WO2023231889A1

WO2023231889A1 - Antibody conjugate and use thereof

Info

Publication number: WO2023231889A1
Application number: PCT/CN2023/096265
Authority: WO
Inventors: 钟志荣; 陈彬; 林芳博; 王静文; 党海滨; 刘青昀; 孟媛
Original assignee: 菲鹏生物股份有限公司
Priority date: 2022-05-29
Filing date: 2023-05-25
Publication date: 2023-12-07
Also published as: CN117147826A; CN117147824A

Abstract

An antibody conjugate and the use thereof, which relates to the technical field of antibody conjugation. Provided is a technique for directional modification of a disulfide bond of an antibody. The antibody is efficiently conjugated to a conjugation partner via a linker. The prepared antibody conjugate has good uniformity, has obvious advantages, such as high detection sensitivity and good stability compared with an antibody conjugate prepared by means of a conventional process, and provides a route for the development and application of diagnostic reagents.

Description

An antibody conjugate and its application

Cross-references to related applications

This disclosure requires a Chinese patent application with application number CN202210595445.3 and titled "Antibody Conjugate and Applications" submitted to the China Patent Office on May 29, 2022. The Chinese patent application was submitted on September 27, 2022. The application number of the China Patent Office is CN202211181319. The Chinese patent application "Antibody conjugate and its application" and the Chinese patent application with the application number CN202310508396.X and the name "Antibody conjugate and its application" submitted to the China Patent Office on May 6, 2023 priority, the entire contents of which are incorporated by reference into this disclosure.

Technical field

The present disclosure relates to the field of antibody conjugation technology, specifically, an antibody conjugate and its application.

Background technique

In the diagnostic field, EDC, NHS or maleimide (MAL) are usually used to couple the carboxyl, amino or sulfhydryl groups of proteins. This protein coupling technology has two problems, which affect the sensitivity and precision of the reagents. First, the randomness of the coupling site: Taking the IgG1 type antibody as an example, it contains an average of 80 lysines, 20 of which are in solvent-accessible parts, and some solvent-accessible lysines are in the antigen recognition region of Fab . Therefore, when the amino group of an antibody is used as the coupling site, the random distribution of amino groups in the antibody will lead to inhomogeneity of the antibody conjugate and inactivation of the antibody. Second, the coupling efficiency is low: the secondary reaction kinetics of NHS and amino groups is 10 ^-1 ~ 10 ² M ^-1 S ^-1 , and the conjugate yield is 40% when equilibrium is reached. The product yield is low, resulting in the loss of raw materials. Waste and affect the sensitivity of reagents.

The secondary reaction kinetics of maleimide and thiol groups reaches 10 ² ~ 10 ³ M ^-1 S ^-1 , and the conjugate yield is 80%, but the cysteine residue of the antibody exists in the form of a disulfide bond , there are very few free sulfhydryl groups accessible to the solvent. Another method is to use Traut's reagent (2-iminothiolane) to convert the amino group of the antibody into a thiol group, but there are still problems with low conversion efficiency and random coupling sites.

Contents of the invention

The purpose of this disclosure is to provide an antibody conjugate and its application.

This disclosure is implemented as follows:

The present disclosure provides an antibody conjugate suitable for immunodiagnosis, which includes the following structure:

From left to right are conjugation partners and linkers (i.e. ) and antibodies;

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.

The connection method between the conjugation partner and the oxygen atom in the linker is not limited. The conjugation partner may be directly coupled to the oxygen atom with certain inherent groups, or may be indirectly coupled to the oxygen atom through a connecting functional group. Whether direct or indirect coupling is used, it is for the purpose of connection.

Optionally, the conjugation partner and the linker are connected via a linking functional group, including but not limited to pairs of NHS (N-hydroxysuccinimide) and amino groups, or pairs of MAL (horse imide) and sulfhydryl groups, or pairs of orthogonal click chemical groups; if orthogonal click chemical groups are used, the reaction efficiency of obtaining the antibody conjugate of the present disclosure from the raw material reaction coupling will be higher, while using The reaction rate of other coupling methods is slower and may produce more by-products, but those skilled in the art can also make corresponding adjustments through purification and separation to obtain the same final product.

Optionally, the conjugation partner and the linker are connected via pairs of orthogonal click chemistry groups, and the antibody conjugate includes the following structure:

From left to right are conjugation partners, linkers and antibodies;

Wherein, the R ₁ is a first click chemical group, and the R ₂ is a second click chemical group.

Optionally, there is an amplification carrier between the conjugate partner and the antibody, and the antibody conjugate includes the following structure:

Optionally, the amplification carrier is a substance having both a disulfide bond and an amino group. Optionally, the amplification carrier is selected from bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

Optionally, the amplification carrier is a carrier modified by a re-bridging agent. The re-bridging agent can be the reconstituted cross-linking agent disclosed in the present disclosure for coupling an antibody with reduced disulfide bonds, or it can be a current cross-linking agent. Any disulfide bond rebridging agent known in the art, such as a: mono/disulfone reagent, b: 3,4-disubstituted maleimide, c: dibromopyridazinedione and d: dibromopyridazinedione Vinylpyrimidine;

At this time, the antibody conjugate includes the following structure:

Among them, L can represent absence, that is, the X group is directly connected to the oxygen atom, or L can represent a connecting group, that is, the X group and the oxygen atom are indirectly connected through the connecting group;

Optionally, a structure in which the X group and two adjacent S atoms jointly form Includes one of the following structures:

The present disclosure provides an antibody-linker suitable for immunodiagnosis, which includes the following structure:

Among them, R ₁ is the first click chemical group, 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.

For the antibody conjugates or antibody-linkers described in the present disclosure, the number of linkers in the structure is not limited and may have one or more linkers. That is, the antibody can have multiple disulfide bonds, and each disulfide bond can be independently reduced to a sulfhydryl group and then reconstituted with the linker to form a sulfur-carbon bridge. In this way, an antibody can be connected to multiple linkers, and each linker can be connected to a conjugation partner independently, that is, a (conjugation partner-linker) x-antibody structure is formed, where x is an integer greater than or equal to 1.

The present disclosure provides a method for preparing an antibody conjugate as described in the previous embodiment, which includes: performing an addition reaction between an antibody-linker and an R ₂ -conjugation partner to obtain an antibody conjugate; the antibody-linker The sub has the following structure:

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.

The present disclosure provides the use of antibody conjugates or antibody-linkers as described in the preceding embodiments in the preparation of diagnostic reagents or kits.

The present disclosure provides a kit comprising an antibody conjugate or antibody-linker as described in the preceding examples.

For the "linker" described in the present disclosure, it exists in a form without forming a conjugate, that is, it has the following structure There are also forms that form conjugates that have the following structure

For the "conjugation partner-R ₂ (also written as R ₂ -conjugation partner)" described in the present disclosure, or the "conjugation partner-R ₂ (also written as R 2 -conjugation partner) contained in the antibody conjugate structure described in the present disclosure It can be written as R ₂ -conjugation partner)" unit. The horizontal bar "-" between R ₂ and the conjugation partner does not limit whether R ₂ is directly bonded to the conjugation partner. R ₂ can be directly bonded to the conjugation partner. R ₂ The conjugation partner can be indirectly connected through small molecule chemical structural units, or indirectly through macromolecular biological materials. Experimental data later proves that these will not affect the detection sensitivity of the final prepared antibody conjugate.

For antibody conjugates described in the present disclosure, if there is no other specific description of the structure (e.g., the conjugation partner-amplification carrier-linker structure is explicitly stated), the conjugation in the "conjugation partner-linker" structure is considered There may be no amplification carrier between the partner and the linker, or there may be an amplification carrier. Therefore, for the "conjugation partner-linker" described in the present disclosure, the number of conjugation partners connected to the same linker in the structure is not limited, and the same linker can be connected to one conjugation partner (no amplification vector) or multiple conjugation partners (with amplification vectors). That is, the conjugation partner can be indirectly connected to the linker through the amplification carrier to form (conjugation partner) y-amplification carrier-linker-antibody structure. y is an integer greater than or equal to 1. One amplification carrier can connect multiple conjugation partners. In this way, the same linker can connect to multiple conjugation partners, which can amplify the detection signal of the sample by the conjugated antibody. If the antibody is also connected to multiple linkers at this time, ((conjugation partner)y-amplification carrier-linker)x-antibody structure is formed, x and y are integers greater than or equal to 1, and x (conjugation partners) The value of y for each (conjugation partner) y-amplifying vector-linker structure in the y-amplifying vector-linker structure is independent of each other.

For the "reconstructing cross-linking agent" (or disulfide bond reconstructing agent) and "heavy bridging agent" (or disulfide bond rebridging agent) described in this disclosure, their chemical functions are the same. Reconstruct the disulfide bonds opened by reduction to form sulfur-carbon bridge bonds. Just for the sake of differentiation and understanding, the reagents that act on antibodies are called reconstituting cross-linking agents, and the reagents that act on amplification carriers are called re-bridging agents . The invention of the present disclosure relates to the structure of the remodeling cross-linking agent acting on the antibody, but the invention has nothing to do with the structure of the re-bridging agent acting on the amplification carrier. Therefore, the structure of the re-bridging agent of the present disclosure can be related to the structure of the re-bridging agent of the present disclosure. The structure of the reconstructed cross-linking agent is the same, and it can also be other known heavy bridging agents in the prior art (such as mono/bis sulfone reagents, 3,4-disubstituted maleimide, dibromopyridazine di Ketones, divinylpyrimidines, etc.).

