CN118496347A - Method for coupling antibody oligonucleotides - Google Patents
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- CN118496347A CN118496347A CN202410963473.5A CN202410963473A CN118496347A CN 118496347 A CN118496347 A CN 118496347A CN 202410963473 A CN202410963473 A CN 202410963473A CN 118496347 A CN118496347 A CN 118496347A
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- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 46
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000010168 coupling process Methods 0.000 title claims abstract description 32
- 230000008878 coupling Effects 0.000 title claims abstract description 27
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title claims abstract description 14
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for coupling an antibody oligonucleotide. The method comprises coupling an antibody to a thiol-modified oligonucleotide using a heterobifunctional crosslinker (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester. The present invention has found that compounds of formula (I) are particularly suitable for use as heterobifunctional cross-linkers for coupling antibodies and oligonucleotides together and, more importantly, can be coupled in a single step without unwanted polymerization or self-coupling, which simplifies handling and reduces the number of treatments of the antibody, thereby avoiding the risk of the antibody changing structure during the treatment leading to reduced activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for coupling an antibody oligonucleotide.
Background
The development of bioconjugate technology has become a cornerstone in the fields of chemical proteomics, biomaterial synthesis, biomolecular imaging, single molecule analysis, single cell multigroup chemistry, multispecific drugs, and the like. Modification of natural or unnatural amino acids on proteins is a common method of bioconjugate
Antibodies have multiple primary amine groups present at the N-terminus of each polypeptide chain (known as the α -amino group) and on the side chains of lysine residues. Since primary amines are positively charged under physiological conditions, they are typically directed outwards, i.e. on the outer surface of the antibody, which facilitates coupling without altering the protein structure. The current oligonucleotide synthesis technology is mature and low in cost, and the synthesized oligonucleotide can be selectively added with various types of modified terminals, wherein the technology of adding primary amine or sulfhydryl group to the terminals for modification is mature and low in cost, and the synthesis period is faster.
There are various chemical reagents available for antibody coupling oligonucleotides, such as the sulfo-SMCC of thermofisher company, the antibody coupling agent of MCE company, etc., and the main principle is based on modifying the functional groups on the antibody to couple with the crosslinking agent, and then performing chemical reaction or affinity reaction with the functional groups of other oligonucleotides to form the coupling substance. However, these methods typically involve a two-step treatment of the antibody, i.e., activation followed by coupling to the oligonucleotide. The structure of antibodies is at risk of denaturing failure during such complex and prolonged procedures.
There is also a common antibody coupling strategy that utilizes cysteine (Cys) in an antibody to specifically modify Cys through its high nucleophilicity and low abundance to couple with other small molecules. To date, 7 out of the 12 antibody conjugated drugs approved by the U.S. food and drug administration are constructed by the michael addition of Cys and maleimide (Mal). Although Mal has been widely used for protein modification since the 50 s of the 20 th century, the addition of sulfhydryl groups and Mal has several problems that limit its use. First, mal may react with lysine at high pH (pH > 7.5), which can produce heterogeneous conjugates; second, mal is susceptible to hydrolysis in alkaline solutions to form unreactive maleimido acids, which requires that the Mal reagent be stored under stringent conditions; thirdly, the sulfhydryl-Mal adduct is unstable under physiological conditions and is easy to undergo reversible reaction, and the application of the sulfhydryl-Mal adduct in antibody-coupled drugs can cause off-target effect. Furthermore, the thiol groups on antibodies are usually present in disulfide form, requiring their reduction to active thiol groups, which leads to complex operations and may disrupt the structure of the antibody, and furthermore, the thiol groups in the residual reducing agent compete for the reaction of maleimide with thiol groups on antibodies.
The prior art discloses a linker for antibody drug conjugates and applications thereof, such as CN111433188A, wherein the linker can be simultaneously coupled with sulfhydryl or amino groups on an antibody or a functional fragment of the antibody, especially can be coupled with 2,3 or 4 sulfhydryl groups of the functional fragment of the antibody, and the coupled product is uniform and stable in structure.
