CN118496347A - Method for coupling antibody oligonucleotides - Google Patents

Abstract

The present invention belongs to the field of biotechnology, and in particular to a method for antibody oligonucleotide coupling. 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 the compound of (Formula I) is particularly suitable for coupling an antibody and an oligonucleotide as a heterobifunctional crosslinker, and more importantly, a single-step coupling can be performed without unwanted polymerization or self-coupling, which simplifies the operation and reduces the number of times the antibody is treated, thereby avoiding the risk of reduced activity due to structural changes in the antibody during the treatment process.

Description

A method for antibody oligonucleotide coupling

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a method for antibody oligonucleotide coupling.

Background Art

The development of bioconjugation technology has become the cornerstone of chemical proteomics, biomaterial synthesis, biomolecular imaging, single molecule analysis, single cell multi-omics and multi-specific drugs. Modification of natural or unnatural amino acids on proteins is a common method of bioconjugation.

There are multiple primary amine groups on antibodies, present at the N-terminus (called α-amino) of each polypeptide chain and on the side chains of lysine residues. Because primary amines carry a positive charge under physiological conditions, they are usually facing outward, that is, on the outer surface of the antibody, which makes it easy to couple without changing the protein structure. Currently, oligonucleotide synthesis technology is very mature and inexpensive. Synthesized oligonucleotides can also selectively add various types of modified ends, among which the technology of adding primary amine or thiol group modification to the end is mature, inexpensive, and has a fast synthesis cycle.

There are currently a variety of chemical reagents that can be used to couple antibodies to oligonucleotides, such as sulfo-SMCC from ThermoFisher and antibody coupling agents from MCE. The main principle is to modify the functional groups on the antibody and couple them to the cross-linking agent, which then reacts chemically or affinity with the functional groups of other oligonucleotides to form a coupling substance. However, these methods usually involve two-step treatment of the antibody, namely activation first and then coupling with the oligonucleotide. In such a complex and time-consuming process, the structure of the antibody is at risk of denaturation and failure.

There is also a commonly used antibody conjugation strategy, which uses cysteine (Cys) in antibodies to specifically modify Cys through its high nucleophilicity and low abundance, thereby conjugating it to other small molecules. So far, 7 of the 12 antibody-drug conjugates approved by the US Food and Drug Administration are constructed by Michael addition of Cys and maleimide (Mal). Although Mal has been widely used in protein modification since its discovery in the 1950s, there are some problems with the addition of thiol and Mal that limit its application. First, Mal may react with lysine at high pH values (pH>7.5), which will produce heterogeneous conjugates; second, Mal is easily hydrolyzed in alkaline solutions to generate non-reactive maleimide acid, which requires the Mal reagent to be stored under strict conditions; third, the thiol-Mal adduct is unstable under physiological conditions and is prone to reversible reactions, and its application in antibody-drug conjugates will cause off-target effects. In addition, the thiol groups on antibodies usually exist in the form of disulfide bonds, which need to be reduced to active thiol groups, which leads to complicated operations and may damage the structure of the antibody. In addition, the thiol in the residual reducing agent will compete with the reaction of maleimide with the thiol groups on the antibody.

The prior art discloses a type of linker for antibody-drug conjugates and its application, such as CN111433188A. The linker can be coupled to the thiol or amino group on the antibody or the functional fragment of the antibody at the same time, especially to 2, 3 or 4 thiol groups of the functional fragment of the antibody. The coupled product is uniform and has a stable structure.

However, there is still a need to develop an antibody oligonucleotide coupling method that can replace the use of maleimide and is easy to operate and has less impact on the antibody structure.

Summary of the invention

In order to solve the above problems, the present invention provides a method for antibody-oligonucleotide coupling, which comprises coupling an antibody and a thiol-modified oligonucleotide together using a heterobifunctional cross-linking agent (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester (hereinafter referred to as the heterobifunctional cross-linking agent).

Further, the structural formula of the (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimide ester is shown in the following (Formula I):

(Formula I).

Furthermore, the (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester can be commercially available, customized by a chemical synthesis company, or prepared using conventional methods well known in the art, for example, as described in the examples of the present invention.

Furthermore, the heterobifunctional cross-linker is linked to the thiol-modified oligonucleotide via a thiol Michael addition reaction.