For the schematic structures of antibody conjugates depicted in this disclosure, both well-defined chemical moieties (e.g., linkers) and undefined chemical moieties (e.g., conjugate partners, amplification vectors, antibodies) are included in the structure, and the schematic structures are indicative of the invention. A simplified representation of the points, omitting the "linking groups" that exist between parts of definite chemical structure and parts of ambiguous chemical structure, and between parts of ambiguous chemical structure and parts of unclear chemical structure. A "linking group" may therefore be implicit in the schematic structures of the antibody conjugates of the present disclosure.

"Linking group" as used in this disclosure refers to a chemical moiety that may contain about 2 to about 50 atoms or 4 to about 30 atoms (excluding hydrogen), and may contain 2 to about 30 atoms, or a chain of 3 to about 20 atoms, each independently selected from the group consisting generally of carbon, oxygen, sulfur, nitrogen and phosphorus. In some examples, some or all of the linking groups may be part of the linked molecule, such as, but not limited to, for example, amino acid residues on a poly(amino acid). The number of heteroatoms in the linking group can be from 0 to about 20, or from 1 to about 15, or from about 2 to about 10. The linking group may be aliphatic or aromatic. When heteroatoms are present, oxygen is usually present as an oxo or oxy group bonded to carbon, sulfur, nitrogen, or phosphorus, and nitrogen is usually present as a nitro, nitroso, or amino group usually bonded to carbon, oxygen, sulfur, or phosphorus present; sulfur is similar to oxygen; and phosphorus is typically bonded to carbon, sulfur, oxygen, or nitrogen as phosphonate mono- or diesters and phosphate mono- or diesters. Common functional groups that form covalent bonds between the linking group and the molecule to be conjugated are alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azos, thioethers and carboxylates, sulfonates Acid and phosphate esters, amides and thioesters.

In most cases, the linking group further has linking functional groups (functional groups for reaction with the group), including non-oxycarbonyl groups of nitrogen and sulfur analogs, phosphate ester groups, amino groups, alkylation agents such as halogenated or tosylalkyl, oxy (hydroxy or sulfur analogs, thiol) oxycarbonyl (e.g., aldehydes or ketones), or reactive olefins such as vinyl sulfone or α-, β-unsaturated esters , these functional groups can be attached to amine groups, carboxyl groups, reactive olefins, alkylating agents such as bromoacetyl groups. When linking amines and carboxylic acids or their nitrogen derivatives or phosphoric acids, amides, amidines and phosphoramides can be formed. When mercaptans and activated alkenes are linked, thioethers are formed. When a thiol and an alkylating agent are linked, a thioether is formed. When aldehydes and amines are linked under reducing conditions, alkylamines are formed. When connecting a ketone or aldehyde and a hydroxylamine (including derivatives thereof in which a substituent replaces the hydrogen of the hydroxyl group), an oxime functionality (=N-O-) is formed. When a carboxylic acid or phosphoric acid and an alcohol are linked, an ester is formed. When the linking functional group is, for example, a click chemical group, including methyltetrazine, trans-cyclooctene, azide, dibenzocyclooctyne, tetrazine, alkyne, cyclopropanecyclooctyne, cyclopropene, etc., it Can be linked to pairs of orthogonal click chemistry groups.

The disclosure has the following beneficial effects:

The present disclosure provides a technology for directional modification of the disulfide bonds of antibodies for coupling, which includes using a disulfide bond-reconstructing cross-linking agent (i.e., 2-(toluene) with a linker of the disclosure as shown in the following structure Sulfonyl)methacrylamide-(PEG) _m -amide-(PEG) _n ),

As follows Reactive coupling of antibodies with reduced disulfide bonds to high Effectively coupled antibodies and conjugation partners, the antibody conjugates prepared by it have good uniformity, and have obvious advantages such as high detection sensitivity and good stability compared with antibody conjugates prepared by conventional processes, and are ideal for diagnostic reagents. development and application.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present disclosure more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present disclosure and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the antibody disulfide bond being reconstructed again after being reduced by a reducing agent;

Figure 2 shows the results based on gel electrophoresis showing the number of disulfide bond reductions RDAR (lanes 1 to 3) of the antibody and the number of disulfide bond reductions after religation by TSMA (lanes 4 to 6);

Figure 3 shows the effect of TCEP concentration on the number of antibody disulfide bond reduction based on gel electrophoresis;

Figure 4 is a chromatogram of 2-(tosyl)methacrylamide-PEG4-amide-methyltetrazine;

Figure 5 is a mass spectrum of 2-(tosyl)methacrylamide-PEG4-amide-methyltetrazine;

Figure 6 is a mass spectrum peak analysis diagram of 2-(tosyl)methacrylamide-PEG4-amide-methyltetrazine;

Figure 7 is a nuclear magnetic resonance carbon spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-methyltetrazine;

Figure 8 is a hydrogen nuclear magnetic resonance spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-methyltetrazine.

Detailed ways

In order to make the purpose, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Embodiments of the present disclosure provide an antibody conjugate comprising the following structure:

The structure includes sequentially connected conjugation partners, linkers and antibodies;

Wherein, n and m are integers and independently selected from 0 to 24. Optionally, 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, optionally m is not zero, optionally, n is not zero, optionally, both m and n are Non-zero, optionally, m+n≥4.

In some embodiments, there is a linking functional group between the conjugation partner and the linker, the linking functional group is selected from a pair of NHS (N-hydroxysuccinimide) and an amino group, or a pair of MAL (male imide) and sulfhydryl groups, or pairs of orthogonal click chemistry groups.

In some embodiments, there are pairs of orthogonal click chemistry groups between the conjugation partner and the linker, and in this case, the conjugation partner has the following structure:

Wherein, the R ₁ is a first click chemical group, the R ₂ is a second click chemical group, and the first click chemical group and/or the second click chemical group are selected from: methyl Any of tetrazine, trans-cyclooctene, azide, dibenzocyclooctyne, tetrazine, alkyne, cyclopropanecyclooctyne and cyclopropene, The definitions of m and n are the same as above;

In some embodiments, the first click chemistry group and the second click chemistry group are mutually exclusive selected from any one of methyltetrazine and trans-cyclooctene.

The antibody conjugate provided by the present disclosure consists of a conjugation partner, a linker and an antibody chemical coupling. The conjugation partner can be modified with N-hydroxysuccinimide, amino, maleimide, sulfhydryl or click chemistry. The group R ₂ is optionally modified with a click chemistry group R ₂ ; and the linker can also be modified with N-hydroxysuccinimide, amino, maleimide, thiol or click chemistry group R ₂ , optionally modified with a click chemistry group R ₁ , in which case the linker is 2-(tosyl)methacrylamide-(PEG) _m -amide-(PEG) _n -R ₁ , which is as follows:

R ₁ and R ₂ are orthogonal pairs of click chemical groups that can be combined and connected based on click chemical reactions. The 2-(toluenesulfonyl)methacrylamide structure is a reconstructed cross-linking agent that can free the antibody. The sulfhydryl groups are re-bridged together to form the carbon-sulfur bond of SCS, see Figure 1. The number of PEG in the linker, that is, the value of m+n is preferably 0 to 24. The number of PEG in the linker can adjust the distance between the conjugation partner and the antibody. If the distance is too close, it will affect the spatial structure of the antibody. If the distance is too far, it will affect the spatial structure of the antibody. Can affect the stability of antibody conjugates.

In some embodiments, n and m are both integers and are independently selected from 0 to 24, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Any one of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or the range between any two. Commonly used reconstituting cross-linkers and click chemical groups are highly hydrophobic organic compounds, which are difficult to access the disulfide bond positions of water-soluble antibodies. For example, the divinylsulfonamide disclosed in CN107400072B as a reconstruction cross-linking agent has poor water solubility, which affects the efficiency of disulfide bond reconstruction. This disclosure introduces PEG molecules into the reconstituted cross-linking agent through design, which improves the water solubility, increases the efficiency of disulfide bond reconstruction, and also greatly improves the detection signal intensity of the final conjugated product.

In some embodiments, the sulfur-carbon bridge in the structural formula of the antibody conjugate is located between the antibody hinge region, between the antibody light and heavy chains, or between the antibody heavy chains. The distance between the heavy chains of the antibody includes between CH1 and between Fc regions.

In some embodiments, the conjugation partner and the linker are indirectly connected through an amplification carrier selected from the group consisting of polysaccharides, polylysine, protein carriers, PEG (polyethylene glycol), polyethylenimine, and dendrites. at least one of the polymers. Specifically, polysaccharides include cross-linked polysucrose and dextran; protein carriers include at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin; dendrimers Includes polyethylene glycol and/or polypropyleneimine. In some embodiments, the amplification vector includes at least one of bovine serum albumin, human serum albumin, ovalbumin, keyhole hemocyanin, and thyroglobulin.

The antibodies described herein are construed 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, so long as they exhibit the desired biological activity and have The disulfide bond can cross-link with the 2-(toluenesulfonyl)methacrylamide structure after being reduced to form a sulfur-carbon bridge.

In some embodiments, R ₁ and R ₂ are click chemistry groups capable of orthogonal pairing. Click chemistry is an orthogonal connection reaction, and there are two commonly used types: the first is copper-catalyzed azide-alkynyl cycloaddition reaction (CuAAC) or copper-free azido-dibenzocyclooctyne Strain-promoted cycloaddition reaction (SPAAC); the second is tetrazine-trans-cyclooctene inversion electron demand addition reaction, which is the fastest orthogonal reaction discovered so far, and the second-order reaction kinetics can reach 30000M ^- 1S ^-1 . The stability of tetrazine with a strong electron-withdrawing group is lower than that of hydrogen-substituted tetrazine and lower than that of methyl-substituted tetrazine. Hydrogen-substituted tetraazines exhibit exceptionally fast kinetics (10000 M ^-1 S ^-1 ), typically at least 10 times faster than methyl-substituted tetraazines. Therefore, the selection of tetrazine groups needs to strike a balance between reaction rate and stability. The inventor found that 1 mg/mL methyltetrazine-modified IgG antibody and TCO-modified AP (alkaline phosphatase) can be completely converted into IgG-AP conjugates within 90 minutes, and the methyltetrazine-modified IgG antibody was The activity will only decrease by 10-20% after one month at 4°C. Based on these results and combined with application scenarios in the field of IVD, methyltetrazine (MTz) and trans-cyclooctene (TCO) are optionally used as click chemistry orthogonal pairs in this disclosure, and other click chemistry orthogonal pairs can also be achieved Similar effect.