There remains a need to develop an antibody oligonucleotide coupling method that replaces the use of maleimide and is simple to operate with less impact on antibody structure.
Disclosure of Invention
In order to solve the above-mentioned problems, the present invention provides a method for coupling an antibody oligonucleotide, which comprises coupling an antibody with a thiol-modified oligonucleotide using a hetero-bifunctional crosslinking agent (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester (hereinafter abbreviated as hetero-bifunctional crosslinking agent).
Further, the structural formula of the (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester is shown in the following formula (I):
(formula I).
Further, the (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester can be obtained commercially, custom made by chemical synthesis companies, or prepared using conventional methods well known in the art, for example, as described in the examples of the present invention.
Further, the heterobifunctional crosslinking reagent is linked to the thiol-modified oligonucleotide by a thiol-michael addition reaction.
Further, the heterobifunctional crosslinking reagent is linked to the antibody by reaction of a succinimidyl ester with a primary amino group on the antibody.
Further, the method for antibody oligonucleotide coupling comprises: (1) Coupling reaction is carried out on the abnormal bifunctional crosslinking agent and the sulfhydryl modified oligonucleotide to obtain a coupling intermediate; (2) And then carrying out a coupling reaction on the coupling intermediate and the antibody to obtain the antibody oligonucleotide conjugate. Further, the reaction route of the method is as follows:
。
further, the method for antibody oligonucleotide coupling comprises: and (3) carrying out coupling reaction on the heterobifunctional crosslinking agent, the antibody and the sulfhydryl modified oligonucleotide in the same reaction system to obtain the antibody oligonucleotide conjugate. Further, the reaction route of the method is as follows:
。
further, the coupling reactions were all performed in phosphate buffer at pH 8.0.
Further, the heterobifunctional cross-linking reagent is dissolved in N, N-dimethylformamide and then added to a phosphate buffer comprising antibodies and/or thiol-modified oligonucleotides.
Further, the molar ratio of the antibody, thiol-modified oligonucleotide, and heterobifunctional crosslinking reagent is 1:5:20.
Further, it includes first activating the thiol-modified oligonucleotide with a reducing agent to reduce disulfide bonds in the thiol-modified oligonucleotide to active thiols.
Further, the reducing agent is selected from dithiothreitol.
The invention also provides an antibody oligonucleotide conjugate prepared by the methods described herein.
The beneficial effects of the invention are that
As is known in the art, heterobifunctional crosslinkers have different reactive groups at both ends to couple molecules with respective target functional groups, and sequential (two-step) coupling is typically performed using heterobifunctional crosslinkers in order to minimize unwanted polymerization or self-coupling. The present invention has found that compounds of formula (I) are particularly suitable for use as heterobifunctional cross-linkers for coupling antibodies and oligonucleotides together and, more importantly, can be coupled in a single step process without unwanted polymerization or self-coupling. The principle of the invention is that a compound shown in the formula I is utilized to carry out a sulfhydryl Michael addition reaction with sulfhydryl modified oligonucleotide, and simultaneously carry out amidation reaction with a natural antibody with primary amino group, so that the antibody and the oligonucleotide are coupled together. The compounds selected for use as heterobifunctional cross-linkers according to the invention (formula I) are very specific, since the methylsulfonyl vinyl groups carried by them can undergo selective thiol-michael addition reactions with thiol-modified oligonucleotides without reacting with small amounts of free thiol groups that the antibody itself may carry (although in theory all cysteines within the antibody molecule should form intra-or inter-chain disulfide bonds with other cysteines, there has been much evidence of the presence of free thiol structures on the antibody molecule) thereby causing self-coupling of the antibody. The method of the invention simplifies the operation steps of coupling the antibody and the oligonucleotide, and reduces the treatment times of the antibody, thereby avoiding the risk of activity reduction caused by structural change of the antibody in the treatment process.
