CLDN18.2-targeted antibody coupling drug as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a CLDN18.2-targeted antibody coupling medicine as well as a preparation method and application of the CLDN18.2-targeted antibody coupling medicine. According to the present invention, the prepared new antibody coupling drug intermediate LND1001 (K-lock-VC-DUO-5) is utilized, and the DUO-5 is subjected to site-specific coupling on the antibody through the K-lock connexon, such that the homogeneity of the ADC drug can be improved, the stability of the antibody can be improved, the enzyme specific cleavage of the Linker can be improved, and the toxic-side effect of the ADC can be reduced. After verification, the antibody-coupled drug intermediate LND1001 is found to have good tumor targeting ability and good antitumor activity after being coupled with the antibody h1D6.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an antibody-drug conjugate targeting CLDN18.2, and a preparation method and application thereof. Background Art

[0002] Antibody drug conjugate (ADC) is a new type of anti-tumor drug. Its principle is to connect cytotoxins to antibodies. Through the recognition of specific antigens on the surface of cancer cells by antibodies, ADCs enter cancer cells through endocytosis, thereby transporting cytotoxins to the target site, achieving the purpose of targeted treatment of malignant tumors. Compared with traditional small molecule anti-tumor drugs, ADCs are more specific and effective because they can use the targeted recognition of antibodies and the high activity of toxins.

[0003] ADC consists of three different components, namely antibodies, linkers and cytotoxins. Antibodies achieve targeting, linkers ensure the stability of ADC during blood transport, and after reaching the target site, the toxins play a killing effect on cancer cells. According to different mechanisms of action, toxins suitable for ADC are divided into microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors, etc. At present, the toxins used in ADCs sold on the market and in clinical trials are mainly microtubule inhibitors, mainly including compounds designed based on Dolastatin, such as MMAE, MMAE and MMAD, and compounds designed based on Maytansine, such as DM1 and DM4. In terms of linkers, the main ones used are cleavable types, such as Valine-Citriline and cyclohexylcarboxylic acid (MCC). After lysosomal hydrolysis, the drug is still active and is combined with an amino acid residue through the linker region.

[0004] Traditional ADCs use 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 80-90 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, traditional ADCs are highly heterogeneous mixtures with poor uniformity (i.e., the drug-to-antibody ratio (DAR) is 1-8), low stability, and poor efficacy.

[0005] Based on the above, there is an urgent need to obtain an antibody-drug conjugate that can improve the uniformity of ADC drugs, improve the stability of antibodies, improve the enzyme-specific cleavage of linkers, and reduce the toxic side effects of ADCs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an antibody-drug conjugate targeting CLDN18.2 and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention site-specifically couples DUO-5 to the antibody through a K-lock linker, which can improve the uniformity of the ADC drug, improve the stability of the antibody, improve the enzyme-specific cleavage of the Linker, and reduce the toxic and side effects of the ADC.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an antibody-drug conjugate h1D6-LDN1001, wherein the antibody-drug conjugate h1D6-LDN1001 is obtained by conjugating an antibody-drug conjugate intermediate LDN1001 with an antibody h1D6.

[0009] Furthermore, the chemical structural formula of the antibody-drug conjugate intermediate LDN1001 is:

[0010]

[0011] The present invention also provides a method for preparing the antibody-drug conjugate h1D6-LDN1001, comprising the following steps:

[0012] (1) The antibody h1D6 was replaced with the buffer required for the experiment to obtain an antibody solution, and LND1001 was added to the antibody solution and mixed well, and then stirred for reaction;

[0013] (2) Then, His buffer was added to the system, stirring was continued, and then the mixture was washed and filtered twice with His buffer containing 20% ​​PG, and finally washed and filtered with His buffer to remove excess small molecules and organic solvents, thereby obtaining the antibody-drug conjugate h1D6-LDN1001.

[0014] Furthermore, in step (1), the buffer required for the experiment is a buffer containing 5 mM PB, 5 mM EDTA, pH 7.2.

[0015] Furthermore, in step (1), the parameters of the antibody solution are 7.56 mg / mL, 3 mL.

[0016] Furthermore, in step (1), the parameters of LND1001 are 5 mM, DMA, 6 eq.

[0017] Furthermore, in step (2), the parameters of the His buffer are 20 mM, pH 6.0.

[0018] The present invention also provides an application of the antibody-coupled drug h1D6-LDN1001 in the preparation of drugs for targeted tumor treatment.