In some embodiments, the first click chemistry group and/or the second click chemistry group is selected from: methyltetrazine (MTz), transcyclooctene (TCO), azide (N ₃ ), any one of dibenzocyclooctyne (DBCO), tetrazine (Tz), alkynes, cyclopropanecyclooctyne (BCN) and cyclopropene.

Optionally, the first click chemical group and the second click chemical group are selected from any one of methyltetrazine (MTz) and trans-cyclooctene (TCO).

The addition reaction process of TCO and MTz is as follows:

Antibodies and conjugation partners modified with click chemistry groups can be rapidly cross-linked to form antibody conjugates. The antibody conjugates prepared by this disclosure are better than conventional processes, mainly in terms of high yield, clear coupling sites and good uniformity. In addition, using the antibody conjugate of the present disclosure to form a detection kit is superior to conventionally produced antibody conjugates in terms of sensitivity and correlation.

In some embodiments, the conjugation partner is a label selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels; optionally, the fluorescent dye At least one selected from the group consisting 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; can be Optionally, the enzyme is selected from any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose-6-phosphate deoxygenase. Optionally, the radioactive isotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru , at least one of 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F; optionally, the chemiluminescence reagent is selected from luminol and its derivatives , lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lopranine and its derivatives and peroxygen At least one of acid salts and their derivatives; optionally, the nanoparticle marker is selected from any one of colloids, organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles; optionally Preferably, the colloid is selected from at least one of colloidal metals, disperse dyes, dye-labeled microspheres and latex; optionally, the colloidal metal is selected from at least one of colloidal gold, colloidal silver and colloidal selenium. .

The embodiments of the present disclosure provide a method for preparing an antibody conjugate as described in any of the preceding embodiments, which includes: coupling a linker reagent to an antibody whose disulfide bonds have been opened by reduction to obtain an antibody-linker, and then converting the antibody -The linker is coupled to the conjugation partner to obtain the antibody conjugate;

The linker reagent has the following structure:

The antibody-linker has the following structure:

In some embodiments, the linker reagent carries a click chemical group R ₁ , and the coupling process of the linker reagent and the antibody whose disulfide bond is opened by reduction is as follows:

In some embodiments, the feeding molar ratio of the linker and the antibody whose disulfide bond is opened by reduction is (1-30):1. Specifically, the molar ratio can be 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, Any one of 22:1, 24:1, 26:1, 28:1, 30:1 or the range between any two.

In some embodiments, the coupling reaction conditions of the linker and the antibody whose disulfide bond is opened by reduction include: 2-37°C, 0.5-48 h. The reaction temperature can specifically be 2°C, 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℃, 34℃, 36℃, 37℃, any one or the range between any two; the reaction time of this reaction can be specifically 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h , 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h , any one of 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h or the range between any two.

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

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

2-Mercaptoethylamine·HCl, referred to as 2-MEA, is a mild reducing agent that can specifically reduce the disulfide bonds in the hinge region of the antibody without reducing the disulfide bonds at other sites in the antibody. key. TCEP is another mild disulfide bond reducing agent. At a certain TCEP concentration, taking IgG as an example, the order of reduction of antibody disulfide bonds is: inter-light and heavy chain disulfide bonds > upper hinge region disulfide bonds > lower hinge region Disulfide bond > intrachain disulfide bond.

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

In some embodiments, the feeding molar ratio of the reducing agent and the antibody is (0.5-50):1. Specifically, the molar ratio can be 0.5:1, 1:1, 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, 20:1, 25:1, 30:1, Any one of 35:1, 40:1, 45:1, 50:1 or the range between any two.

In some embodiments, the antibody-linker with click chemistry group _R1 has the following structure:

At the same time, the conjugation partner carries a click chemical group R ₂ ,

In the process of coupling the antibody-linker to the conjugation partner, the feeding molar ratio of the antibody-linker to the R ₂ -conjugation partner is (0.5-4): (0.5-4), specifically it can be 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 : Any one of 1.5, 3:1, 3:1, 4:3.5, 4:3, 4:2.5, 4:2, 4:1.5, 4:1 or the range between any two.

In some embodiments, the reaction conditions for coupling the antibody-linker to the conjugation partner include: 4-37°C, 0.5-16h; optionally, the reaction temperature can be 4°C, 6°C, 8°C, Any one of 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 37℃ or The range between any two; the reaction time can be specifically 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h , any one or two of 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 range between species.

For the convenience of demonstration, the "antibody-specific click chemical group" described below is used to represent the antibody-linker structure with a specific click chemical group of the present disclosure.

In some embodiments, the preparation method further includes preparation of R ₂ -conjugation partner, including mixing and reacting R ₂ -NHS ester and the conjugation partner to obtain R ₂ -conjugation partner.

In some embodiments, the molar ratio of R ₂ -NHS ester to conjugation partner is (5-25):1. Specifically, the molar ratio can be 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, 17:1, 19:1, 21:1, 23:1, 25:1, any one of them or a range between any two.

In some embodiments, the reaction conditions of R ₂ -NHS ester and conjugation partner include: 10～30°C, 0.5～1.5h. The reaction temperature can specifically be any one of 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C or any two of them. Range; the reaction time of this reaction can specifically be any one of 0.5h, 0.7h, 0.9h, 1.1h, 1.3h, 1.5h or a range between any two.

In some embodiments, the preparation method also includes preparation of the linker: mixing and reacting TSMA and R ₁ -(PEG) _n -NHS ester, where the TSMA is 2-(tosyl)methacrylamide-( PEG) _m -amide, its structural formula is:

In some embodiments, the molar ratio of TSMA and R ₁ -(PEG) _n -NHS ester is (0.5˜1.5): (0.5˜1.5). Specifically, the molar ratio may be any one of 0.5:1.5, 0.5:1, 0.5:1:1:0.5, 1.5:1, or a range between any two.

In some embodiments, the reaction conditions of TSMA and R ₁ -(PEG) _n -NHS ester include: 10 to 30°C, 10 to 14 hours. The reaction temperature can specifically be any one of 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C or any two of them. Range; the reaction time of this reaction can specifically be any one of 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h or a range between any two.

In some embodiments, the TSMA and R ₁ -(PEG) _n -NHS ester are reacted in an organic solvent or water.

In some embodiments, the organic solvent of the organic phase includes methylene chloride.

In some embodiments, the TSMA and R ₁ -(PEG) _n -NHS ester are reacted in the presence of DIEA and/or triethylamine.

In some embodiments, the molar ratio of DIEA and TSMA is 1: (1˜4). Specifically, the molar ratio may be any one of 1:1, 1:2, 1:3, 1:4 or a range between any two.

In some embodiments, the molar ratio of triethylamine and TSMA is 1: (1˜4). Specifically, the molar ratio may be any one of 1:1, 1:2, 1:3, 1:4 or a range between any two.

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

Using Compound A and Compound B as starting materials, the Compound A and the Compound B are reacted to obtain Compound C;

Compound C and compound D are subjected to a substitution reaction to obtain TSMA;

Wherein, the compound A is tert-butoxycarbonyl-imino-polyethylene glycol-amine, and the structural formula is: The compound B is methacrylic acid methacryloyl halide (where X is halogen) and methacrylic anhydride At least one of; the compound C is tert-butoxycarbonyl-imino-polyethylene glycol-methacrylamide, and the structural formula is The compound D is 4-toluenesulfonyl chloride and sodium 4-toluenesulfinate At least one of; wherein, m is 0 to 24. The specific selection of m is the same as that described in the corresponding embodiment above, and will not be described again.

In some embodiments, the molar ratio of compound A and compound B is (0.35~0.85): (0.58~0.98); the molar ratio can specifically be 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: Any one of 0.58, 0.85:0.78, 0.85:0.98 or the range between any two.

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

In some embodiments, the reaction of compound A and compound B includes: dissolving compound A in a first organic solvent, adding compound B, and reacting under the action of a dehydrating agent and/or a hydroxyl activator, The solvent was removed, and the precipitate was removed by filtration and then purified to obtain the compound C.

In some embodiments, the first organic solvent includes at least one of DMF and methylene chloride;

In some embodiments, the dehydrating agent includes dicyclohexylcarbodiimide.

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

In some embodiments, the molar ratio of compound C and compound D is (0.33~0.73): (0.60~1.00). Specifically, the molar ratio can be any one or two of 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, 0.73:1.00 range between.

In some embodiments, the reaction conditions of the substitution reaction include: stirring at 20-30°C for 20-28 hours. The reaction temperature can be any one of 20°C, 22°C, 24°C, 26°C, 28°C, 30°C or the range between any two; the reaction time of the reaction can be 20h, 21h, 22h. , any one of 23h, 24h, 25h, 26h, 27h, 28h or the range between any two.

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

The stirred product is washed and dried, and the solvent is removed to obtain a crude product; the crude product is dissolved in ethyl acetate, triethylamine is added to reflux, the solvent is removed by rotary evaporation, and the compound E is purified.

In some embodiments, the second organic solvent includes methylene chloride.

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.

