Coupling compound of oligonucleotide medicine and carrier protein and preparation method thereof

Abstract

The invention discloses a coupling compound of an oligonucleotide drug and a carrier protein and a preparation method of the coupling compound, and belongs to the field of biological medicines.The method for preparing the coupling compound comprises the steps that firstly, the oligonucleotide drug is subjected to maleimide activation, and the carrier protein is subjected to sulfhydrylation modification; the oligonucleotide drug activated by maleimide is coupled with the carrier protein modified by sulfhydrylation, the preparation method is simple to operate and convenient to widely use, and the prepared coupling compound is high in coupling efficiency and high in antibody titer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a coupling complex of an oligonucleotide drug and a carrier protein and a preparation method thereof. Background Art

[0002] Compared with macromolecular biologics (such as monoclonal antibodies), oligonucleotide drugs have a simple structure and a small molecular weight. Because they have fewer potential epitopes, they are generally considered to have lower immunogenicity. However, the complex interactions between nucleic acid molecules and the immune system have the potential to induce innate immunity. In order to improve the stability, safety, absorption and efficacy of nucleic acid drugs, they are usually chemically modified during the development of new-generation oligonucleotide drugs, and these modifications are very likely to increase the immunogenicity of the drugs and enhance the body's immune response. When analyzing the immunogenicity of oligonucleotide drugs, oligonucleotide drug antibodies are used as positive control antibodies. In order to establish a fast, accurate and reliable analysis method, oligonucleotide drug antibodies need to have high specificity and high titer.

[0003] Although the new generation of oligonucleotide drugs have a certain degree of chemical modification, the overall structure is still relatively simple, and the immunogenicity is still poor compared with macromolecules such as proteins. Directly using them as antigens to immunize animals for antibody preparation usually does not produce high-quality positive quality control antibodies. The currently widely used method is to first couple the oligonucleotide drug with a certain carrier protein (such as hemocyanin KLH, bovine serum albumin BSA, ovalbumin OVA, etc.) to form a complex, so that it forms a complete antigen before animal immunization and antibody preparation. Conventional coupling methods usually couple the primary amine group (-NH2) on the oligonucleotide drug with the primary amine group (-NH2) on the carrier protein with the help of glutaraldehyde crosslinkers, or couple with the carboxyl group (-COOH) or sulfhydryl group (-SH) on the carrier protein with crosslinkers such as EDC and SMCC. However, the former is prone to self-linking of the carrier or oligonucleotide, while the latter is due to the limited number of free sulfhydryl groups on the carrier protein and the poor reactivity of the -NH2 group on the oligonucleotide, resulting in low coupling efficiency. The above conventional methods are difficult to obtain highly efficient coupling products, resulting in poor subsequent animal immunization effects and low antibody titers of approximately 1:100,000. In the establishment of drug immunogenicity analysis methods, its sensitivity is difficult to meet compliance requirements. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a coupling complex of an oligonucleotide drug and a carrier protein and a preparation method thereof, wherein the coupling complex has good animal immune effect, the prepared antibody has high titer, and can be used for immunogenicity analysis; at the same time, the preparation method of the coupling complex improves the coupling efficiency of the oligonucleotide drug and the carrier protein.

[0005] In order to solve the above technical problems, the first aspect of the present invention is to provide a coupling complex of an oligonucleotide drug and a carrier protein, the structure of which is shown in Formula I:

[0006]

[0007] In the formula, the 5 ， The terminal phosphate group is connected to R2 through carbon-oxygen bonding, and the lysine side chain of the carrier protein is connected to R1 through sulfur-oxygen bonding;

[0008] In the formula, R1 and R2 are independently selected from a C1-C10 straight carbon chain and / or a PEG chain with a degree of polymerization of 1-10.

[0009] The coupled complex provided by the present invention has a complete structure, good stability, is easy to synthesize and purify, and is convenient to prepare. The antibody obtained by using the coupled complex has high antibody titer, strong specificity and high sensitivity, providing a favorable guarantee for the smooth establishment of immunogenicity analysis and pharmacokinetic detection of oligonucleotide drugs.

[0010] In order to solve the above technical problems, the second aspect of the present invention is to provide a method for preparing a coupling complex of an oligonucleotide drug and a carrier protein, comprising the following steps:

[0011] Step S1, maleimide activation of the oligonucleotide drug;

[0012] Step S2, modifying the carrier protein with thiol groups;

[0013] Step S3, coupling the oligonucleotide drug activated by maleimide in step S1 with the carrier protein modified by thiol in step S2.