Drawings
FIG. 1 shows the results of non-reducing SDS-PAGE Coomassie blue and EB staining of the antibody-oligonucleotide conjugates prepared according to the present invention.
FIG. 2 shows the antigen binding activity of the antibody-oligonucleotide conjugates prepared according to the present invention.
FIG. 3 shows the synthetic route for (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: (E) Preparation of N-succinimidyl-6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoate
(E) The preparation of N-succinimidyl-6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoate was carried out with reference to published literature, the synthetic route of which is shown in FIG. 3.
In short, compound S2 was synthesized based on the previously disclosed procedure. 1H NMR. Delta. 7.54 (2H), 6.69 (2H), 5.37 (1H), 3.98 (2H), 1.90 (2H), 1.78 (2H), 1.62 (2H), 1.45 (2H).
Synthesis of compound S3: to a dry reaction tube (20 mL) was added S2 (501 mg,1.5mmol,1 eq), tBuOH (5 eq), anhydrous DCM (2 mL). The mixture was cooled to 0deg.C and DMAP (0.1 eq.) was added dropwise. DCC (1.1 eq) was then added. The resulting mixture was stirred at room temperature overnight. Purifying the crude product by silica gel column chromatography to obtain S3.1H NMR: δ 7.55 (2H), 6.67 (2H), 3.98 (2H), 1.92 (2H), 1.75 (2H), 1.63 (2H), 1.45 (2H), 1.43 (9H).
Synthesis of compound S4: to a dry reaction tube (20 mL) was added AgTFA (0.6 mmol,1.2 eq), pd (OAc) 2 (5.5 mg,5 mol%), acetone (2 mL), S3 (1.02 g,0.5 mmol) and (methylsulfonyl) ethylene (212 mg,2 eq). The resulting mixture was refluxed at 60 ℃ for 12h. Purifying the crude product by silica gel column chromatography to obtain S4.1H NMR: δ 7.89 (1H), 7.41 (2H), 6.91 (2H), 6.58 (1H), 3.98 (2H), 3.18 (3H), 1.88 (2H), 1.80 (2H), 1.60 (2H), 1.46 (2H), 1.42 (9H).
Synthesis of compound S5: to a dry reaction tube (20 mL) was added S4 (0.25 mmol), TFA (2 mL) and DCM (2 mL). The resulting mixture was stirred at room temperature for 4h. Purifying the crude product by silica gel column chromatography to obtain S5.1H NMR: δ 7.87 (1H), 7.40 (2H), 6.86 (2H), 6.61 (1H), 5.37 (1H), 3.95 (2H), 3.22 (3H), 1.90 (2H), 1.77 (2H), 1.61 (2H), 1.47 (2H).
(E) -synthesis of N-succinimidyl-6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoate (compound S6): to a dry reaction tube (20 mL) were added S5 (0.2 mmol), N-hydroxysuccinimide (1.2 eq), EDC (1.2 eq) and CH 2Cl2 (4 mL). The resulting mixture was stirred at room temperature for 12h. After completion of the reaction, the reaction was quenched with 20mL of water and extracted 3 times with 20mL of DCM, the organic phases were combined, dried over anhydrous Na 2SO4 and concentrated to give the compound S6.1H NMR: δ 7.91 (1H), 7.42 (2H), 6.89 (2H), 6.59 (1H), 4.01 (2H), 3.17 (3H), 2.84 (4H), 2.36 (2H), 1.77 (2H), 1.62 (2H), 1.43 (2H).
Examples
This example provides a method for coupling an antibody oligonucleotide comprising coupling an antibody with a thiol-modified oligonucleotide using a heterobifunctional cross-linker (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester.
The antibodies used in this example were commercial anti-CD 44 monoclonal antibodies (Abcam, ab 157107).
Thiol-modified oligonucleotides are obtained by reduction of commercially available thiol-modified oligonucleotides.
