Cyclopeptide toxin Amanitin ADCs as well as preparation method and application thereof

Abstract

The invention relates to the technical field of organic synthesis, in particular to cyclopeptide toxin Amanitin ADCs as well as a preparation method and application thereof. The Amanitin ADCs have very promising cell activity, the anti-tumor activity of the Amanitin ADCs is in HeLa and SKOV3 cell lines, the two antibody-drug conjugates both show a selective cell killing effect, the efficacy is within a two-digit nanomole range, and the activity in HeLa cells is higher than that in SKOV3 cells.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a cyclic peptide toxin Amanitin-type ADCs, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are one of the fastest-growing technologies in cancer treatment, combining the precision of monoclonal antibodies (mAbs) with the potency of cytotoxic drugs. Traditional therapies suffer from limited efficacy due to a lack of tumor specificity, significant systemic toxicity, and a high risk of disease relapse after initial treatment, highlighting the need for more targeted treatments. The initial series of ADCs failed to achieve a balance between efficacy and off-target toxicity. Amanitin toxins possess a unique mechanism of action. While most common ADCs primarily target microtubules or DNA, α-Amanitin can efficiently bind to eukaryotic RNA polymerase II at nanomolar concentrations, thereby reducing transcription and protein synthesis efficiency by thousands of times. This allows amanitin-based ADCs to kill not only dividing tumor cells but also dormant cells, including cancer stem cells. These ADCs also exhibit low resistance because RNA polymerase II is a key pathway in cellular metabolism, making it very difficult for tumor cells to develop resistance by bypassing this pathway. Simultaneously, the high hydrophilicity of amanitin molecules effectively reduces ADC drug aggregation and minimizes off-target toxicity. According to ample literature, this toxin is highly stable in plasma and can be rapidly cleared by the kidneys. Existing ADCs prepared using amanitin suffer damage during the conjugation process, which affects the pharmacodynamic activity of the resulting ADCs. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a cyclic peptide toxin Amanitin-based ADC, its preparation method, and its application.

[0004] The first aspect of this invention provides a cyclic peptide toxin amanitin-type ADC, specifically having the structure of any one of the following compounds: ; In the formula, Me represents methyl and antibody trastuzuma represents antibody.

[0005] This invention bypasses modification of the critical carboxyl domain, essential for toxin activity, by linking the toxin to a specific site at the Asn residue of asparagine. This ensures that the toxin retains its intact native conformation and full bioactivity after release into target cells, solving the core problem of impaired efficacy during conjugation. Finally, in terms of construction and targeting strategy, the clinically validated HER2 antibody trastuzumab is used, combined with cysteine-maleimide site-directed conjugation technology, achieving efficient, controllable assembly and precise targeted delivery of the ADC. The inherent high hydrophilicity of the amanitin molecule further optimizes the physicochemical properties of the ADC, reducing aggregation tendency and the risk of nonspecific toxicity.

[0006] The second aspect of this invention provides a method for preparing the aforementioned amanitin-type cyclic peptide toxin ADCs, wherein the specific preparation method of compound 1 is as follows: The antibody is reduced. Drug-linker-1 was coupled with cysteine ​​to obtain compound 1; The structure of drug-linker-1 is shown below: .

[0007] In another preferred embodiment, the specific process of cysteine ​​coupling of drug-linker-1 to obtain compound 1 is as follows: The drug-linker-1 stock solution was mixed with the reduced antibody solution, wherein the molar ratio of drug-linker-1 to antibody was 6-8:1; the coupling reaction was carried out at room temperature in the dark for 1-2 hours, and the mixture was purified to obtain compound 1.

[0008] In another preferred embodiment, the specific preparation method of compound 2 is as follows: The antibody is reduced. The drug-linker-2 was coupled with cysteine ​​to obtain compound 2; The structure of drug-linker-2 is shown below: .

[0009] In another preferred embodiment, the specific process of compound 2 is as follows: The drug-linker-2 stock solution was mixed with the reduced antibody solution, wherein the molar ratio of drug-linker-2 to antibody was 6-8:1; the coupling reaction was carried out at room temperature in the dark for 1-2 hours, and the mixture was purified to obtain compound 1.

[0010] In another preferred embodiment, the specific process of the antibody reduction reaction is as follows: Trastuzumab and reaction buffer were mixed at a mass-volume ratio of 2 mg to 5 mg to 1 L to obtain a mixture. Tris(2-carboxyethyl)phosphonic acid hydrochloride was added to the mixture at 8 times the molar amount of trastuzumab, and the mixture was incubated at room temperature of 20°C to 23°C for 2 to 4 hours.

[0011] In another preferred embodiment, the reaction buffer contains the following components: 25 mM L-histidine, 1×PBS, 0.15 M sodium chloride and 2.5 mM ethylenediaminetetraacetic acid.

[0012] The third aspect of this invention provides the use of the aforementioned cyclic peptide toxin Amanitin antibody-drug conjugate in the preparation of an antitumor drug, wherein the antitumor drug is an anti-cervical cancer drug or an anti-ovarian cancer drug.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention bypasses modification of the critical carboxyl domain, essential for toxin activity, by linking to the asparagine Asn residue at a specific site. This ensures the toxin retains its intact native conformation and full bioactivity after release into target cells, solving the core problem of impaired efficacy during conjugation. The amanitin-based antibody-drug conjugates of this invention exhibit good cellular activity. Experiments on the antitumor activity of compounds 1 and 2, evaluated in HeLa and SKOV3 cell lines, revealed that both compounds 1 and 2 exhibited selective cell-killing activity in the double-digit nanomolar range, with higher activity in HeLa cells than in SKOV3 cells. In both HeLa and SKOV3 cell lines, the IC50 of compound 1... 50 The values ​​are 8.5 nm and 20 nm, respectively, and the IC50 of compound 2 is... 50 The values ​​are 7.5 nm and 27 nm, respectively. Compounds 1 and 2 in this invention have maleimide chemical bonds for binding to antibody thiol groups, and also have the characteristic bicyclic octapeptide structure of Amanitin and use asparagine residues as linker attachment sites, thereby giving the obtained cyclic peptide toxin Amanitin-like antibody-drug conjugates good drug activity and expanding the types of ADCs drugs. Attached Figure Description