[0014] In the present invention, a maleimide group is introduced into the structure of the oligonucleotide drug, and the carrier protein is thiol-modified, and then the oligonucleotide drug is coupled to the carrier protein through coupling of the maleimide group and the thiol group, which can effectively avoid self-linking of the carrier protein or the oligonucleotide. At the same time, by thiol-modifying the carrier protein, the number of thiol groups of the carrier protein is increased, thereby improving the coupling efficiency.

[0015] In the present invention, the oligonucleotide drug may be, for example, siRNA, mRNA, and ASO; the carrier protein may be, for example, bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA), hemocyanin (KLH), human IgG constant region protein (Fc), etc.

[0016] Specifically, in step S1, at 5 ，The maleimide-containing group is introduced into the terminal phosphate group to realize maleimide activation of the oligonucleotide drug. Preferably, the introduced maleimide group includes an amide bond. The activation step of the maleimide of the oligonucleotide drug can be, for example, firstly at the 5 ， The terminal phosphate group is introduced into a terminal amino group to form a product of formula II, which is then subjected to an amidation reaction with a maleimide derivative of formula III to produce an activated oligonucleotide drug of formula IV. It should be noted that the maleimide activation step of the oligonucleotide drug is not limited to this. ， Any method of introducing a terminal phosphate group into a maleimide-containing group is applicable to the present invention.

[0017]

[0018] Wherein, R1 and R2 are independently selected from C1-C10 straight carbon chains and / or PEG chains with a degree of polymerization of 1-10. R1 is preferably a C1-C4 straight carbon chain and / or a PEG chain with a degree of polymerization of 1-4, and R2 is preferably a C3-C6 straight carbon chain and / or a PEG chain with a degree of polymerization of 4-8.

[0019] Specifically, in step S2, the carrier protein is thiolated, including: incubating the carrier protein with a compound that can generate thiol groups in a first coupling buffer to obtain a thiol-modified carrier protein. Preferably, the compound that can generate thiol groups is selected from 2-iminothiolane, N-succinic acid S-acetylthioglycol ester or 3-[2-pyridyldithio]succinimidyl propionate.

[0020] Specifically, the thiolation site of the carrier protein is located at the amino group on the lysine side chain, preferably bonded via an amidine bond or an amide bond.

[0021] Specifically, the molar ratio of the carrier protein to the thiol-containing compound is 1:40 to 80;

[0022] The first coupling buffer is a PBS-EDTA solution with a pH value of 7.0-9.0.

[0023] Specifically, in step S3, the carrier protein modified by thiol group and the oligonucleotide drug activated by maleimide are coupled in the second coupling buffer.

[0024] Specifically, the molar ratio of the carrier protein modified by thiol group to the oligonucleotide drug activated by maleimide is 1:5-20; the second coupling buffer is a PBS-EDTA solution with a pH value of 6.5-7.5.

[0025] In the present invention, the coupling efficiency of the oligonucleotide drug activated by maleimide and the carrier protein modified by thiol is increased by at least 250% compared with the coupling efficiency of the conventional method, and the coupling method is highly operable and convenient for wide use.

[0026] The antibody titer of the coupled complex prepared by the aforementioned oligonucleotide drug and carrier protein coupling method provided by the present invention can reach above 1:1000000, with strong specificity and high sensitivity, providing favorable guarantee for the smooth establishment of oligonucleotide drug immunogenicity analysis and pharmacokinetic detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 is the SDS-PAGE analysis electrophoresis diagram of the oligonucleotide drug-BSA coupling complex obtained in Example 1;

[0029] Figure 2 This is the SDS-PAGE analysis electrophoresis diagram of the oligonucleotide drug-OVA coupling complex obtained in Example 2;

[0030] Figure 3 This is a graph showing the antibody titer results obtained by ELISA analysis of the oligonucleotide drug-KLH coupling complex obtained in Example 3; DETAILED DESCRIPTION

[0031] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In the examples of the present invention, BSA was purchased from Shanghai Aladdin, OVA and KLH were purchased from Sigma-Aldrich, and centrifugal desalting columns and 2-iminothiolane were purchased from Thermo fisher scientific.

