity was further increased by 84.6%.

[0187] Table 1. Sensitivity of acridine ester labeled compounds (CA153)

[0188]

[0189]

[0190] Performance testing

[0191] The antibody conjugates of the present invention and those produced by conventional processes were diluted with enzyme-labeled diluent to prepare enzyme-labeled working solutions. These solutions were then combined with magnetic bead working solutions to form detection reagents. The reagents were tested on a Phytonishine series fully automated chemiluminescence immunoassay analyzer to evaluate their sensitivity.

[0192] Internal reference samples for different projects were tested twice to obtain relative luminescence (RLU) values, and the mean RLU was calculated. The sensitivity (P / N) was calculated by the ratio of the mean RLU values ​​of different samples.

[0193] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody conjugate, characterized in that, The system comprises a sequentially linked antibody-linker-vector-conjugation partner, wherein the linker is directionally coupled to the antibody via interchain disulfide bonds, and the linker and antibody are connected via interchain sulfur-carbon bridges, the sulfur-carbon bridges having an SCCCS structure, which includes the following structures: ； Where n and m are both integers and neither is zero, and 4≤m+n≤24.

2. An antibody-linker-vector, characterized in that, It includes the following structure: ； The linker and the antibody are directionally coupled by interchain disulfide bonds, and the linker and the antibody are connected by a sulfur-carbon bridge, which has an SCCCS structure. Where n and m are both integers and neither is zero, and 4≤m+n≤24.

3. The antibody conjugate according to claim 1 or the antibody-linker-vector according to claim 2, characterized in that, The sulfur-carbon bridges in the structure are located between antibody hinge regions, between antibody light and heavy chains, or between antibody heavy chains.

4. The antibody conjugate according to claim 1 or the antibody-linker-vector according to claim 2, characterized in that, The carrier is selected from at least one of protein carriers, polysaccharides, polylysine, polyethylene glycol, polyethyleneimine, and polypropyleneimine.

5. The antibody conjugate or antibody-linker-vector according to claim 4, characterized in that, The polysaccharide includes cross-linked sucrose or dextran.

6. The antibody conjugate or antibody-linker-vector according to claim 4, characterized in that, The protein carrier includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, thyroglobulin, horseradish peroxidase, and alkaline phosphatase.

7. The antibody conjugate according to claim 1, characterized in that, The conjugation partner is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle labels.

8. A method for preparing the antibody conjugate according to any one of claims 1, 3, 4, 5, 6, and 7, characterized in that, It includes: The antibody-linker and R2-vector-conjugation partner were subjected to an addition reaction to obtain the antibody conjugate; The antibody-linker has the following structure: R1 is the first click chemical group, and R2 is the second click chemical group; The preparation method of antibody-linker is as follows: The linker and the antibody whose disulfide bond has been reduced and opened are mixed and reacted to obtain the antibody-linker; the linker has the following structure: 。 9. The use of the antibody conjugate according to any one of claims 1, 3, 4, 5, 6, and 7 in the preparation of diagnostic reagents or kits.

10. The use of the antibody-linker-vector according to any one of claims 2, 3, 4, 5, and 6 in the preparation of diagnostic reagents or kits.

11. A reagent kit, characterized in that, It includes the antibody conjugates according to any one of claims 1, 3, 4, 5, 6, and 7.

12. A reagent kit, characterized in that, It includes the antibody-linker-vector according to any one of claims 2, 3, 4, 5, and 6.

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