Targeting CLDN18.2 antibody coupling medicine, preparation method and application

Abstract

The invention discloses an antibody coupled drug targeting CLDN18.2. The antibody coupled drug is obtained by coupling an antibody coupled drug intermediate LND1025 and an antibody h1D6, the chemical structural formula of the antibody coupled drug intermediate LND1025 is # imgabs0, and the antibody coupled drug intermediate LND1025 and the antibody h1D6 are coupled, so that the antibody coupled drug shows very good tumor cell killing ability, and the antibody h1D6 can be used for treating tumor cells. For example, for BGC1823 / CLDN18.2 (high expression CLDN18.2 human gastric cancer cells), the antibody coupling drug shows good killing ability, which shows that after the antibody h1D6 is coupled with the LND1025, the anti-tumor effect is significantly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemical synthesis and relates to an antibody-drug conjugate, and in particular to an antibody-drug conjugate targeting CLDN18.2, a preparation method and an application thereof. Background Art

[0002] Antibody drug conjugate (ADC) includes three different components, namely antibody, linker and cytotoxin. Traditional ADC uses the amino group of antibody lysine or the thiol group of cysteine ​​obtained by opening the interchain disulfide bond for coupling. The amino group of lysine is connected to the activated carboxylate linker through an amide bond, and the thiol group of cysteine ​​reacts with the maleimide group. An antibody molecule contains 80190 lysines, and coupling may occur on nearly 40 different lysine residues. Opening the interchain disulfide bond will obtain multiple cysteine ​​residues and destroy the integrity of the antibody molecule. Therefore, the traditional ADC is a highly heterogeneous mixture with poor uniformity (i.e., the drug-to-antibody ratio (DAR) is 1-8), low stability, and poor efficacy.

[0003] The Chinese invention patent with publication number CN117567625A discloses an anti-Claudin18.2 monoclonal antibody and its application. The heavy chain variable region of the monoclonal antibody includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.4 to SEQ ID NO.6. The anti-Claudin18.2 monoclonal antibody has a higher affinity than the existing commercial IMB362 antibody, and the anti-Claudin18.2 monoclonal antibody can specifically bind to tumor cells and can target and bind to tumors with high expression of Claudin18.2 in mice. Therefore, it is necessary to design a suitable linker to combine the anti-Claudin18.2 monoclonal antibody with the toxin.

[0004] The Chinese invention patent with application number 202311605048.0 discloses an antibody-drug conjugate intermediate SET0570 for treating tumors and its application in the production of biopharmaceuticals. The intermediate is characterized by using dolastatin-5 (DUO-5) as a cytotoxin. Through the innovatively designed C-lock-CC linker, DUO-5 is site-specifically coupled to the antibody, which can improve the uniformity of the ADC drug and significantly improve the killing effect of the antibody on tumor cells. However, the binding effect of the C-lock-CC linker with such a structure and the anti-Claudin18.2 monoclonal antibody is not ideal. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an antibody-drug conjugate targeting CLDN18.2 in order to overcome the deficiencies of the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is: an antibody-drug conjugate targeting CLDN18.2, which is obtained by conjugating an antibody-drug conjugate intermediate LND1025 with an antibody h1D6, and the chemical structure of the antibody-drug conjugate intermediate LND1025 is:

[0007]

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned antibody-drug conjugate targeting CLDN18.2, comprising the following steps:

[0009] replacing the antibody h1D6 into a buffer to obtain an antibody solution;

[0010] Take the antibody solution, add a thiol reducing agent and stir, then add the antibody-drug conjugate intermediate LND1025 and stir to react to obtain a reaction solution;

[0011] The reaction solution can be washed and filtered with His buffer.

[0012] Optimally, the buffer solution is a buffer solution containing 5 mM PB, 5 mM EDTA, pH 7.2, the parameters of the antibody solution are 7.56 mg / mL, 3 mL, and the parameters of the LND1025 are 5 mM, 60% ACN / H2O, 6 eq.

[0013] Furthermore, the ratio of the antibody solution to the LND1025 is 2-5 ml: 180-200 μL.