The embodiments of the present disclosure provide a linker compound, the structure of which is 2-(toluenesulfonyl)methacrylamide-(PEG) _m -amide-(PEG) _n- R ₁ ;

Wherein, m and n are both integers and independently selected from 0 to 24, optionally 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; optionally m+n=4, optionally m is not zero, optionally n is not zero, optional Neither m nor n is zero;

R ₁ is the first click chemical group, which can be quickly and easily coupled with substances with the second click chemical group R ₂ ; while the 2-(toluenesulfonyl)methacrylamide structure can be coupled with two free thiol groups The reaction connects to form a sulfur-carbon bridge structure.

It can be understood that the specific selection of the linker compound is the same as that described in the foregoing corresponding embodiments, and will not be described again.

The embodiments of the present disclosure provide a method for preparing a linker as described in the previous embodiments, which includes the preparation of TSMA as described in any of the foregoing embodiments and/or the preparation of the linker as described in any of the foregoing embodiments.

The embodiments of the present disclosure provide the use of the antibody conjugate as described in any of the preceding embodiments in the preparation of diagnostic reagents or kits.

In addition, the embodiments of the present disclosure provide the use of the antibody conjugate as described in any of the preceding embodiments in the preparation of reagents or kits for improving detection sensitivity.

The features and performance of the present disclosure will be described in further detail below with reference to examples.

The disclosed research and development idea is to anchor the disulfide bond of the antibody, first reduce and open the disulfide bond to obtain two free sulfhydryl groups, and then use a heavy bridging reagent to connect the sulfhydryl groups to form a sulfur-carbon bridge. The main compounds commonly used in the prior art that can be used as heavy bridging reagents include bisulfone compounds, maleimide compounds, pyridazine compounds, and bisethylene sulfonamide compounds. Other small reports include allyl sulfone compounds, bromopyridinedione, divinylpyridine, N-substituted-3-bromo-5-methylenepyrrole-2-one compounds, etc. This disclosure designed an allyl sulfone compound linker and found that it was used to couple antibodies to prepare labeled antibodies. The resulting labeled antibodies had unexpected sensitivity effects in immunodetection.

Example 1

Preparation of 2-(toluenesulfonyl)methacrylamide-(PEG) _m -amide and 2-(toluenesulfonyl)methacrylamide-(PEG) _m -amide-(PEG) _n -R ₁ .

Unless otherwise specified, all chemical reagents used in the synthesis were purchased from McLean Reagent Network.

This example uses m=4 as an example to illustrate the preparation method of the title compound. In fact, in the preparation method, by changing the number of PEG units in Compound A used, titles with other m values (positive integers including zero) can be obtained accordingly. Compound (Other compound A with different m values can also be purchased from McLean Reagent Network).

The following is an exemplary description of the preparation process of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine.

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

①

a. Dissolve tert-butoxycarbonyl-imino-tetrapolyethylene glycol-amine (compound A, 200 mg, 0.65 mmol) in 10 mL DMF (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), stirred magnetically at 25°C for 12 hours.

b. Remove the solvent by rotary evaporation, dissolve the crude product in 10 mL of deionized water, filter to remove the precipitate, and purify using column chromatography (ethyl acrylate:n-hexane=1:1) to obtain tert-butoxycarbonyl- Imino-tetrapolyethylene glycol-methacrylamide (compound C), the yield is about 90%.

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

a. Dissolve tert-butoxycarbonyl-imino-tetrapolyethylene glycol-amine (compound A, 200 mg, 0.65 mmol) in 10 mL dichloromethane, and add triethylamine (78.8 mg, 108 μL, 0.78 mmol), methacryloyl halide or methacrylic anhydride (0.78 mmol), stirred magnetically for 12 hours.

b. Remove the solvent by rotary evaporation, dissolve the crude product in 30 mL of methylene chloride, wash with 1M HCl and saturated sodium chloride solution, dry the organic layer with anhydrous sodium sulfate, and then remove the solvent by rotary evaporation. Purify using column chromatography (ethyl acrylate: n-hexane = 1:1) to obtain tert-butoxycarbonyl-imino-tetrapolyethylene glycol-methacrylamide (compound C) with a yield of about 90%.

②

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

b. Then add triethylamine (162 mg, 223 μL, 1.60 mmol) and stir for 12 hours. The crude product was washed with 1M HCl, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation.

c. Dissolve the crude product in 10 mL of ethyl acetate, add triethylamine (162 mg, 223 μL, 1.60 mmol) at 0°C and reflux for 12 hours. After the solvent was removed by rotary evaporation, column chromatography (ethyl acrylate: n-hexane = 3:1) was used to purify to obtain tert-butoxycarbonyl-2-(toluenesulfonyl) methacrylate (compound E), with a yield of about 70%. .

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

a. Dissolve compound C (200 mg, 0.53 mmol) in 5 mL of methylene chloride, add sodium 4-toluenesulfinate (143 mg, 0.80 mmol) and iodine (204 mg, 0.80 mmol), and stir magnetically at 25°C for 72 hours.

b. Then add triethylamine (162 mg, 223 μL, 1.60 mmol) and stir for 12 hours. The crude product was washed with 1M 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.

Step c is the same.

③ Dissolve compound E (26.7 mg, 0.05 mmol) in 5 mL of methylene chloride, add trifluoroacetic acid (278 mg, 2.50 mmol), stir magnetically at 25°C for 12 hours, and rotary evaporate to remove the solvent to obtain TSMA with a yield of about 98%.

(2) Preparation of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine (TSMA-MTz)

① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of methylene chloride, and add DIEA (N, N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), stir to dissolve. Dissolve methyltetrazine-PEG4-NHS ester (Compound F, purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 16.7 mg, 0.03 mmol) in 1 mL of methylene chloride, add it to the above solution of TSMA after being dissolved.

② Stir the mixture magnetically at 25°C for 12 hours, remove the solvent by rotary evaporation, dissolve the crude product in 20 mL of methylene chloride, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and use Purify by column chromatography (dichloromethane: methanol = 20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine (compound G, TSMA-MTz), with a yield of approximately 50%.

The product was subjected to liquid chromatography-mass spectrometry detection and nuclear magnetic resonance detection. The detection results are shown in Figure 4-8, which proves that this method has indeed successfully prepared 2-(tosyl)methacrylamide-PEG4-amide-PEG4- Methyltetrazine.

The above is an example of the preparation method of the title compound using n=4. In fact, methyltetrazine-PEGn-NHS esters containing different numbers of PEG units can be used to synthesize titles with different n values (positive integers including zero). compound. Moreover, the methyltetrazine here can also be replaced by other tetrazine groups, such as tetrazine (if not otherwise stated, the various click chemical group-PEGn-NHS compounds used in this article can be obtained from Xi'an Kangfunuo Biotech Technology Co., Ltd. customized). For example, when preparing, refer to the previous steps, and other synthetic steps remain unchanged. You only need to replace methyltetrazine-PEG4-NHS ester with equimolar amounts of methyltetrazine-NHS ester and tetrazine-PEG4-NHS ester respectively. and tetrazine-NHS ester, which can be synthesized respectively to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-amide- Methyltetrazine, 2-(tosyl)methacrylamide-PEG4-amide-PEG4-tetrazine, and 2-(tosyl)methacrylamide-PEG4-tetrazine.

The compounds of this embodiment can also be synthesized in aqueous phase. The exemplary steps for aqueous phase synthesis are as follows:

Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir to dissolve. Dissolve methyltetrazine-PEG4-NHS ester (Compound F, purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 16.7 mg, 0.03mmol) or an equimolar amount of tetrazine-PEG4-NHS ester in 1 mL of deionized water. After dissolving, add to the above solution of TSMA. The mixture was magnetically stirred at 25°C for 12 hours to obtain 2-(tosyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine or 2-(tosyl)methacrylamide-PEG4- Aqueous solution of amide-PEG4-tetrazine.

(3) Preparation of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-(4E)-trans-cyclooctene (TSMA-TCO).

① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of methylene chloride, and add DIEA (N, N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), stir to dissolve. Dissolve (4E)-trans-cyclooctene-PEG4-NHS ester (purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 15.4 mg, 0.03 mmol) in 1 mL of methylene chloride, and then add it to the above solution of TSMA .

② Stir the mixture magnetically at 25°C for 12 hours, remove the solvent by rotary evaporation, dissolve the crude product in 20 mL of methylene chloride, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and use Purify using column chromatography (dichloromethane:methanol=20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-(4E)-trans-cyclooctene (TSMA-TCO).

The above is an example of the preparation method of the title compound using n=4. (4E)-trans-cyclooctene-PEGn-NHS ester containing different numbers of PEG units can be used to synthesize 2-(toluenesulfonate) with different n values. Acyl)methacrylamide-PEG4-amide-PEG4-(4E)-trans-cyclooctene, and (4E)-trans-cyclooctene can also be replaced by (2E)-trans-cyclooctene. For example, when preparing, refer to the previous steps, other synthetic steps remain unchanged, only need to replace (4E)-trans-cyclooctene-PEG4-NHS ester with an equal molar amount of (4E)-trans-cyclooctene-NHS. ester, and (2E)-trans-cyclooctene-PEG4-NHS ester, which can be synthesized respectively to obtain 2-(tosyl)methacrylamide-PEG4-amide-(4E)-trans-cyclooctene, and 2 -(Toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-(2E)-trans-cyclooctene.

The compounds of this example can also be synthesized in aqueous phase. Exemplary steps for aqueous synthesis are as follows:

Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir to dissolve. Add (4E)-trans-cyclooctene-PEG4-NHS ester (purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 15.4 mg, 0.03 mmol) or an equimolar amount of (2E)-trans-cyclooctene-PEG4- NHS ester was dissolved in 1 mL of deionized water, and then added to the above solution of TSMA. The mixture was magnetically stirred at 25°C for 12 hours to obtain 2-(tosyl)methacrylamide-PEG4-amide-PEG4-(4E)-trans-cyclooctene or 2-(tosyl)methyl Aqueous solution of acrylamide-PEG4-amide-PEG4-(2E)-trans-cyclooctene.