Commercially available thiol-modified oligonucleotides: HO- (CH 2)6-S-S-(CH2)6 -ACGTACGTACGTACGTACGTACGT).
The reduction scheme for thiol-modified oligonucleotides is: thiol-modified oligonucleotides were reduced by adding 50mM tris (2-carboxyethyl) phosphine (TCEP) and incubating for 1 hour at 25 ℃. Excess TCEP was removed by ethanol precipitation.
Antibody-oligonucleotide coupling: anti-CD 44 monoclonal antibody and thiol-modified oligonucleotide were added to PBS buffer at pH 8.0 to give concentrations of 20. Mu.M and 100. Mu.M, respectively, followed by addition of an equal volume of DMF solution of (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester (400. Mu.M concentration). The mixture was incubated overnight at 30℃with shaking. Unreacted (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester was removed from the reaction mixture using a Zeba desalting spin column equilibrated in PBS+5mM EDTA pH 7.2, following the instructions of the supplier, excess unreacted sulfhydryl modified oligonucleotide was removed from the reaction mixture by sulfophilic adsorption chromatography, the fractions containing the conjugate were pooled and concentrated using an Amicon 10kDa MWCO spin filter. The final protein concentration of the antibody-oligonucleotide conjugate was determined using the (biquinolinecarboxylic acid) BCA protein assay kit with bovine gamma globulin standard.
Comparative example 1
Following the procedure described in example 1, using vinylsulfonyl fluoride instead of (methylsulfonyl) ethylene in the synthesis of compound S4, the final preparation of (E) -6- (4- (2- (fluorosulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester was performed as follows:
。
The hetero-bifunctional cross-linker was coupled to the thiol-modified oligonucleotide using (E) -6- (4- (2- (fluorosulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester as hetero-bifunctional cross-linker according to the method described in example 2.
Test example 1:
Non-reducing SDS-PAGE analysis was performed on 2. Mu.g of the antibody-oligonucleotide conjugate prepared in example 2 and comparative example 1, and the gel after electrophoresis was stained with Coomassie brilliant blue and EB, as shown in FIG. 1, wherein the left panel shows the imaging result of EB staining, the right panel shows the imaging result of Coomassie brilliant blue staining, lane 1 shows a protein Marker, lane 2 shows an unconjugated anti-CD 44 monoclonal antibody, lane 3 shows the antibody-oligonucleotide conjugate prepared in example 2, and lane 4 shows the antibody-oligonucleotide conjugate prepared in comparative example 1.
From the results of fig. 1, it can be seen that the sample prepared in example 2 has a significantly increased molecular weight when compared to the unconjugated antibody, indicating successful preparation of the antibody-oligonucleotide conjugate. The results of comparative example 1 also indicate that an antibody-oligonucleotide conjugate was successfully prepared, but lane 4 was seen to have one more protein band above the antibody-oligonucleotide conjugate band, but this band was not shown in EB staining imaging, indicating that it was not a conjugate of an antibody and an oligonucleotide, but rather a conjugate between antibody molecules, such conjugate being due to the reaction of the highly reactive (fluorosulfonyl) vinyl group in the (E) -6- (4- (2- (fluorosulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimide ester used in comparative example 1 with the free thiol group on the antibody, resulting in cross-linking between antibody molecules, resulting in the formation of a conjugate of an antibody-cross-linker-antibody structure. The (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester used in example 2 of the present invention did not exist, probably because of the low activity of the (methylsulfonyl) vinyl group, which was insufficient to react with the free thiol group on the antibody.
Test example 2
The antibody-oligonucleotide conjugate prepared in example 2 was tested for its ability to recognize antigen by ELISA to demonstrate that there was no loss of antibody activity during the conjugation of antibody and oligonucleotide.