[0014] Optimally, the antibody-drug conjugate intermediate LND1025 is prepared by the following method:

[0015] Add LND1025-2 and DMF to a reactor, stir until clear, and then add DBBK to the reaction mixture; stir the obtained mixture for at least 15 minutes, and take a sample to test the completion of the reaction; add the reaction solution to methyl tert-butyl ether and stir; filter and recover the obtained precipitate; perform rotary evaporation on the obtained product to remove the solvent to obtain a crude product; dissolve the crude product in a solution composed of DMF, acetonitrile and water, purify the solution by medium-pressure reverse phase purification using a column machine, collect fractions and perform detection; combine qualified fractions and then freeze-dry them, and perform detection on the freeze-dried solid.

[0016] Furthermore, the LND1025-2 is prepared by the following method:

[0017] The LND1025-1 and DMF are added to a reactor and stirred until clear; piperidine is added to the reaction mixture and stirred for not less than 1 hour, and then the completion of the reaction is determined by HPLC; the reaction solution is added to methyl tert-butyl ether and stirred to form a suspension; the obtained precipitate is filtered and recovered; the obtained product is subjected to rotary evaporation to remove the solvent to obtain a crude product; the crude product is dissolved in a solution consisting of acetonitrile and water; the solution is purified by column reverse phase chromatography and the collected fractions are tested; qualified fractions are collected together and freeze-dried, and the freeze-dried product is subjected to release inspection to obtain the intermediate LND1025-2.

[0018] Furthermore, the LND1025-1 is prepared by the following method:

[0019] Add G5-FPDA, DUO-5 and DMF into the reactor and mix, stirring until clear;

[0020] Then, ethyl 2-oximecyanoacetate and DIC are added to the reaction mixture, and the reaction mixture is heated to 30-35° C. and stirred for not less than 3 hours, and the reaction process is monitored by HPLC to determine the completion of the reaction;

[0021] Methyl tert-butyl ether was added to the reaction mixture and stirred to form a suspension; the obtained precipitate was filtered and recovered, and the obtained product was subjected to rotary evaporation to remove the solvent. After the product passed the release test, the intermediate LND1025-1 was obtained.

[0022] Another object of the present invention is to provide a use of the above-mentioned antibody-drug conjugate targeting CLDN18.2 in the preparation of drugs for targeted tumor treatment.

[0023] Due to the application of the above technical scheme, the present invention has the following advantages compared with the prior art: the antibody-drug conjugate targeting CLDN18.2 of the present invention couples the antibody-drug conjugate intermediate LND1025 with the antibody h1D6, so that the antibody-drug conjugate exhibits good tumor cell killing ability. For example, for BGC-823 / CLDN18.2 (human gastric cancer cells that highly express CLDN18.2), the antibody-drug conjugate exhibits good killing ability, indicating that the anti-tumor effect is significantly improved after the antibody h1D6 is coupled to LND1025. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The H-NMR spectrum of the antibody-drug conjugate intermediate LND1025 of the present invention;

[0025] Figure 2 The mass spectrum of the antibody-drug conjugate intermediate LND1025 of the present invention;

[0026] Figure 3The synthetic route of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0027] Figure 4 This is a HIC-HPLC chart of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0028] Figure 5 This is the SEC-HPLC chart of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0029] Figure 6 The activity of the antibody-drug conjugate targeting CLDN18.2 of the present invention on cells with different CLDN18.2 expression levels;

[0030] Figure 7 The tumor targeting ability of the antibody-drug conjugate targeting CLDN18.2 of the present invention in BGC823-CLDN18.2 tumor-bearing mice (0h, 0.5h, 6h, 12h, 24h, 48h, respectively);

[0031] Figure 8 The tumor targeting ability of the antibody-drug conjugate targeting CLDN18.2 of the present invention in BGC823-CLDN18.2 tumor-bearing mice (left: control, right: treatment group; the organs in each figure are heart, liver, spleen, lung, kidney and tumor, respectively);

[0032] Fig. 9 The average body weight during administration of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0033] Fig.10 The average tumor volume during administration of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0034] Fig.11 The tumor weight after administration of the antibody-drug conjugate targeting CLDN18.2 of the present invention;

[0035] Fig.12 It is the HIC spectrum of the antibody conjugate LN474-64 in Comparative Example 1;

[0036] Fig.13 This is the SEC spectrum of the antibody conjugate LN474-64 in Comparative Example 1. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below.