(4) Preparation of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-azide (TSMA-N3)

① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of methylene chloride, and add DIEA (N, N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), stir to dissolve. Dissolve azide-PEG4-NHS ester (0.03mmol) in 1 mL of methylene chloride, add it to the above solution of TSMA after mixing thoroughly.

② Stir the mixture magnetically at 25°C for 12 hours, remove the solvent by rotary evaporation, dissolve the crude product in 20 mL of methylene chloride, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and use Purify using column chromatography (dichloromethane:methanol=20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-azide (TSMA-N3).

The above is taking n=4 as an example to illustrate the preparation method of the title compound of this example. In fact, azide-PEGn-NHS esters containing different numbers of PEG units can be used (the azide-PEGn-NHS ester here can be purchased from Shanghai Peng Customized by Shuo Biotechnology Company) to synthesize 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEGn-azide with different n values. For example, when preparing, refer to the previous steps, and other synthetic steps remain unchanged. You only need to replace the azide-PEG4-NHS ester with an equal molar amount of azide-PEG2-NHS ester and azide-PEG8-NHS ester. 2-(tosyl)methacrylamide-PEG4-amide-PEG2-azide and 2-(tosyl)methacrylamide-PEG4-amide-PEG8-azide were synthesized respectively.

Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir to dissolve. Dissolve azide-PEG4-NHS ester (0.03mmol) in 1 mL of deionized water, add it to the above solution of TSMA after mixing thoroughly. The mixture was magnetically stirred at 25°C for 12 hours to prepare an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-azide.

(5) Preparation of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-DBCO (TSMA-DBCO)

① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of methylene chloride, and add DIEA (N, N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), stir to dissolve. Dissolve DBCO-PEG4-NHS ester (purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 0.03 mmol) in 1 mL of methylene chloride, add it to the above solution of TSMA after being dissolved.

② Stir the mixture magnetically at 25°C for 12 hours, remove the solvent by rotary evaporation, dissolve the crude product in 20 mL of methylene chloride, wash with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, remove the solvent by rotary evaporation, and use Purify using column chromatography (dichloromethane:methanol=20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-DBCO (TSMA-DBCO).

The above is an example of the preparation method of the title compound of this example using n=4. In fact, DBCO-PEGn-NHS esters containing different numbers of PEG units can be used to synthesize 2-(tosyl)methane with different n values. Acrylamide-PEG4-amide-PEGn-DBCO. For example, when preparing, refer to the previous steps, other synthetic steps remain unchanged, only need to replace DBCO-PEGn-NHS ester with equimolar amounts of DBCO-NHS ester and DBCO-PEG2-NHS ester, and 2- can be synthesized respectively. The above raw materials of (tosyl)methacrylamide-PEG4-DBCO and 2-(tosyl)methacrylamide-PEG4-amide-PEG2-DBCO can be purchased from Xi'an Kangfunuo Biotechnology Co., Ltd. .

The compounds of this example can also be synthesized in aqueous phase. The exemplary steps during aqueous phase synthesis are as follows: Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of deionized water, adjust the pH to 7.0-8.0, and stir to dissolve. Dissolve DBCO-PEG4-NHS ester (purchased from Xi'an Kangfunuo Biotechnology Co., Ltd., 0.03 mmol) in 1 mL of deionized water, add it to the above solution of TSMA after being dissolved. The mixture was magnetically stirred at 25°C for 12 hours to prepare an aqueous solution of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-DBCO.

Example 2

Preparation of conjugation partners modified with click chemical groups.

For the click chemical group-modified alkaline phosphatase prepared in this example, there is a PEG4 spacer arm between the click chemical group and the alkaline phosphatase, but in fact the click chemical group and the alkaline phosphatase can Direct connection can also be connected through some spacer arms. Experimental data later proves that whether there is a spacer arm between the click chemical group and alkaline phosphatase has no substantial impact on the detection signal intensity of the final antibody conjugate.

(1) Preparation of alkaline phosphatase-trans-cyclooctene (AP-TCO)

Alkaline phosphatase (5 mg/mL) was displaced into 100 mM PBS buffer pH 7.4 using a Zeba ^™ desalting column (10K MWCO). Add 10mM TCO-PEG4-NHS (10eq, dissolved in DMSO) to 40uM alkaline phosphatase solution, and react at 25°C for 1 hour. Then, the excess TCO-PEG4-NHS was desalted using a Zeba ^TM desalting column (10K MWCO) and replaced into PB (50 mM, pH 7.4) buffer. Prepared AP-TCO is diluted to 4mg/mL with PB (50mM, pH7.4) buffer and stored at 4°C for later use.

(2) Preparation of alkaline phosphatase-methyltetrazine (AP-MTz)

Alkaline phosphatase (5 mg/mL) was displaced into 100 mM PBS buffer pH 7.4 using a Zeba ^™ desalting column (10K MWCO). Add 10mM methyltetrazine-PEG4-NHS (10eq, dissolved in DMSO) to 40uM alkaline phosphatase solution, and react at 25°C for 1 hour. Then, the excess methyltetrazine-PEG4-NHS was desalted using a Zeba ^TM desalting column (10K MWCO) and replaced into PB (50 mM, pH 7.4) buffer. Prepare AP-MTz, dilute it to 4mg/mL with PB (50mM, pH7.4) buffer, and store it at 4°C for later use.

(3) Preparation of alkaline phosphatase-dibenzocyclooctyne (AP-DBCO)

Alkaline phosphatase (5 mg/mL) was displaced into 100 mM PBS buffer pH 7.4 using a Zeba ^™ desalting column (10K MWCO). Add 10mM DBCO-PEG4-NHS (10eq, dissolved in DMSO) to 40uM alkaline phosphatase solution, and react at 25°C for 1 hour. Then, the excess DBCO-PEG4-NHS was desalted using a Zeba ^TM desalting column (10K MWCO) and replaced into PB (50 mM, pH 7.4) buffer. Prepare AP-DBCO, dilute it to 4mg/mL with PB (50mM, pH7.4) buffer, and store it at 4°C for later use.

(4) Preparation of acridinium ester-(unmodified) bovine serum albumin-trans-cyclooctene (AE-BSA-TCO)

In this example, bovine serum albumin as an amplification carrier is modified to connect acridinium ester and trans-cyclooctene at the same time. One amplification carrier molecule will be connected to multiple acridinium ester molecules, and subsequent antibodies are coupled to the amplification carrier. An antibody molecule can be indirectly linked to multiple acridinium ester molecules.

Bovine serum albumin (5mg/mL) was dissolved in 100mM PBS buffer with pH 7.4, 10mM TCO-PEG4-NHS (10eq, dissolved in DMSO) was added, and the reaction was carried out at 25°C for 1 hour. The excess TCO-PEG4-NHS was desalted using a Zeba ^TM desalting column (10K MWCO) and replaced with 100 mM PBS buffer of pH 7.4. Then add 10mM acridinium ester-NHS (20eq, DMSO dissolved), and react at 25°C for 1 hour. The excess acridinium ester-NHS was desalted using a Zeba ^TM desalting column (10K MWCO) and replaced into PB (50 mM, pH 7.4) buffer. Prepare AE-BSA-TCO-, dilute it with PB (50mM, pH7.4) buffer to 4mg/mL, and store it at 4°C for later use.

(5) Preparation of acridinium ester-rebridged modified bovine serum albumin-trans-cyclooctene (AE-rebridgedBSA-TCO)

When coupling acridinium ester molecules and antibody molecules to bovine serum albumin molecules, the amino group of bovine serum albumin is usually anchored as the connecting functional group site, as described in the above Example 2.(4), in which acridinium ester molecules -NHS molecule is connected to the amino group of bovine serum albumin, and TCO-PEG4-NHS molecule is also connected to the amino group of bovine serum albumin. In the antibody conjugate obtained by coupling in the subsequent process (Example 5), acridinium ester Both the molecule and the antibody molecule actually anchor the amino group of bovine serum albumin as the connecting functional group site. However, the number of available amino groups on the surface of bovine serum albumin does not exceed 30. Acridinium ester molecules and antibody molecules compete for limited amino sites, and the ratio of acridinium ester molecules to antibody molecules in the final antibody conjugate will also be limited.

In view of this, this embodiment provides an optional solution, which is to anchor the disulfide bond of bovine serum albumin, reduce and open the disulfide bond of bovine serum albumin, and then add a heavy bridging agent to reconstruct it to form a sulfur-carbon bridge. , thus introducing a heavy bridging agent as a connecting functional group site for the antibody molecule, while the acridinium ester still fixes the amino group of bovine serum albumin as a connecting functional group site. Therefore, the ratio of acridinium ester molecules to antibody molecules in the final antibody conjugate is improved. The experimental operation is as follows:

① Use Zeba ^TM desalting column (7K MWCO) to replace bovine serum albumin (4mg/mL) into PBS (10mM, pH7.4, 150mM NaCl) buffer. Prepare TCEP (Sigma) with PBS buffer to make a 100mM solution. Add 20 eq of TCEP to the bovine serum albumin solution, react with gentle shaking (400 rpm) at 25°C for 2 hours, and then replace it with PB (50 mM, pH 7.8) buffer.

② Prepare TSMA-TCO into a 60mM solution using DMSO under dry conditions. Add 20eq of TSMA-TCO to the BSA solution obtained in step ① and react at 4°C for 16 hours. Excess chemical reagents in the BSA solution were replaced into PBS buffer using a Zeba ^TM desalting column (7K MWCO). The rebridged modified bovine serum albumin-trans-cyclooctene (rebridgedBSA-TCO) was prepared and stored at 4°C for later use.