1. Antigen coating: diluting antigen (human CD44 recombinant protein) to 10 [ mu ] g/mL by using a1 Xcarbonate coating buffer (Na 2CO3（1.59g）+ NaHCO3 (2.93 g), adjusting the pH value and then fixing the volume to 50 mL), and coating 96-well plates (a positive control group, a negative control group and an experimental group are arranged, wherein 3 compound wells are arranged in each group) by using 50 [ mu ] L of diluted antigen to 0.5 [ mu ] g/200 [ mu ] L;
2. Closing: the wells were pipetted and washed 3 times with wash buffer (0.05% solution of Tween-20 in 1 XPBS) and 100. Mu.L of blocking solution (skimmed milk powder (1 g) +1XTBST (20 mL)) was added to each well and incubated for 1h at 37 ℃;
3. Incubation of antibody to be tested: washing the 96-well plate in a plate washer for 5 times, adding antibodies to be detected with different concentrations according to different groups, standing at 37 ℃ for 1h, discarding liquid in the holes, washing for 3min by using a washing buffer solution table, and repeating washing for three times;
4. And (3) incubation of enzyme-labeled secondary antibodies: 100 mu L of diluted enzyme-labeled secondary antibody (diluted 1:5000) is added into each well of a 96-well plate, and the mixture is incubated for 30min at 37 ℃ in a dark place;
5. color development: placing the 96-well plate into a plate washer for washing for 6-7 times, adding 100 TMB color development liquid into each well, and developing the color of the liquid in the well to be blue at 37 ℃ in a dark place for 5-15min;
6. Detecting the absorbance: 50. Mu.L of stop solution (the liquid in the well turns yellow) was added to each well, and the absorbance was measured at 450nm by a microplate reader.
The results are shown in FIG. 2, which shows that the antibody-oligonucleotide conjugate prepared in example 2 has substantially equivalent binding activity to CD44 protein as the unconjugated CD44 monoclonal antibody. It was demonstrated that the conjugation method of the present invention did not lose the antigen binding activity of the antibodies.
It should be noted that while the present invention has been described in connection with the preferred embodiments thereof, it should be understood that the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but are to be construed as providing a full breadth of the disclosure. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A method for coupling an antibody oligonucleotide comprising coupling an antibody to a thiol-modified oligonucleotide using a heterobifunctional cross-linker (E) -6- (4- (2- (methylsulfonyl) vinyl) phenoxy) hexanoic acid-N-succinimidyl ester.
2. The method of claim 1, wherein the heterobifunctional crosslinking reagent is attached to the thiol-modified oligonucleotide via a thiol-michael addition reaction and to the antibody via reaction of a succinimidyl ester with a primary amine group on the antibody.
3. The method according to claim 1, characterized in that it comprises:
(1) Coupling reaction is carried out on the abnormal bifunctional crosslinking agent and the sulfhydryl modified oligonucleotide to obtain a coupling intermediate;
(2) And then carrying out a coupling reaction on the coupling intermediate and the antibody to obtain the antibody oligonucleotide conjugate.
4. The method according to claim 1, characterized in that it comprises: and (3) carrying out coupling reaction on the heterobifunctional crosslinking agent, the antibody and the sulfhydryl modified oligonucleotide in the same reaction system to obtain the antibody oligonucleotide conjugate.
5. The method according to claim 3 or 4, wherein the coupling reactions are each carried out in phosphate buffer at a pH of 8.0.
6. The method according to claim 5, wherein the heterobifunctional cross-linking reagent is dissolved in N, N-dimethylformamide and then added to a phosphate buffer comprising antibodies and/or thiol-modified oligonucleotides.
7. The method of claim 1, wherein the molar ratio of the antibody, thiol-modified oligonucleotide, and heterobifunctional crosslinking reagent is 1:5:20.
8. The method of claim 1, further comprising first activating the thiol-modified oligonucleotide with a reducing agent to reduce disulfide bonds in the thiol-modified oligonucleotide to active thiols.
9. The method of claim 8, wherein the reducing agent is selected from dithiothreitol.
10. An antibody oligonucleotide conjugate prepared by the method of any one of claims 1-9.
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