[0038] Example 1

[0039] This example provides a method for preparing an antibody-drug conjugate intermediate LND1025, which is as follows:

[0040]

[0041] Step 1 Synthesis of LND1025-1

[0042] G5-FPDA (8.81 g, 1 eq), DUO-5 (7.72 g, 1 eq) and DMF (50 ml) were added to the reactor and mixed, and stirred until clear; ethyl 2-oxime cyanoacetate (1.42 g, 1 eq) and DIC (3.72 g, 2 eq) were added to the reaction mixture; the reaction mixture was heated to 30-35 ° C and stirred for not less than 3 hours, and the reaction process was monitored by HPLC to determine the completion of the reaction; methyl tert-butyl ether (100 mL) was added to the reaction mixture and stirred to form a suspension. The obtained precipitate was filtered and recovered. The obtained product was subjected to rotary evaporation to remove the solvent. The intermediate LND1025-1 (10.72 g) was obtained after the product was qualified by release inspection.

[0043] Step 2 Synthesis of LND1025-2

[0044] LND1025--1 (10.72 g, 1 eq) and DMF (50 mL) were added to the reactor and stirred until clear. Piperidine was added to the reaction mixture and stirred for not less than 1 hour, and then the completion of the reaction was determined by HPLC. The reaction solution was added to methyl tert-butyl ether (100 mL) and stirred to form a suspension. The obtained precipitate was filtered and recovered. The obtained product was subjected to rotary evaporation to remove the solvent to obtain a crude product. The crude product was dissolved in a solution consisting of acetonitrile (amount: crude mass × 3 ml / g) and water (5% TFA aqueous solution, amount: crude mass × 7 ml / g). The solution was purified by column reverse phase chromatography and the collected fractions were tested. The qualified fractions were collected together and freeze-dried. The freeze-dried product was released and tested to obtain the intermediate LND1025-2 (5.62 g).

[0045] Step 3 Synthesis of LND1025

[0046] LND1025-2 (5.62 g, 1 eq) and DMF (20 mL) were added to the reactor, stirred until clear, and DBBK (3.42 g, 2 eq) was added to the reaction mixture. The resulting mixture was stirred for at least 15 minutes, and a sample was taken to test the completion of the reaction. The reaction solution was added to methyl tert-butyl ether (100 mL) and stirred. The resulting precipitate was filtered and recovered. The obtained product was subjected to rotary evaporation to remove the solvent to obtain a crude product. The crude product was dissolved in a solution consisting of DMF (amount: crude mass × 3 ml / g), acetonitrile (amount: crude mass × 3 ml / g) and water (amount: crude mass × 6 ml / g). The solution was purified by medium-pressure reverse phase column machine. The fractions were collected and tested. The qualified fractions were combined and freeze-dried, and the freeze-dried solid was tested. The final solid was LND1025 (4.56 g), and the results are shown in Figure 1 and Figure 2 shown.

[0047] Example 2

[0048] This embodiment provides a method for preparing an antibody-drug conjugate targeting CLDN18.2 (abbreviated as h1D6-LND1025), such as Figure 3 As shown, the following steps are included:

[0049] Replace the antibody XM001AB03 (i.e., antibody h1D6) into the buffer required for the experiment (5 mM PB, 5 mM EDTA, pH 7.2);

[0050] Take 22.7 mg of antibody (7.56 mg / mL, 3 mL), add 154.8 μL of TCEP (10 mM, 10 eq), stir at room temperature for 2 h, add 186 μL of LND1025 (5 mM, 60% ACN / H2O, 6 eq), mix well, stir at room temperature for 30 min, detect by HIC-HPLC, complete coupling, and filter the reaction solution with 20 mM His buffer (pH 6.0) to remove excess small molecules and organic solvents to obtain the product (19.4 mg). The final product was tested at the same time, such as Figure 4 and Figure 5 shown.