Furthermore, the heterobifunctional cross-linking agent is linked to the antibody through the reaction of succinimidyl ester with primary amine groups on the antibody.

Furthermore, the method for antibody-oligonucleotide coupling comprises: (1) coupling the heterobifunctional crosslinker with a thiol-modified oligonucleotide to obtain a coupling intermediate; (2) then coupling the coupling intermediate with an antibody to obtain an antibody-oligonucleotide conjugate. Furthermore, the reaction route of the method is as follows:

.

Furthermore, the method for antibody-oligonucleotide coupling comprises: coupling the heterobifunctional crosslinker with an antibody and a thiol-modified oligonucleotide in the same reaction system to obtain an antibody-oligonucleotide conjugate. Furthermore, the reaction route of the method is as follows:

.

Furthermore, the coupling reactions were all carried out in a phosphate buffer solution at a pH of 8.0.

Further, the heterobifunctional cross-linker is dissolved in N,N-dimethylformamide and then added to a phosphate buffer containing the antibody and/or thiol-modified oligonucleotide.

Furthermore, the molar ratio of the antibody, the thiol-modified oligonucleotide and the heterobifunctional cross-linking agent is 1:5:20.

Furthermore, the method also includes first activating the thiol-modified oligonucleotide using a reducing agent to reduce the disulfide bonds in the thiol-modified oligonucleotide to active thiol groups.

Furthermore, the reducing agent is selected from dithiothreitol.

The present invention also provides an antibody-oligonucleotide conjugate prepared by the method described herein.

Beneficial Effects of the Invention

As is known in the art, heterobifunctional crosslinkers have different reactive groups at both ends to couple molecules with respective target functional groups, and in order to minimize unwanted polymerization or self-coupling, heterobifunctional crosslinkers are usually used for sequential (two-step) coupling. The present invention has found that the compound of (Formula I) is particularly suitable for use as a heterobifunctional crosslinker to couple antibodies and oligonucleotides together, and more importantly, can be coupled in a single step without unwanted polymerization or self-coupling. The principle of the present invention is to use the compound of (Formula I) to react with a thiol-modified oligonucleotide by a thiol Michael addition reaction, and to react with an antibody naturally bearing a primary amine group by an amidation reaction, thereby coupling the antibody and the oligonucleotide together. The compound (Formula I) selected as the heterobifunctional cross-linking agent of the present invention is very special because the methylsulfonyl vinyl group carried by it can react with the thiol-modified oligonucleotide by selective thiol Michael addition reaction, but will not react with the small amount of free thiol groups that may be carried by the antibody itself (although in theory all cysteines in the antibody molecule should form intrachain or interchain disulfide bonds with other cysteines, there is a lot of evidence showing that free thiol structures exist on antibody molecules), thereby causing the antibody to self-couple. The method of the present invention simplifies the operation steps of coupling the antibody and the oligonucleotide, and reduces the number of times the antibody is treated, thereby avoiding the risk of reduced activity due to structural changes in the antibody during the treatment process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the non-reducing SDS-PAGE Coomassie Brilliant Blue and EB staining results of the antibody-oligonucleotide conjugate prepared by the present invention.

FIG2 shows the antigen binding activity of the antibody-oligonucleotide conjugates prepared in the present invention.

FIG3 shows the synthetic route of (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester.

DETAILED DESCRIPTION

The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Example 1: Preparation of (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester

The preparation of (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester was carried out with reference to published literature, and its synthetic route is shown in FIG3 .

Briefly, compound S2 was synthesized based on a previously published procedure. 1H NMR: δ 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: S2 (501 mg, 1.5 mmol, 1 eq.), tBuOH (5 eq.), and anhydrous DCM (2 mL) were added to a dry reaction tube (20 mL). The mixture was cooled to 0 ° C and DMAP (0.1 eq.) was added dropwise. Then DCC (1.1 eq.) was added. The resulting mixture was stirred at room temperature overnight. The crude product was purified by silica gel column chromatography to obtain S3. 1H NMR: δ7.55 (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: 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.) were added to a dry reaction tube (20 mL). The resulting mixture was refluxed at 60 ° C for 12 h. The crude product was purified by silica gel column chromatography to give 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: S4 (0.25 mmol), TFA (2 mL) and DCM (2 mL) were added to a dry reaction tube (20 mL). The resulting mixture was stirred at room temperature for 4 h. The crude product was purified 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).