[0019] The present invention discloses the following technical effects:

[0020] The present invention utilizes the prepared new antibody-drug conjugate intermediate LND1001 (K-lock-VC-DUO-5) to site-specifically couple DUO-5 to an antibody through a K-lock linker, thereby improving the uniformity of ADC drugs, improving the stability of antibodies, improving the enzyme-specific cleavage of Linker, and reducing the toxic side effects of ADC. After verification, it was found that the antibody-drug conjugate intermediate LND1001 coupled with the antibody h1D6 has good tumor targeting ability and good anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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.

[0022] Figure 1 The synthetic route of the antibody-drug conjugate intermediate K-lock-VC-DUO-5 in Example 1 of the present invention;

[0023] Figure 2 This is the HNMR graph of the antibody-drug conjugate intermediate K-lock-VC-DUO-5 in Example 1 of the present invention;

[0024] Figure 3 The mass spectrum of the antibody-drug conjugate intermediate K-lock-VC-DUO-5 in Example 1 of the present invention;

[0025] Figure 4 This is a graph showing the HIC-HPLC results of the antibody-drug conjugate h1D6-LND1001 in Example 2 of the present invention;

[0026] Figure 5 This is a graph showing the SEC-HPLC results of the antibody-drug conjugate h1D6-LND1001 in Example 2 of the present invention;

[0027] Figure 6 This is a graph showing the activity of the antibody-drug conjugate h1D6-LND1001 on cells with different CLDN18.2 expression levels in Example 3 of the present invention;

[0028] Figure 7 This is a diagram of the tumor targeting ability of the antibody-drug conjugate h1D6-LND1001 in Example 4 of the present invention in BGC823-CLDN18.2 tumor-bearing mice, wherein from left to right and from top to bottom are 0h, 0.5h, 6h, 12h, 24h, and 48h;

[0029] Figure 8 This is a diagram showing the tumor targeting ability of the antibody-drug conjugate h1D6-LND1001 in Example 4 of the present invention in BGC823-CLDN18.2 tumor-bearing mice, wherein the left figure is the control group, the right figure is the treatment group, and the organs in each figure are the heart, liver, spleen, lung, kidney and tumor, respectively;

[0030] Fig. 9 This is a graph showing the average body weight of BGC823-CLDN18.2 tumor-bearing mice during administration of the antibody-drug conjugate h1D6-LND1001 in Example 4 of the present invention;

[0031] Fig.10 This is a graph showing the average tumor volume of BGC823-CLDN18.2 tumor-bearing mice during administration of the antibody-drug conjugate h1D6-LND1001 in Example 4 of the present invention;

[0032] Fig.11 This is a picture of the tumor re-appearance of BGC823-CLDN18.2 tumor-bearing mice after administration of the antibody-drug conjugate h1D6-LND1001 in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field;

[0039] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0040] The structure of the antibody-drug conjugate intermediate (K-lock-VC-DUO-5) prepared by the inventors of the present invention is shown below:

[0041]

[0042] in, It is DUO-5, which is a cytotoxin designed based on Aplysia toxin and has the function of killing tumor cells.

[0043] K-Lock-VC-DUO-5 is a new type of K-lock linker used by the inventors, which can site-specifically couple cytotoxins to antibodies. At the same time, the linker has good plasma stability, which can avoid the cleavage of ADC drugs in the blood circulation, reduce the toxic side effects of ADC, and improve safety.

[0044] The K-lock structure can be coupled to a specific lysine site on the antibody light chain through a stable covalent irreversible reaction with high specificity.

[0045] h1D6 is an antibody designed by the inventors, which is recorded in the related patent with patent number CN117567625A. K-Lock-VC-DUO-5 is coupled with CLDN18.2 target antibody (h1D6) to obtain ADC sample h1D6-K-Lock-VC-DUO-5, which shows good tumor cell killing ability. For BGC-823 / CLDN18.2 (human gastric cancer cells with high expression of CLDN18.2), h1D6-K-Lock-VC-DUO-5 shows good killing ability, indicating that the antibody has obvious anti-tumor effect after coupling with K-Lock-VC-DUO-5.

[0046] The present invention will be further described below by way of specific embodiments.