③ Prepare acridinyl ester-NHS (AE-NHS) with DMSO under dry conditions to form an 8mM solution. Add 20eq of acridinium ester-NHS to the rebridged BSA-TCO solution obtained in step ②, react at 25°C for 60 minutes, add 200eq of glycine solution (100mM), continue the reaction for 30 minutes, and use Zeba ^TM desalting column (7K MWCO ) into PBS buffer. Acridinium ester-rebridged modified bovine serum albumin-trans-cyclooctene (AE-rebridgedBSA-TCO) was prepared and stored at 4°C for later use.

Example 3

Reduction of antibody disulfide bonds.

The embodiments of the present disclosure select IgG1, IgG2a, and IgG2b mouse monoclonal antibodies for research, in which the IgG1 type antibodies include AFP antibodies, CA153 antibodies, CA724 antibodies, CA199 antibodies, and HIV P24 antibodies; the IgG2a type antibodies are CA125 antibodies; and the IgG2b antibodies are cTnI Antibody.

Two free sulfhydryl groups can be obtained by reduction at the disulfide bond of the antibody, and then a linker molecule with a 2-(tosyl)methacrylamide structure is added. The sulfhydryl linkage reaction reconnects to form a sulfur-carbon bridge bond. The above-mentioned antibodies have 2 to 4 disulfide bonds in the hinge region, and there are also disulfide bonds before the light and heavy chains. The reduction process can be adjusted to make it more likely to reduce the disulfide bonds in the hinge region of the antibody or between the light and heavy chains of the antibody. Reduction of disulfide bonds.

(1) Reduction of disulfide bonds in the antibody hinge region

The antibody (5 mg/mL) was displaced into PBS (100 mM PB, 50 mM sodium chloride, pH 7.4, 1 mM EDTA) buffer using a Zeba ^™ desalting column (10K MWCO). 2-MEA (2-mercaptoethylamine hydrochloride) was prepared into a 10mM solution using PBS (100mM, 50mM sodium chloride, pH 7.4, 1mM EDTA). Add 10 eq of 2-MEA to the antibody solution and react with gentle shaking (400 rpm) at 25°C for 1 hour. Store the reduced antibodies at 4°C for later use.

(2) Reduction of disulfide bonds between light and heavy chains

The antibody (5 mg/mL) was displaced into PB (50 mM, pH 7.4, 1 mM EDTA) buffer using a Zeba ^™ desalting column (10K MWCO). TCEP (Sigma) was prepared into a 10mM solution using PB (50mM, pH7.4, 1mM EDTA). Add 2 eq of TCEP to the antibody solution and react with gentle shaking (400 rpm) at 37°C for 2 hours. Store the reduced antibodies at 4°C for later use.

Example 4

Reconstruction of antibody disulfide bonds.

(1) Introduction of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-methyltetrazine (TSMA-MTz) linker molecule

TSMA-MTz was prepared into a 10mM solution using DMSO under dry conditions. Add 20eq of TSMA-MTz to the reduced antibody solution and react at 4°C for 16 hours. The excess chemical reagents in the antibody solution were replaced into PB (50mM, pH7.4) buffer using a Zeba ^TM desalting column (10K MWCO). Prepare the antibody-linker-methyltetrazine (antibody-MTz), dilute it with PB (50mM, pH7.4) to 4mg/mL, and store it at 4°C for later use.

(2) Introduction of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-trans-cyclooctene (TSMA-TCO) linker molecule

TSMA-TCO is prepared into a 10mM solution using DMSO under dry conditions. Add 20eq of TSMA-TCO to the reduced antibody solution and react at 4°C for 16 hours. The excess chemical reagents in the antibody solution were replaced into PB (50mM, pH7.4) buffer using a Zeba ^TM desalting column (10K MWCO). Prepare the antibody-linker-trans-cyclooctene (antibody-TCO), dilute it with PB (50mM, pH7.4) to 4mg/mL, and store it at 4°C. spare.

(3) Introduction of 2-(tosyl)methacrylamide-PEG4-amide-azide (TSMA-N3) linker molecule

TSMA-N3 is prepared into a 10mM solution using DMSO under dry conditions. Add 20eq of TSMA-N3 to the reduced antibody solution and react at 4°C for 16 hours. The excess chemical reagents in the antibody solution were replaced into PB (50mM, pH7.4) buffer using a Zeba ^TM desalting column (10K MWCO). Prepare the antibody-linker-azide (antibody-N3), dilute it with PB (50mM, pH7.4) to 4mg/mL, and store it at 4°C for later use.

Example 5

Preparation of antibody conjugates with conjugation partner-directed cross-linking.

(1) Take the antibody-MTz (4mg/mL) and AP-TCO (4mg/mL) from the previous example, perform cross-linking at a molar ratio of 1:1, and react at 25°C for one hour to obtain the alkaline solution of the present disclosure. Phosphatase-labeled antibody conjugates. After the reaction is completed, store it at 4°C for later use.

(2) Take the antibody-TCO (4 mg/mL) and AP-MTz (4 mg/mL) from the previous example, perform cross-linking at a molar ratio of 1:1, and react at 25°C for one hour to obtain the alkaline solution of the present disclosure. Phosphatase-labeled antibody conjugates. After the reaction is completed, store it at 4°C for later use.

(3) Take the antibody-N3 (4mg/mL) and AP-DBCO (4mg/mL) of the previous example, perform cross-linking at a molar ratio of 1:1, and react at 25°C for one hour to obtain the alkaline solution of the present disclosure. Phosphatase-labeled antibody conjugates. After the reaction is completed, store it at 4°C for later use.

(4) Take the antibody-MTz (4 mg/mL) and AE-BSA-TCO (4 mg/mL) of the previous example, conduct cross-linking at a molar ratio of 1:1, and react at 25°C for one hour to obtain the disclosed product Acridinium ester-labeled antibody conjugates via (unmodified) bovine serum albumin as amplification carrier. After the reaction is completed, store it at 4°C for later use.

(5) Take the antibody-MTz and AE-rebridgedBSA-TCO from the previous example, carry out cross-linking at a molar ratio of 1.5:1 and react at 25°C for one hour to obtain the (rebridged transformation) bovine serum albumin of the present disclosure. Amplify the acridinium ester-labeled antibody conjugate of the carrier and store it at 4°C for later use.

Comparative example 1

A conventional method for preparing antibody conjugates is provided, as follows.

The conventional preparation principle of antibody conjugates in the IVD field is to use the amino or sulfhydryl groups of protein amino acid side chains as coupling sites.

The conventional method for preparing alkaline phosphatase antibody conjugates is as follows:

(1) Thiolization modification of antibody (IgG-SH):

Antibody (5mg/mL) was exchanged into PBS (100mM PB, 50mM sodium chloride, pH 8.0, 5mM EDTA) using a Zeba ^™ desalting column (10K MWCO). Traut's reagent (Pierce) was prepared into a 10mM solution in PBS (100mM PB, 50mM sodium chloride, pH 7.4, 1mM EDTA). Add 10 eq of Traut's reagent to the antibody solution and react with gentle shaking (400 rpm) at 25°C for 2 hours. Desalt to remove excess reagents, and store the thiolated antibody at 4°C for later use.

(2) Maleimide modification of alkaline phosphatase (AP-MAL):

Alkaline phosphatase (5mg/mL) was displaced into PBS (10mM PB, 50mM sodium chloride, pH7.4, 5mM EDTA) buffer using a Zeba ^™ desalting column (10K MWCO). 10mM SMCC (10eq, dissolved in DMSO) was added to 40uM alkaline phosphatase solution and reacted at 25°C for 1 hour. Desalt to remove excess reagents, and store the maleimide-modified AP at 4°C for later use.

(3)IgG-SH and AP-MAL cross-linking:

Thiol-modified antibody (4mg/mL) and maleimide-modified AP (4mg/mL) were reacted in PBS (10mM PB, 50mM sodium chloride, pH7.4) buffer at 25°C for 1 hour. After the reaction is completed, store it at 4°C for later use.

The method for preparing acridinium ester antibody conjugates by conventional techniques is as follows:

Antibody (5mg/mL) was replaced into PBS (10mM PB, 50mM sodium chloride, pH7.4, 5mM EDTA) buffer using Zeba ^TM desalting column (10K MWCO). 10mM NHS-AE (10eq, dissolved in DMSO) was added to the 30uM antibody solution and reacted at 25°C for 1 hour. After the reaction is completed, desalt to remove excess reagents and store at 4°C for later use.

Verification example 1

Validation of reduction and modification of antibody hinge regions.

2-Mercaptoethylamine·HCl, referred to as 2-MEA, is a mild reducing agent that can specifically reduce the disulfide bonds in the hinge region of the antibody without reducing the disulfide bonds at other sites in the antibody. Sulfur bonds.

Three concentrations of 2-MEA, 10eq, 5eq, and 3eq, were used to reduce the AFP monoclonal antibody (mouse IgG1 type) (refer to Example 3 for the process). It was found that 3eq of 2-MEA had the best reduction specificity, opening only one disulfide bond in the hinge region, and the antibody's disulfide bond reduction number RDAR was 1.1 (Figure 2 and Table 1).

Table 1 Disulfide bond reduction number RDAR of antibodies

Under the reduction of 10eq and 5eq of 2-MEA, the disulfide bonds between the light and heavy chains of the AFP antibody were opened to varying degrees, and the number of disulfide bond reductions RDAR of the antibody were 3.4 and 2.5 (numbers 1 to 2).

Under the action of TSMA-MTz linker molecules, the opened disulfide bonds of the three reduced AFP antibodies are reconnected to form sulfur-carbon bridge bonds (refer to Example 4 for the process), forming a complete antibody again, and the antibody disulfide bonds are reduced The number is 0.01 to 0.02 (numbers 4 to 6), indicating that the broken disulfide bonds of most antibodies form sulfur-carbon bridges again.