[0033] In the embodiment of the present invention, the nucleotide sequence (siRNA) used in the oligonucleotide drug is as follows:

[0034] Sense oligonucleotide: 5'-AGAAGAUGCUUCAGACAGAUT-3';

[0035] Antisense oligonucleotide: 5'-UCUGUCUGAAGCAUCUUCUUT-3';

[0036] In the embodiment of the present invention, the structure of the oligonucleotide drug after maleimide activation is shown in Formula V:

[0037]

[0038] Example 1: Conjugation of oligonucleotide drugs with BSA

[0039] Step S1, maleimide activation of the oligonucleotide drug, the structure after activation is shown in Formula V;

[0040] Step S2, BSA thiol modification: BSA solid powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of BSA was 10 mg / mL; 2-iminothiolane powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of 2-iminothiolane was 10 mg / mL; 1.0 mL of BSA solution was mixed with 0.1 mL of 2-iminothiolane solution (the molar ratio of BSA to 2-iminothiolane was 1:50), and the mixture was shaken at 25° C. for 1 hour; the mixture was then treated with a desalting column, and the flow-through solution was collected;

[0041] Step S3, coupling the maleimide-activated oligonucleotide drug with thiol-modified BSA: dissolve the maleimide-activated oligonucleotide drug obtained in step S1 in PBS-EDTA coupling buffer with a pH value of 7.4 at a concentration of 10 mg / mL, take 1.0 mL, and mix with 1.0 mL of the flow-through solution (thiol-modified BSA solution) collected in step S2 (the molar ratio of thiol-modified BSA to maleimide-activated oligonucleotide drug is 1:10), incubate at room temperature for 4 hours, then add 50 mM cysteine ​​solution to terminate the reaction, and use a 50 kDa ultrafiltration tube to separate and purify the conjugate to obtain an oligonucleotide drug and BSA coupling complex, as shown in Formula VI, and its electrophoresis diagram is shown in Figure 1 As shown in lane 6.

[0042]

[0043] Example 2: Conjugation of oligonucleotide drugs with OVA

[0044] Step S1, maleimide activation of the oligonucleotide drug, the structure after activation is shown in Formula V;

[0045] Step S2, OVA thiolation modification: OVA solid powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of OVA was 10 mg / mL; 2-iminothiolane powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of 2-iminothiolane was 10 mg / mL; 1.0 mL of OVA solution was mixed with 0.15 mL of 2-iminothiolane solution (the molar ratio of OVA to 2-iminothiolane was 1:50), and the mixture was shaken at room temperature for 1 hour; then the mixture was treated with a desalting column, and the flow-through solution was collected;

[0046] Step S3, coupling the maleimide-activated oligonucleotide drug with the thiol-modified OVA: dissolve the maleimide-activated oligonucleotide drug obtained in step S1 in a PBS-EDTA coupling buffer with a pH value of 7.4 at a concentration of 10 mg / mL, take 0.85 mL, and mix with 1.0 mL of the flow-through solution (thiol-modified OVA solution) collected in step S2 (the molar ratio of thiol-modified OVA to maleimide-activated oligonucleotide drug is 1:10), incubate at room temperature for 4 hours under shaking conditions, then add 50 mM cysteine ​​solution to terminate the reaction, and use a 50 kDa ultrafiltration tube to separate and purify the conjugate to obtain an oligonucleotide drug and OVA coupling complex, as shown in Formula VII, and its electrophoresis diagram is shown in Figure 2 As shown in lane 2.

[0047]

[0048] Example 3: Conjugation of oligonucleotide drugs with KLH

[0049] Step S1, maleimide activation of the oligonucleotide drug, the structure after activation is shown in Formula V;

[0050] Step S2, KLH thiol modification: KLH solid powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of KLH was 10 mg / mL; 2-iminothiolane powder was dissolved in PBS-EDTA coupling buffer with a pH value of 7.4, wherein the concentration of 2-iminothiolane was 10 mg / mL; 1 mL of KLH solution was mixed with 0.035 mL of 2-iminothiolane solution (the molar ratio of KLH to 2-iminothiolane was 1:500), and the mixture was shaken at 25° C. for 1 hour; then the mixture was treated with a desalting column, and the flow-through solution was collected;

[0051] Step S3, coupling the maleimide-activated oligonucleotide drug with thiol-modified KLH: the maleimide-activated oligonucleotide drug obtained in step S1 was dissolved in a PBS-EDTA coupling buffer with a pH value of 7.4 at a concentration of 10 mg / mL, 0.25 mL was taken, and mixed with 1 mL of the flow-through solution (thiol-modified KLH solution) collected in step S2 (the molar ratio of thiol-modified KLH to maleimide-activated oligonucleotide drug was 1:200), incubated at room temperature for 4 hours, and then 50 mM cysteine ​​solution was added to terminate the reaction, and the conjugate was separated and purified using a 50 kDa ultrafiltration tube to obtain an oligonucleotide drug and KLH coupling complex, as shown in Formula VIII.