[0047] Example 1 Synthesis of K-Lock-VC-DUO-5 (LDN1001)

[0048] The synthetic route of K-Lock-VC-DUO-5 (named as LDN1001) in this embodiment is as follows Figure 1 As shown, its specific synthesis method comprises the following steps:

[0049] Step 1, add 40.10g LND1001-1 (Nanjing Lianning Biopharmaceutical Co., Ltd.) to a 1000mL reaction bottle; add 200mL of methanol, control the temperature at 10-20°C, and stir thoroughly until clear; add 400mL of hydrochloric acid / dioxane solution, continue stirring at 25-30°C for not less than 1 hour, and take samples for testing. After the test is qualified (i.e. the remaining raw material is less than 1%), the reaction solution is concentrated, the solvent is removed, 400ml of dichloromethane is added to the residue and ultrasonically dissolved, and then concentrated to remove the solvent, the water bath temperature is less than 30°C, and the distillation is rotary evaporated until no fraction is distilled out, and 37.0-48.1g of off-white solid is obtained, and the yield is calculated (100-130%). The solid LND1001-2 obtained above.

[0050] Step 2, add 47.07g LND1001-2 crude product to a 2000mL reaction bottle, add 240mL methanol and 160.4mL water, control the temperature at 10-20°C, and stir until clear. Add N, N-diisopropylethylamine (18mL), adjust the pH value of the solution to 7-8, weigh 5.96g sodium cyanate and add it to the reaction solution, stir continuously at 25-30°C, react for 12 hours and take a sample for HPLC detection. After the test is qualified, the reaction solution is not further processed to obtain a reaction solution containing LND1001-3, which is directly used for the next step reaction.

[0051] Step 3, weigh 6.12g sodium hydroxide and dissolve it in 80mL water, add the above sodium hydroxide solution to the reaction solution of LND1001-3, control the temperature at 25-30℃, stir for at least 2h, take samples for HPLC detection, until the reaction end point. After the test is qualified, add acetic acid (10mL) dropwise to the reaction solution, adjust the pH value of the solution to 6-7, transfer the reaction solution to a 2L single-mouth round-bottom flask, the temperature is less than 30℃, and rotate until no fraction is evaporated, and rotate until oil or solid precipitation occurs. Weigh the crude product, dissolve it in acetonitrile water (each 1g crude product is dissolved in acetonitrile 1-2mL, water 1-2mL), filter, and purify the filtrate with a column machine for medium pressure reverse phase. Collect the product components and freeze-dry to obtain LND1001-4 (36.7g).

[0052] Step 4, add 36.7g LND1001-4 crude product to a 2L reaction bottle, add 1100mL dichloromethane and 7.66g DIPEA, stir well, control the temperature at 20-30°C, and protect with nitrogen. Add 16.36g pentafluorophenol (PFP) and 28.37g 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCl) respectively, stir for not less than 1 hour, and take samples for LND1001-IPC-4 detection. After the test is qualified, the organic phase is added to the separatory funnel, washed once with 734mL water, washed once with 734mL saturated NaCl aqueous solution, and the organic phase is dried with 112.32g anhydrous sodium sulfate; after drying for 30 minutes, filter, the water bath temperature of the vacuum rotary evaporator does not exceed 30°C, concentrate to obtain an off-white solid, and weigh. Dissolve 110 mL of dichloromethane until the solution is clear, then add dropwise to 1470 mL of methyl tert-butyl ether and 735 mL of n-heptane, stir for at least 20 minutes, filter, rinse the filter cake with methyl tert-butyl ether, and dry to obtain an off-white powder. The obtained product is weighed and named LND1001-C, and a sample is taken for release inspection of LND1001-C. LND1001-C is dissolved in acetonitrile and water, then filtered, and the filtrate is purified by medium-pressure reverse phase purification using a column machine. The product components are collected and lyophilized to obtain LND1001 (23.4 g). The HNMR spectrum of the obtained LND1001 is as follows: Figure 2 As shown, the mass spectrum Figure 3 shown.

[0053] Example 2 Preparation of antibody conjugate h1D6-LND1001

[0054] The antibody h1D6 was replaced with the buffer required for the experiment (5mM PB, 5mM EDTA, pH7.2), 22.7 mg of the antibody (7.56 mg / mL, 3 mL) was taken, 186 μL of LND1001 (5mM, DMA, 6eq) was added, mixed, and stirred at room temperature for 3 h, 1.5 mL of His buffer (20mM, pH6.0) was added to the system, and stirring was continued for 2 h, and then washed and filtered twice with His buffer (20mM, pH6.0) containing 20% ​​PG (propylene glycol), and finally washed and filtered with His buffer (20mM, pH6.0) to remove excess small molecules and organic solvents to obtain the antibody conjugate h1D6-LND1001 (18.4 mg). The HIC-HPLC results of the antibody conjugate h1D6-LND1001 obtained are as follows: Figure 4 As shown, the SEC-HPLC results are as follows Figure 5 shown.