Verification example 2

The three antibodies with MTz tags in Verification Example 1 were cross-linked with alkaline phosphatase respectively (refer to Example 5 for the process), configured into AFP detection reagents, and sensitivity tested. The test results showed that the sensitivity of low-value samples (CL) was 23.2% and 26.7% higher for antibody conjugates prepared from 3eq and 10eq of 2-MEA respectively (Table 2).

Table 2 AFP project sensitivity test

This result shows that the disulfide bond in the antibody hinge region is a better coupling site than the disulfide bond between the antibody light and heavy chains. Coupling the conjugation partner at the disulfide bond position in the antibody hinge region has less impact on the structure and function of the antibody.

Compared with the conventional process, the antibody conjugates prepared using the embodiments of the present disclosure have a low-value sample (CL) sensitivity that is 127%, 120%, and 179% higher respectively, which has obvious advantages.

Verification example 3

Control of disulfide bond reduction sites.

There are three subtypes of mouse IgG, IgG1, IgG2a and IgG2b. Different subtypes have differences in the number of interchain disulfide bonds. Both IgG2a and IgG2b have 6 interchain disulfide bonds, while IgG1 has 4. Mouse IgG antibodies of two subtypes, CA153 (IgG1) and cTnI (IgG2b), were selected for study.

Divide CA153 and cTnI antibodies at a concentration of 1 mg/mL into 7 equal parts. TCEP is prepared into a 10mM solution using PB (50mM, pH7.4, 1mM EDTA). Add 0eq, 5eq, 10eq, 15eq, 20eq, 30eq and 50eq TCEP solution to 7 antibody solutions respectively, and react with gentle shaking (400rpm) at 37°C for 2 hours. After the reaction, protein samples were taken for SDS-PAGE and antibody disulfide bond reduction number analysis. The results are shown in Figure 3.

The results show that there is a positive correlation between TCEP concentration and the number of antibody disulfide bond reductions (RDAR). The higher the TCEP concentration, the greater the number of antibody disulfide bond reductions. By adjusting the concentration of TCEP, the number of reduced disulfide bonds between antibody chains can be controlled. When the TCEP concentration is 10 eq, the number of open disulfide bonds of the IgG1 antibody is 3.1, and the number of open disulfide bonds of the IgG2b antibody chain is 1.6.

Further, 10 eq TECP-reduced CA153 antibody and cTnI antibody were used for cross-linking with alkaline phosphatase (using TSMA-MTz in Example 1). The obtained antibody conjugate was subjected to sensitivity testing, and the results are shown in Table 3.

Table 3 Sensitivity of different items

Table 3 shows that the sensitivity of low-value samples (CL) is 96% and 220% higher respectively for cTnI antibody conjugates and CA153 antibody conjugates prepared using the disclosed technology compared with antibody conjugates prepared by conventional processes. At the same time, the difference in sensitivity improvement between the two antibodies suggests a positive correlation between sensitivity and the number of antibody disulfide bond reductions (RDAR). That is, the greater the number of reduced disulfide bonds, the higher the sensitivity.

Verification example 4

Click Chemical Reaction Orthogonality Assessment.

AFP monoclonal antibody (IgG1), 10eq TCEP-reduced AFP monoclonal antibody, 10eq TCEP-reduced 20eq TSMA-MTz (Example 1) reconstituted AFP monoclonal antibody were cross-linked with AP-TCO respectively, with a molar ratio of 1:1. After reacting at 25°C for one hour, keep it at 4°C for later use.

At the same time, 20eq TSMA-MTz reconstituted AFP monoclonal antibody was cross-linked with AP after 10eq TCEP reduction, with a molar ratio of 1:1. After reaction at 25°C for one hour, it was kept at 4°C for use.

Prepare IgG-SH and AP-MAL according to the conventional process, perform cross-linking according to the molar ratio of 1:1, react at 25°C for one hour, and keep at 4°C for use.

Five kinds of AFP monoclonal antibody conjugates were prepared, diluted into a conjugate working solution according to a certain ratio, and combined with the magnetic bead working solution to form a detection reagent for sensitivity testing. Judging from the sensitivity test results (Table 4).

Table 4 Click chemistry reaction orthogonality assessment

The antibody conjugates obtained by cross-linking AP-TCO with IgG and TCEP-reduced IgG have no activity. The antibody conjugates prepared by AP and antibody-MTz also have no signal. Only the antibodies prepared by AP-TCO and antibody-MTz are conjugated. The results indicate that the click chemistry groups are active. It also shows that the click chemical group does not react with bioactive groups such as amino, sulfhydryl or carboxyl groups of the protein, and has good biocompatibility.

Verification example 5

Evaluation of the versatility of disulfide bond reconstruction directional cross-linker technology.

Two IgG1 type antibodies and one IgG2a project were tested to evaluate the versatility of the technology provided by the embodiments of the present disclosure in the field of immunodiagnosis.

The IgG1 type selects monoclonal antibodies of the CA724 and HIV P24 projects, the IgG2a type selects the monoclonal antibodies of the CA125 project, and uses the disclosed technology to prepare alkaline phosphatase antibody conjugates (using the linker TSMA-MTz), with each The magnetic bead coating is combined with the detection reagent for sensitivity testing. Judging from the sensitivity test results (Table 5).

Table 5 Evaluation of the versatility of disulfide bond reconstruction directional cross-linking technology

Table 6 Versatility evaluation of disulfide bond reconstruction directional cross-linking technology

The IgG2a type antibody CA125 project applies the disclosed technology, and the sensitivity of low-value samples (CL) can be increased by 108.1%. The IgG1 type antibody CA724 project applies the disclosed technology, and the sensitivity of low-value samples (CL) can be increased by 63.9%. The detection sensitivity of the P24 antigen of the fourth-generation HIV detection reagent can improve the detection window period, which is of great significance for the clinical diagnosis of HIV. As can be seen from Table 6, when the technology of the present disclosure is used to detect the international standard product (NIBSC, British National Institute for Biological Products Control) of 1.25 IU/mL, the P/N ratio reaches 30.3, and the sensitivity can be increased by 105.3% compared to the conventional process.

Verification example 6

Disulfide reduction sites as a function of sensitivity and correlation.

According to the aforementioned verification results, it can be seen that the disulfide bond in the hinge region is a better coupling site than the disulfide bond between light and heavy chains. The CA153 antibody was reduced with 10eq concentration of TCEP and 2-MEA respectively, and then 20eq TSMA-MTz (Example 1) was used to reconstruct the disulfide bond, and AP-TCO was added for cross-linking. The molar ratio of antibody-MTz to AP-TCO is 1:1. Two directional cross-linked conjugates were prepared and subjected to SEC-HPLC analysis, sensitivity and correlation tests together with the conjugate prepared by conventional techniques.

Table 7 CA153 project sensitivity

For conjugates prepared by two reduction methods of TCEP and 2-MEA, that is, the reconstruction of the disulfide bonds of the light and heavy chains and the disulfide bonds of the hinge region, the sensitivity of the low value sample (CL) is 189% and 200% higher than the conventional process respectively ( Table 7). Hinge region disulfide bond reconstruction has no obvious advantage in sensitivity compared to light and heavy chain disulfide bond reconstruction.

The antibody conjugate composition detection reagents were prepared by the above three different processes, and 57 Roche clinical samples with a concentration range of 4.22 to 367.4U/mL were tested respectively. Samples of each concentration were tested once. The test data are shown in the supplementary material. 1. The RLU obtained from the Feipeng instrument test was used as the y-axis variable, and the concentration value of Roche's clinical sample was used as the x-axis variable. Linear regression analysis and data fitting were performed. Using conventional processes, the correlation coefficients of the conjugates obtained by reducing the antibody with TCEP and reducing the antibody with 2-MEA were 0.9524, 0.9257, and 0.9789 respectively (Table 7). Hinge region disulfide bond reconstruction has obvious advantages over light and heavy chain disulfide bond reconstruction in terms of correlation.

SEC-HPLC analysis showed that the yields of the conventional process, TCEP reduction process and 2-MEA reduction process were 43.78%, 87.67%, and 91.16% respectively. The antibody conjugate yield was consistent with its sensitivity results. The antibody conjugate obtained by conventional processes is a mixture of dimers, trimers and tetramers, while the antibody conjugate obtained by the disclosed technology is a homogeneous substance mainly composed of trimers.

Verification example 7

Effect of 2-(tosyl)methacrylamide-(PEG) _m -amide-(PEG) _n -MTz linker with different PEG amounts on detection sensitivity.

The linkers of the title compound with six values of m+n=0, 2, 3, 4, 8, and 16 were synthesized and coupled with the CA153 antibody to obtain the CA153 antibody-MTz, which was then coupled with trans-cyclooctene-AP to obtain the CA153 antibody. -AP conjugate (refer to Example 5), and then perform a detection sensitivity test on the CA153 sample. The test results show that the P/N ratios of six CA153 antibody-AP conjugates with m+n=0, 2, 3, 4, 8, and 16 in the test CA153 low value sample (CL) are 1.4, 1.9, 3.6, and 3.1, 2.1 (Table 8), in the table, C0 represents the background value sample, CL represents the low value sample, and CH represents the high value sample, the same below.

Table 8 Sensitivity of different PEG numbers in the linker (CA153)

In the absence of PEG molecules, the linker has low solvent accessibility, low disulfide bond reconstruction efficiency, and significantly reduced sensitivity. Too many PEG molecules affect the structure and stability of the antibody conjugate, which is not conducive to the immune reaction and reduces sensitivity. The most suitable number of PEG molecules for the linker is between 4 and 8, of which 4 is optimal.