[0052]

[0053] Example 4 Test Example

[0054] Sample control group settings: conventional glutaraldehyde method to prepare BSA-oligonucleotide conjugates, conventional EDC method to index BSA-oligonucleotide conjugates, conventional glutaraldehyde method to prepare OVA-oligonucleotide conjugates, conventional EDC method to index OVA-oligonucleotide conjugates.

[0055] Protein control groups were set up: BSA protein control group and OVA protein control group.

[0056] 1. Coupling efficiency test

[0057] Test principle: Utilize non-reducing SDS-PAGE electrophoresis to detect, according to the protein molecular weight marker, determine the molecular weight of BSA-oligonucleotide conjugate, calculate its molecular weight difference with BSA, and then according to the molecular weight of oligonucleotide, the coupling ratio of the two can be obtained.

[0058] Test method: The coupling complexes obtained in Example 1 and Example 2, as well as the sample control group and the blank control group were tested by SDS-PAGE electrophoresis; Figure 1 The electrophoresis results of the coupled complex of Example 1, as well as the protein standard, BSA protein control group, BSA-oligonucleotide conjugate control group prepared by conventional glutaraldehyde method, and conventional EDC method index BSA-oligonucleotide conjugate control group are shown, wherein lane M is the BSA protein standard, lanes 1, 3, and 5 are the BSA protein control group, lane 2 is the BSA-oligonucleotide conjugate control group prepared by conventional glutaraldehyde method, lane 4 is the conventional EDC method index BSA-oligonucleotide conjugate control group, and lane 6 is the coupled complex of Example 1; Figure 2The electrophoresis results of the coupled complex of Example 2, as well as protein standards, an OVA protein control group, an OVA-oligonucleotide conjugate control group prepared by a conventional glutaraldehyde method, and an OVA-oligonucleotide conjugate control group indicated by a conventional EDC method are shown, wherein lane M is a BSA protein standard, lanes 1, 3, and 5 are an OVA protein control group, lane 2 is the coupled complex of Example 2, lane 4 is an OVA-oligonucleotide conjugate control group indicated by a conventional EDC method, and lane 6 is an OVA-oligonucleotide conjugate control group prepared by a conventional glutaraldehyde method.

[0059] according to Figure 1 The calculated molecular weight of the coupled complex in Example 1 is 120-180 kDa. The molecular weight of the oligonucleotide in Example 1 is 7.7 kDa, and the molecular weight of BSA is 66 kDa. It is calculated that 7-13 moles of oligonucleotide molecules are successfully coupled per mole of BSA molecule. Similarly, in the BSA-oligonucleotide conjugate prepared by the conventional glutaraldehyde method and the BSA-oligonucleotide conjugate prepared by the conventional EDC method, 1-2 moles of oligonucleotide molecules are successfully coupled per mole of BSA molecule. It can be seen that the coupling efficiency of the coupled complex of the oligonucleotide drug and the BSA carrier protein provided by the present invention is much higher than that of the coupled complex in the prior art.

[0060] according to Figure 2 The calculated molecular weight of the coupled complex in Example 2 is 150 kDa. The molecular weight of the oligonucleotide in Example 2 is 7.7 kDa, and the molecular weight of OVA is 45 kDa. It is calculated that at least 13 moles of oligonucleotide molecules are successfully coupled to each mole of OVA molecules. Similarly, in the conventional glutaraldehyde method for preparing OVA-oligonucleotide conjugates and the conventional EDC method index OVA-oligonucleotide conjugates, 1-2-XXX moles of oligonucleotide molecules are coupled to each mole of OVA molecules. It can be seen that the coupling efficiency of the coupled complex of the oligonucleotide drug and the OVA carrier protein provided by the present invention is much higher than that of the coupled complex in the prior art.