[0051] Example 3

[0052] This embodiment provides an application of an antibody-drug conjugate targeting CLDN18.2, as follows:

[0053] BGC823-CLDN18.2 was added to each well at 100 μL and 3.0×10 3 The cells were evenly inoculated into a 96-well cell culture plate at a density of 10 cells / well and cultured overnight. The next day, the following concentration gradients were used: 0, 10 -7 , 10-6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 h1D6-LND1025 was added to the cells at concentrations of 0.1, 0.1, and 1 μM, with 3 replicates for each concentration. After 72 hours of drug uptake, 10 μL of 5 mg / mL MTT solution was added to each well in the dark, and the cells were cultured at 37°C for 4 hours. After the culture was completed, the drug-containing culture medium was discarded, 150 μL DMSO was added to each well, and the absorbance of each well at 570 nm was measured after shaking the plate for 5 minutes. The cell death rate was calculated according to the following formula:

[0054]

[0055] Panc1-CLDN18.2, NUGC4, and BxPc-3 were operated in the same way to detect the activity of h1D6-LND1025 on cells with different CLDN18.2 expression levels. The expression levels of CLDN18.2 were ranked from high to low as BGC823-CLDN18.2, Panc1-CLDN18.2, NUGC4, and BxPc-3. Figure 6 shown.

[0056] Example 4

[0057] This embodiment provides an application of an antibody-drug conjugate targeting CLDN18.2, as follows:

[0058] h1D6-LND1025 and DyLight680 were incubated at room temperature for 1 h at a molar ratio of 1:10, and then free dye was removed using a Merck microfiltration centrifuge tube to obtain fluorescently labeled h1D6-LND1025. BGC823-CLDN18.2 was inoculated into the right armpit of nude mice in the form of tumor masses. When the tumor volume reached 1000 mm 3 At 48 hours, the mice were sacrificed, and the tumors and major organs were removed and observed and recorded in the same way. Figure 7 and Figure 8 shown.

[0059] Example 5

[0060] This embodiment provides an application of an antibody-drug conjugate targeting CLDN18.2, as follows:

[0061] BGC823-CLDN18.2 was inoculated into the right armpit of nude mice in the form of tumor masses. 3The mice were divided into two groups, 5 in each group. The drug group was injected with 4 mg / kg body weight through the tail vein, and the control group was given an equal amount of saline in the same way. The drug was administered once a week for four weeks, during which the body weight and tumor volume of the nude mice were recorded every 3-4 days. After the fourth administration, the mice were observed for one week, and the mice were killed. The tumors were weighed and photographed. Figure 9-11 shown.

[0062] It can be seen that the ADC sample h1D6-LND1025 was obtained by coupling LND1025 with the CLDN18.2 target antibody (h1D6). The cell activity was compared with that of the simple antibody h1D6. It was found that the antibody h1D6 had basically no cell killing ability, while the ADC sample h1D6-LND1025 showed good tumor cell killing ability. For BGC-823 / CLDN18.2 (human gastric cancer cells with high expression of CLDN18.2), h1D6-LND1025 showed good killing ability, while h1D6 did not kill, indicating that the anti-tumor effect was significantly improved after the antibody was coupled with LND1025.

[0063] Comparative Example 1

[0064] This example provides a preparation of an antibody conjugate LN474-64, as follows:

[0065]

[0066] Buffer 1 configuration: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, commercially available 0.5 M EDTA pH 8.0 aqueous solution 10 mL, adjust pH to 7.2 with phosphoric acid or sodium hydroxide, and dilute to 1000.00 mL with purified water;

[0067] Replace the antibody BY016 into the buffer system Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2);

[0068] Take antibody BY016 (10 μmol), add 10.0 equivalents of TCEP (100 μmol), and stir at room temperature for 2 hours;

[0069] The reduced antibody was replaced with Buffer 1 (50 mM PB, 5 mM EDTA, pH 7.2) through an ultrafiltration tube 3-5 times (equivalent to a total dilution factor of about 1000 times) to remove excess reducing agent;

[0070] The reduced antibody (control concentration 10+5 mg / mL) was taken out, and then 6.0 equivalents of VcMMAE (60 μmol, solvent: acetonitrile / water=60 / 40) were added, stirred at room temperature for 30 minutes, and detected by HIC-HPLC.