Verification example 8

The presence of a spacer between the conjugation partner and the click chemistry has no impact on detection sensitivity.

TCO-modified alkaline phosphatase with TCO-NHS and TCO-PEG4-NHS respectively to obtain AP-PEG4-TCO and AP-TCO, which were then cross-linked with the CA153 antibody-MTz of Validation Example 7, with a molar ratio of 1: 1. Two antibody-AP conjugates were prepared with the number of PEG molecules between the conjugation partner and the click chemical group being 0 and 4, respectively, and the sensitivity test was performed on the CA153 sample. The test results in Table 9 show that the sensitivity of the antibody-AP conjugates with the number of PEG molecules between the conjugation partner and the click chemical group being 0 and 4 respectively when testing CA153 samples with different contents is basically consistent, indicating that the conjugation partner is The presence or absence of spacers between click chemical groups has little effect on the detection sensitivity of antibody conjugates.

Table 9 Effect of the presence or absence of a spacer arm between the conjugation partner and the click chemical group on sensitivity (CA153)

Verification example 9

Evaluation of thermal stability of antibody conjugates obtained by disulfide bond reconstruction technology.

The CA242 antibody was reduced with 10eq of 2-MEA and 10eq of TCEP respectively, and then 20eq of TSMA-MTz (same as Example 4) was used for disulfide bond reconstruction to obtain antibody-MTz, which was then mixed with AP-TCO at a molar ratio of 1:1. Cross-linking is performed to obtain antibody-AP conjugates. The two antibody-AP conjugates prepared in this disclosure and the antibody-AP conjugate prepared by conventional processes are diluted with the conjugation partner diluent respectively to prepare an enzyme label working solution. Three different enzyme-labeled working solutions were divided into three equal parts, and one part was stored at 2 to 8°C as a control sample. The other two were placed in a 37°C incubator as test samples. On the 3rd and 7th days, the test samples were taken out and stored at 2 to 8°C. On the last day, all samples and magnetic bead working solution were used to form detection reagents. Three samples of different concentrations of CA242 were tested. Each sample was tested twice to obtain the relative luminescence value (RLU), and the average relative luminescence value was calculated. Calculate the relative deviation between the experimental group and the control group.

Table 10 Thermal stability evaluation of antibody conjugates obtained by disulfide bond reconstruction technology (CA242)

It can be seen from the experimental results (Table 10) that the conventional process antibody conjugate was accelerated for 3 days and 7 days, and the low-value sample (CL) luminescence value deviation was -12% and 16% respectively. For the directional cross-linked antibody conjugate prepared using two reducing agents, 2-MEA and TCEP, the deviation of the luminescence value after acceleration for 3 days and 7 days was less than 10%. The thermally accelerated stability of the antibody conjugate obtained by the disclosed technology is better than that of conventional processes.

Verification example 10

Assessment of detection sensitivity of acridinium ester-labeled antibody conjugates.

For the acridinium ester-labeled antibody conjugates prepared in the present disclosure (Example 5.(4), using an unmodified amplification vector; Example 5.(5), using a re-bridged modified amplification vector), and conventional The acridinium ester-labeled antibody conjugate (Comparative Example 1) prepared by the process was subjected to sensitivity testing, and the sensitivities of the low-value samples (CL) were 7.4, 17.1, and 26.8 respectively. Compared with the conventional process, the sensitivity of using the disclosed (unmodified) amplification carrier-acridinyl ester-labeled antibody conjugate and (rebridged modified) amplification carrier-acridinyl ester-labeled antibody conjugate is 130% higher respectively. and 262%. The difference between the disclosed (unmodified) amplification carrier-acridinyl ester-labeled antibody conjugate and the (re-bridged modified) amplification carrier-acridinyl ester-labeled antibody conjugate for detecting low-value samples was 57%. The difference in sensitivity shows that adding the secondary solution of a re-bridged modified amplification vector to the main technical solution of the present disclosure can further significantly improve the performance of the final antibody conjugate of the present disclosure.

Table 11 Sensitivity of acridinium ester markers (CA153)

Performance Testing

The antibody conjugate of the present disclosure and the antibody conjugate of the conventional process are diluted with an enzyme-labeled diluent, and configured into an enzyme-labeled working solution, which is combined with the magnetic bead working solution to form a detection reagent. In the Feipengishine series of fully automatic chemiluminescence Tests are conducted on an immunoassay analyzer to evaluate the sensitivity, relevance and stability of the detection reagents.

(1) Sensitivity

The internal reference products C0, CL, and CH of different items were tested. Each sample was tested twice to obtain the relative luminescence value (RLU), and the average RLU value was calculated. The sensitivity (P/N) was calculated as the ratio of the mean RLU values of CL and CH to the mean RLU value of CO.

(2)Relevance

Roche cobas e 411 and Feipeng Shine analyzers were calibrated with their own calibrators, and then at least 40 related samples were tested. Each sample was tested once to obtain the relative luminescence value (RLU). The results of Feipeng system are used as Y-axis variables, and the results of Roche system are used as X-axis variables. Use linear regression analysis to fit the data and obtain the equation Y=KX+B and the correlation coefficient r.

Physical and chemical property analysis

(1)SDS-PAGE

Add 5X Loading Buffer (2.5mM pH7.8 Tris-HCl, 0.1% SDS, 0.005% bromophenol blue) to the protein sample to prepare a protein electrophoresis sample. SDS-PAGE was performed on protein electrophoresis samples using SurePAGE ^TM protein precast gel (GenScript) and Tris-MOPS-SDS Running Buffer Powder (GenScript).

(2)SEC-HPLC

The protein samples were analyzed by size exclusion using Waters high performance liquid chromatography platform. Operate according to the manufacturer's instrument instructions. The protein sample concentration should not be less than 0.1mg/mL, and the total protein amount should not be less than 50ug.

(3) Determination of the number of antibody disulfide bond reductions

Ellman's reagent (DTNB, Pierce) is used to quantitatively measure the molar concentration of free sulfhydryl groups of the antibody. The ratio of the antibody's molar concentration to the antibody's molar concentration can determine the number of antibody disulfide bond reductions (RDAR) (Reduced disulfide bonds antibody ratio). The OD value was measured at a wavelength of 412 nm, and the molar extinction coefficient ε412 of the thiol group was 10mM-1cm-1. The OD value was measured at a wavelength of 280nm, and the molar extinction coefficient ε280 of the antibody was 210mM-1cm-1. According to Lambert-Beer law: Aλ = ελbC, Aλ is the ultraviolet absorption value of the sample at λ wavelength, ελ is the molar extinction coefficient of the sample at λ wavelength, b is the optical path, and C is the molar concentration of the sample. . Therefore, the antibody disulfide bond reduction number RDAR can be calculated by the following formula:

The above are only optional embodiments of the present disclosure and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this disclosure shall be included in the protection scope of this disclosure.

Claims

An antibody conjugate, characterized in that it includes the following structure:

Wherein, n and m are both integers and are independently selected from 0 to 24.
The antibody conjugate according to claim 1, characterized in that, 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.
The antibody conjugate according to claim 1, characterized in that there are pairs of orthogonal click chemical groups R 1 and R 2 connected between the conjugate partner and the antibody,

The antibody conjugates include the following structures:

Among them, R 1 is the first click chemical group, and R 2 is the second click chemical group.
The antibody conjugate according to claim 1, characterized in that there is an amplification carrier between the conjugate partner and the antibody, and the antibody conjugate includes the following structure:
The antibody conjugate according to claim 4, wherein the amplification carrier is a carrier modified by a rebridging agent, and the antibody conjugate includes the following structure:

Among them, L can represent absence, that is, the X group is directly connected to the oxygen atom, or L can represent a connecting group, that is, the X group and the oxygen atom are indirectly connected through the connecting group;

The amplification carrier is a substance having both a disulfide bond and an amino group. Preferably, the amplification carrier is selected from bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
The antibody conjugate according to claim 5, wherein the structural moiety Includes one of the following structures:
An antibody-linker is characterized in that it includes the following structure:

Among them, R 1 is the first click chemical group, and n and m are both integers and independently selected from 0 to 24.
The antibody-linker according to claim 7, characterized in that, 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, both m and n are not zero; preferably, m+n≧4.
The antibody conjugate of claim 1 or the antibody-linker of claim 7, wherein the sulfur-carbon bridge Located between the antibody hinge region, between the antibody light and heavy chains, or between the antibody heavy chains.
The antibody conjugate according to claim 1 or the antibody-linker according to claim 5, characterized in that,

The first click chemical group can be paired and connected with the second click chemical group; the first click chemical group and/or the second click chemical group are selected from: methyltetrazine, trans-cyclooctene, Any of azide, dibenzocyclooctyne, tetrazine, alkyne, cyclopropanecyclooctyne and cyclopropene;

Preferably, the first click chemistry group and the second click chemistry group are mutually exclusive selected from any one of methyltetrazine and trans-cyclooctene.
The antibody conjugate according to claim 1, wherein the conjugation partner is selected from at least one selected from the group consisting of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.
A method for preparing the antibody conjugate of any one of claims 1 to 3, characterized in that it includes: coupling a linker reagent with an antibody whose disulfide bonds have been opened by reduction to obtain an antibody-linker, and then Coupling the antibody-linker with the conjugation partner to obtain the antibody conjugate;

The linker reagent has the following structure:

The antibody-linker has the following structure:
Use of the antibody conjugate according to any one of claims 1-6 or 9-11 in the preparation of diagnostic reagents or kits.
Use of the antibody-linker according to any one of claims 7-10 in the preparation of diagnostic reagents or kits.
A kit, characterized in that it includes the antibody conjugate according to any one of claims 1-6 or 9-11, or the antibody-linker according to any one of claims 7-10.