[0055] Example 3 CLDN18.2 target cell activity test

[0056] BGC823-CLDN18.2 was added to each well at 100 μL, 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-LND1001 was added to the cells at concentrations of 0.1, 0.1, and 1 μM, with three replicate wells for each concentration. After 72 hours of drug uptake, 10 μL of 5 mg / mL MTT (Biyuntian Biotechnology, Shanghai) 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 of 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:

[0057]

[0058] Panc1-CLDN18.2, NUGC4, and BxPc-3 were operated in the same way to detect the activity of h1D6-LND1001 on cells with different CLDN18.2 expression levels. The specific test results are as follows Figure 6 As shown. Figure 6 It can be seen that the expression levels of CLDN18.2 are BGC823-CLDN18.2, Panc1-CLDN18.2, and Panc1-CLDN18.2, respectively. et al. Characterization of zolbetuximab in pancreatic cancer models. Oncoimmunology. 2019, 8(1), e1523096. https: / / doi.org / 10.1080 / 2162402X.2018.1523096) prepared by gene expression, NUGC4 (Kebai Bio, Nanjing), BxPc-3 (Cell Center, Chinese Academy of Medical Sciences).

[0059] Example 4 Tumor targeting ability of antibody conjugate h1D6-LDN1001 in CLDN18.2 positive tumor-bearing mice

[0060] h1D6-LND1001 was incubated with DyLight680 NHS ester (Thermo Fisher Scientific, USA) at a molar ratio of 1:10 for 1 h at room temperature for staining, and then free dye was removed using a Merck microfiltration centrifuge tube to obtain fluorescently labeled h1D6-LND1001. 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 0h, 0.5h, 6h, 12h, 24h, and 48h after injection, the targeting ability of h1D6-LDN1001 was observed using an in vivo imaging device. The results are shown in the figure. Figure 7 The mice were killed at 48 hours, and the tumors and major organs were removed and observed and recorded in the same way. The results are shown in Figure 8 As shown. Figure 7-8 As shown, after administration, the drug was concentrated in the tumor site, demonstrating the good tumor targeting ability of the antibody conjugate h1D6-LDN1001.

[0061] Example 5 Anti-tumor activity of antibody conjugate h1D6-LDN1001 in CLDN18.2 positive tumor-bearing mice

[0062] BGC823-CLDN18.2 was inoculated into the right armpit of nude mice in the form of tumor masses. 3 The 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 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 tumors were weighed and photographed. The average weight of the mice during the h1D6-LDN1001 administration was obtained as follows: Fig. 9 The average tumor volume of mice was as shown in Fig.10 As shown, after the administration of h1D6-LDN1001, the tumor weight of mice was Fig.11 As shown. Figure 9-11 As shown, the antibody conjugate h1D6-LDN1001 prepared by the present invention can effectively delay the growth of tumor volume in mice, reduce tumor weight, and show significant anti-tumor activity.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An antibody-drug conjugate h1D6-LDN1001, characterized in that: The antibody-drug conjugate h1D6-LDN1001 is obtained by conjugating the antibody-drug conjugate intermediate LDN1001 with the antibody h1D6.

2. The antibody-drug conjugate h1D6-LDN1001 according to claim 1, characterized in that: The chemical structural formula of the antibody-drug conjugate intermediate LDN1001 is:

3. The method for preparing the antibody-drug conjugate h1D6-LDN1001 according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) The antibody h1D6 was replaced with the buffer required for the experiment to obtain an antibody solution, and LND1001 was added to the antibody solution and mixed well, and then stirred for reaction; (2) Then, His buffer was added to the system, stirring was continued, and then the mixture was washed and filtered twice with His buffer containing 20% ​​PG, and finally washed and filtered with His buffer to remove excess small molecules and organic solvents, thereby obtaining the antibody-drug conjugate h1D6-LDN1001.

4. The preparation method according to claim 3, characterized in that: In step (1), the buffer required for the experiment is a buffer containing 5 mM PB, 5 mM EDTA, pH 7.

2.

5. The preparation method according to claim 3, characterized in that: In step (1), the parameters of the antibody solution are 7.56 mg / mL, 3 mL.

6. The preparation method according to claim 3, characterized in that: In step (1), the parameters of LND1001 are 5 mM, DMA, 6 eq.

7. The preparation method according to claim 3, characterized in that: In step (2), the parameters of the His buffer are 20 mM, pH 6.

0.

8. Use of the antibody-drug conjugate h1D6-LDN1001 as claimed in claim 1 in the preparation of drugs for targeted tumor treatment.

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