Synthesis of (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester (Compound S6): S5 (0.2 mmol), N-hydroxysuccinimide (1.2 eq.), EDC (1.2 eq.) and CH ₂ Cl ₂ (4 mL) were added to a dry reaction tube (20 mL). The resulting mixture was stirred at room temperature for 12 h. After completion of the reaction, the reaction was quenched with 20 mL of water and extracted three times with 20 mL of DCM. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ and concentrated to give compound S6. 1H NMR: δ7.91 (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).

Example

This embodiment provides a method for antibody-oligonucleotide coupling, which includes 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.

The antibody used in this example is a commercial anti-CD44 monoclonal antibody (Abcam, ab157107).

Thiol-modified oligonucleotides are obtained by reducing commercially available thiol-modified oligonucleotides.

Commercially available thiol-modified oligonucleotide: HO-(CH ₂ ) ₆ -SS-(CH ₂ ) ₆ -ACGTACGTACGTACGTACGTACGT.

The reduction protocol for thiol-modified oligonucleotides was as follows: thiol-modified oligonucleotides were reduced by adding 50 mM tris(2-carboxyethyl)phosphine (TCEP) and incubating for 1 hour at 25° C. Excess TCEP was removed by ethanol precipitation.

Antibody-oligonucleotide conjugation: Anti-CD44 monoclonal antibody and thiol-modified oligonucleotide were added to PBS buffer at pH 8.0 to a concentration of 20 μM and 100 μM, respectively, and then an equal volume of (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester in DMF (400 μM concentration) was added. The mixture was incubated overnight at 30°C with shaking. Unreacted (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester was removed from the reaction mixture using Zeba desalting spin columns equilibrated in PBS + 5 mM EDTA pH 7.2 according to the supplier's instructions, and excess unreacted thiol-modified oligonucleotide was then removed from the reaction mixture using thiophilic adsorption chromatography, and fractions containing the conjugate were pooled and concentrated using an Amicon 10 kDa MWCO spin filter. The final protein concentration of the antibody-oligonucleotide conjugate was determined using a (bicinchoninic acid) BCA protein assay kit with bovine gamma globulin standards.

Comparative Example 1

According to the method described in Example 1, ethylene sulfonyl fluoride was used instead of (methylsulfonyl)ethylene in the synthesis of compound S4, thereby finally preparing (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester, the structural formula of which is shown below:

.

According to the method described in Example 2, (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester was used as a heterobifunctional cross-linker to couple the heterobifunctional cross-linker with the thiol-modified oligonucleotide.

Test Example 1:

2 μg of the antibody-oligonucleotide conjugates prepared in Example 2 and Comparative Example 1 were subjected to non-reducing SDS-PAGE analysis, and the gel after electrophoresis was stained with Coomassie Brilliant Blue and EB. The results are shown in Figure 1, wherein the left figure is the EB staining imaging result, the right figure is the Coomassie Brilliant Blue staining imaging result, lane 1 is the protein Marker, lane 2 is the unconjugated anti-CD44 monoclonal antibody, lane 3 is the antibody-oligonucleotide conjugate prepared in Example 2, and lane 4 is the antibody-oligonucleotide conjugate prepared in Comparative Example 1.

As can be seen from the results of Figure 1, the molecular weight of the sample prepared in Example 2 increased significantly relative to the unconjugated antibody, indicating that the antibody-oligonucleotide conjugate was successfully prepared. The results of Comparative Example 1 also show that the antibody-oligonucleotide conjugate was successfully prepared, but it can be seen that a protein band appears above the antibody-oligonucleotide conjugate band in lane 4, but this band is not shown in the EB staining imaging, indicating that it is not a conjugate of antibody and oligonucleotide, but a conjugate between antibody molecules. Such a conjugate is due to the reaction of the highly active (fluorosulfonyl) vinyl in (E)-6-(4-(2-(fluorosulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester used in Comparative Example 1 with the free thiol on the antibody, resulting in cross-linking between antibody molecules, forming a conjugate of antibody-crosslinker-antibody structure. However, this phenomenon does not exist in (E)-6-(4-(2-(methylsulfonyl)vinyl)phenoxy)hexanoic acid-N-succinimidyl ester used in Example 2 of the present invention, which may be due to the low activity of the (methylsulfonyl)vinyl group therein, which is not enough to react with the free thiol group on the antibody.