[0071] Post-treatment: The coupled ADC sample was replaced with Buffer 1 3-5 times through an ultrafiltration tube (equivalent to a total dilution factor of about 1000 times) to remove excess small molecules and organic solvents. The target ADC product LN474-64 was obtained, and relevant tests were performed and stored at low temperature (such as Fig.12 and Fig.13 ).

[0072] The abbreviations of the above chemical substances are shown in Table 1.

[0073] Table 1 Abbreviations of chemical raw materials

[0074]

[0075] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody-drug conjugate targeting CLDN18.2, characterized in that: It is obtained by coupling the antibody-drug conjugate intermediate LND1025 with the antibody h1D6. The chemical structure of the antibody-drug conjugate intermediate LND1025 is:

2. The method for preparing the antibody-drug conjugate targeting CLDN18.2 according to claim 1, characterized in that: The following steps are involved: replacing the antibody h1D6 into a buffer to obtain an antibody solution; Take the antibody solution, add a thiol reducing agent and stir, then add the antibody-drug conjugate intermediate LND 1025 and stir to react to obtain a reaction solution; The reaction solution can be washed and filtered with His buffer.

3. The method for preparing an antibody-drug conjugate targeting CLDN18.2 according to claim 2, characterized in that: The buffer solution is a buffer solution containing 5 mM PB, 5 mM EDTA, and pH 7.

2. The parameters of the antibody solution are 7.56 mg / mL, 3 mL. The parameters of the LND1025 are 5 mM, 60% ACN / H2O, 6 eq.

4. The method for preparing an antibody-drug conjugate targeting CLDN18.2 according to claim 3, characterized in that: The ratio of the antibody solution to the LND1025 is 2-5 ml: 180-200 μL.

5. The method for preparing an antibody-drug conjugate targeting CLDN18.2 according to claim 2, characterized in that: The antibody-drug conjugate intermediate LND1025 was prepared by the following method: Add LND1025-2 and DMF to a reactor, stir until clear, and then add DBBK to the reaction mixture; stir the obtained mixture for at least 15 minutes, and take a sample to test the completion of the reaction; add the reaction solution to methyl tert-butyl ether and stir; filter and recover the obtained precipitate; perform rotary evaporation on the obtained product to remove the solvent to obtain a crude product; dissolve the crude product in a solution composed of DMF, acetonitrile and water, purify the solution by medium-pressure reverse phase purification using a column machine, collect fractions and perform detection; combine qualified fractions and then freeze-dry them, and perform detection on the freeze-dried solid.

6. The method for preparing an antibody-drug conjugate targeting CLDN18.2 according to claim 5, characterized in that: The LND1025-2 was prepared as follows: The LND1025-1 and DMF are added to a reactor and stirred until clear; piperidine is added to the reaction mixture and stirred for not less than 1 hour, and then the completion of the reaction is determined by HPLC; the reaction solution is added to methyl tert-butyl ether and stirred to form a suspension; the obtained precipitate is filtered and recovered; the obtained product is subjected to rotary evaporation to remove the solvent to obtain a crude product; the crude product is dissolved in a solution consisting of acetonitrile and water; the solution is purified by column reverse phase chromatography and the collected fractions are tested; qualified fractions are collected together and freeze-dried, and the freeze-dried product is subjected to release inspection to obtain the intermediate LND1025-2.

7. The method for preparing an antibody-drug conjugate targeting CLDN18.2 according to claim 6, characterized in that: The LND1025-1 was prepared as follows: Add G5-FPDA, DUO-5 and DMF into the reactor and mix, stirring until clear; Then, ethyl 2-oximecyanoacetate and DIC were added to the reaction mixture, and the reaction mixture was heated to 30-35° C. and stirred for not less than 3 hours, and the reaction process was monitored by HPLC to determine the completion of the reaction; Methyl tert-butyl ether was added to the reaction mixture and stirred to form a suspension; the obtained precipitate was filtered and recovered, and the obtained product was subjected to rotary evaporation to remove the solvent. After the product passed the release test, the intermediate LND1025-1 was obtained.

8. Use of the antibody-drug conjugate targeting CLDN18.2 according to claim 1 in the preparation of a drug for targeted treatment of tumors.

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