[0061] 2. Antibody titer determination

[0062] Experimental group: oligonucleotide drug-KLH coupling complex prepared in Example 3;

[0063] Control group 1: oligonucleotide drug-KLH coupling complex prepared by conventional glutaraldehyde method;

[0064] Control group 2: oligonucleotide drug-KLH coupling complex prepared by conventional EDC method;

[0065] Test method: New Zealand rabbits were immunized with oligonucleotide drug-KLH conjugate complexes, and rabbit polyclonal antibodies were prepared. ELISA was used to determine the antibody titer. During the ELISA test, the conjugate complex was coated at a concentration of 1.25 μg / mL, 100 μL per well; the antibody to be tested was diluted from 1 μg / mL to 0.001 μg / mL, 100 μL per well; the secondary antibody was HRP-goat anti-rabbit IgG, 1:30000 dilution, 100 uL per well, and the color development time was 5 minutes. The test results are as follows: Figure 3 shown. Figure 3 It can be seen that the titer of the rabbit polyclonal antibody prepared by immunization with the oligonucleotide drug-KLH conjugate complex prepared in Example 3 of the present invention can reach 1:1000000 (the antibody is at a concentration of 1 mg / mL, diluted 1000000 times, that is, at 0.001 ug / mL, its OD450nm reading is still greater than 2 times the OD450nm reading of the blank sample), while the titer of the rabbit polyclonal antibody prepared by immunization with the conjugates prepared by the other two conventional conjugation methods can only reach 1:100000 (the antibody is at a concentration of 1 mg / mL, diluted 100000 times, that is, at 0.01 ug / mL, its OD450nm reading is greater than 2 times the OD450nm reading of the blank sample). It can be seen that the antibody titer of the conjugate prepared in Example 3 of the present invention is more than 10 times the antibody titer of the conjugate prepared by the prior art.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A conjugated complex of an oligonucleotide drug and a carrier protein, characterized in that: The coupling complex is shown in Formula I: In the formula, the 5 ， The terminal phosphate group is connected to R2 through carbon-oxygen bonding, and the lysine side chain of the carrier protein is connected to R1 through sulfur-oxygen bonding; In the formula, R1 and R2 are independently selected from a C1-C10 straight carbon chain and / or a PEG chain with a degree of polymerization of 1-10.

2. The coupled complex according to claim 1, characterized in that R1 is selected from a C1-C4 straight carbon chain and / or a PEG chain with a degree of polymerization of 1-4; R2 is selected from a C3-C6 straight carbon chain and / or a PEG chain with a degree of polymerization of 4-8.

3. A method for preparing a coupled complex as claimed in claim 1 or 2, characterized in that: The steps include: Step S1, maleimide activation of the oligonucleotide drug; Step S2, modifying the carrier protein with thiol groups; Step S3, coupling the oligonucleotide drug activated by maleimide in step S1 with the carrier protein modified by thiol in step S2.

4. The method for coupling an oligonucleotide drug with a carrier protein according to claim 3, characterized in that: In step S1, at 5 ， The maleimide-containing group is introduced into the terminal phosphate group to achieve maleimide activation of the oligonucleotide drug.

5. The method for coupling an oligonucleotide drug with a carrier protein according to claim 4, characterized in that: The maleimide-containing group includes an amide bond.

6. The method for coupling an oligonucleotide drug with a carrier protein according to claim 3, characterized in that: In step S2, the thiolation of the carrier protein includes: incubating the carrier protein with a compound capable of generating thiol groups in a first coupling buffer to obtain the thiol-modified carrier protein.

7. The method for coupling an oligonucleotide drug with a carrier protein according to claim 6, characterized in that: The compound capable of generating a thiol group is selected from 2-iminothiolane, N-succinic acid S-acetylmercaptoethylene glycol ester or 3-[2-pyridyldithio]succinimidyl propionate.

8. The method for coupling an oligonucleotide drug with a carrier protein according to claim 6, characterized in that: The site of carrier protein thiolation is located at the amino group on the lysine side chain.

9. The method for coupling an oligonucleotide drug with a carrier protein according to claim 6, characterized in that: The molar ratio of the carrier protein to the compound capable of generating thiol groups is 1:40 to 80; The first coupling buffer is a PBS-EDTA solution with a pH value of 7.0-9.

0.

10. The method for coupling an oligonucleotide drug with a carrier protein according to claim 3, characterized in that: In step S3, the carrier protein modified by thiol group and the oligonucleotide drug activated by maleimide are coupled in a second coupling buffer; The molar ratio of the thiol-modified carrier protein to the maleimide-activated oligonucleotide drug is 1:5-20; The second coupling buffer is a PBS-EDTA solution with a pH of 6.5-7.5.

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