Test Example 2

The antigen recognition ability of the antibody-oligonucleotide conjugate prepared in Example 2 was tested by ELISA to prove that the antibody activity was not lost during the antibody and oligonucleotide conjugation process.

1. Antigen coating: dilute the antigen (human CD44 recombinant protein) to 10µg/mL with 1× carbonate coating buffer (Na ₂ CO ₃ (1.59g) + NaHCO ₃ (2.93g), adjust the pH value and make up to 50mL), take 50µL of the diluted antigen and coat a 96-well plate at 0.5µg/200µL (set up a positive control group, a negative control group and an experimental group, with 3 replicates for each group);

2. Blocking: Aspirate the liquid in the wells, wash three times with washing buffer (1×PBS with Tween-20 to make a 0.05% solution), add 100 μL of blocking solution (skim milk powder (1 g) + 1×TBST (20 mL)) to each well, and incubate at 37°C for 1 hour;

3. Incubation of the test antibody: Wash the 96-well plate in a plate washer for 5 times, add different concentrations of the test antibody according to different groups, place at 37°C for 1 hour, discard the liquid in the wells, wash with washing buffer on a shaker for 3 minutes, and repeat the washing three times;

4. Enzyme-labeled secondary antibody incubation: Add 100 μL of diluted enzyme-labeled secondary antibody (1:5000 dilution) to each well of the 96-well plate and incubate at 37°C in the dark for 30 minutes;

5. Color development: Wash the 96-well plate in a plate washer for 6-7 times, add 100 TMB color development solution to each well, and the liquid in the well turns blue. Incubate the plate in a dark place at 37°C for 5-15 minutes.

6. Detect absorbance: Add 50 μL of stop solution to each well (the liquid in the well turns yellow) and detect absorbance at 450 nm using an ELISA reader.

The results are shown in Figure 2, which show that the binding activity of the antibody-oligonucleotide conjugate prepared in Example 2 to CD44 protein is substantially equivalent to that of the unconjugated CD44 monoclonal antibody, proving that the coupling method of the present invention does not lose the antigen binding activity of the antibody.

It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims (10)

1. A method for antibody-oligonucleotide coupling, characterized in that it comprises 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. 2. The method according to claim 1, characterized in that the heterobifunctional cross-linker is linked to the thiol-modified oligonucleotide through a thiol Michael addition reaction, and is linked to the antibody through the reaction of succinimidyl ester with primary amine groups on the antibody. 3. The method according to claim 1, characterized in that it comprises: (1) coupling the heterobifunctional cross-linking agent with a thiol-modified oligonucleotide to obtain a coupling intermediate; (2) Then, the coupling intermediate is coupled with the antibody to obtain an antibody-oligonucleotide conjugate. 4. The method according to claim 1, characterized in that it comprises: performing a coupling reaction between the heterobifunctional cross-linking agent, an antibody and a thiol-modified oligonucleotide in the same reaction system to obtain an antibody-oligonucleotide conjugate. 5. The method according to claim 3 or 4, characterized in that the coupling reactions are all carried out in a phosphate buffer at a pH of 8.0. 6. The method according to claim 5, characterized in that the heterobifunctional cross-linker is dissolved in N,N-dimethylformamide and then added to the phosphate buffer containing the antibody and/or the thiol-modified oligonucleotide. 7. The method according to claim 1, characterized in that the molar ratio of the antibody, the thiol-modified oligonucleotide and the heterobifunctional cross-linking agent is 1:5:20. 8. The method according to claim 1, further comprising first activating the thiol-modified oligonucleotide using a reducing agent to reduce the disulfide bonds in the thiol-modified oligonucleotide to active thiol groups. 9. The method according to claim 8, characterized in that the reducing agent is selected from dithiothreitol. 10. An antibody-oligonucleotide conjugate, characterized in that it is prepared by the method according to any one of claims 1 to 9.