[0014] Figure 1 The diagram shows the cell activity results of compounds 1 and 2 in this invention; where A is the result diagram of the HeLa cell line, B is the result diagram of the SKOV3 cell line, 1 represents compound 1, and 2 represents compound 2. Detailed Implementation

[0015] The technical solutions of this invention will be clearly and completely described in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0016] Unless otherwise specified, the methods described in the various embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0017] Based on the structure and function of ADCs, this invention designed and synthesized amanitin-based ADC compounds that can be used as cytotoxic loads for ADCs. CTG cell viability assays were performed on these compounds, revealing high activity. Through cysteine ​​conjugation, these compounds were linked to HER2 antibodies, playing a crucial role in the design and synthesis of similar compounds in the future.

[0018] This invention designed and synthesized two drug-linker conjugates with relatively good cellular activity. Next, antibody components were selected. Regarding antibody selection, the key issue lies in the internalization of the antibody-antigen complex, which mainly depends on the binding affinity between the antibody and tumor cell surface antigens. To date, 15 ADCs have been approved, and currently, more than 150 ADCs are undergoing clinical trials for cancer treatment. Among them, HER2 ADCs are receiving increasing attention. HER2 belongs to the epidermal growth factor receptor EGFR family, which includes HER1 (also known as EGFR), HER3, and HER4. Unlike other members, HER2 is frequently overexpressed in cancer, making it a valuable prognostic factor and an ideal target for cancer therapy. HER2-positive and negative tumor models, along with a relatively broad therapeutic index, indicate its potential safety and good tolerability. This invention used HER2 antibodies and then deduced an ADC conjugation method, specifically choosing chemical conjugation: cysteine ​​conjugation. In the field of ADCs, this is one of the most classic conjugation methods. The core principle involves the drug molecule loading reacting with the disulfide bonds of cysteine ​​residues on the antibody surface via a Michael addition reaction, using a linker with a maleimide reactive group. More importantly, this requires no antibody modification, is simple and feasible, and can be achieved through random or site-specific conjugation. Enzyme-mediated conjugation is far more complex than enzyme-mediated conjugation. Trastuzumab is a monoclonal antibody targeting HER2 and has been approved by the FDA for the treatment of HER2-positive cancers.

[0019] The following is a detailed description of an amanitin-type cyclic peptide toxin antibody-drug conjugate, its preparation method, and its application.

[0020] Example 1: Amanitin-type antibody-drug conjugate, the structural formula of which is shown below: .

[0021] The preparation method of compound 1 is as follows: Step 1: The drug-linker has a molecular weight of 1422 Daltons. It was dissolved in anhydrous dimethyl sulfoxide to prepare a 20 mM stock solution. The stock solution was aliquoted and stored at -20°C protected from light until use. The structure of the drug-linker load is shown below: ; Monoclonal antibody mAbs were buffer-exchanged to conjugation buffer using a PD-10 desalting column. Trastuzumab was prepared to a final concentration of 2 mg / mL in reaction buffer containing 25 mM L-histidine, 1×PBS, 0.15 M sodium chloride, and 2.5 mM EDTA. Subsequently, tris(2-carboxyethyl)phosphonic acid hydrochloride was added at 8 times the molar volume of the antibody, and the mixture was incubated at 20°C for 2 hours.

[0022] Step 2: The stock solution was added dropwise to the reduced trastuzumab solution, wherein the molar ratio of drug-linker to trastuzumab was 6:1. The coupling reaction was carried out at 23°C. During the coupling reaction, the mixture was protected from light and gently stirred for 2 hours. Then, the mixture was purified using a desalting column to remove unbound drug-linker molecules, yielding compound 1, denoted as O-3-ADC.

[0023] Example 2: Amanitin-type antibody-drug conjugate with cyclic peptide toxins, the structural formula of which is shown below: .

[0024] The preparation method of compound 2 is as follows: Step 1: The drug-linker has a molecular weight of 1422 Daltons. It is dissolved in anhydrous dimethyl sulfoxide to prepare a 20 mM stock solution. The stock solution is aliquoted and stored at -20°C protected from light until use. The monoclonal antibody is desalted using a PD-10 column and buffer exchanged to the conjugation buffer according to the manufacturer's instructions. Typically, trastuzumab is prepared to a final concentration of 2 mg / mL in reaction buffer containing 25 mM L-histidine, 1×PBS, 0.15 M sodium chloride, and 2.5 mM EDTA. Subsequently, tris(2-carboxyethyl)phosphonic acid hydrochloride is added at 8 times the molar volume of the antibody, and the mixture is incubated at 20°C for 2 hours. The structure of the drug is shown below: .

[0025] Step 2: The stock solution was added dropwise to the reduced trastuzumab solution, wherein the molar ratio of drug-linker to trastuzumab was 6:1. The coupling reaction was carried out at 23°C. During the coupling reaction, the mixture was protected from light and gently stirred for 1 hour. The mixture was then purified using a desalting column to remove unbound drug-linker molecules, yielding compound 2, denoted as P-3-ADC.

[0026] The cell activity experiments of compounds 1 and 2 prepared above are as follows: Human cancer cell lines Hela and SKOV3 were purchased from ProCell, Wuhan, China, and maintained under standard culture conditions at 37°C, 5% CO2 (by volume), and 95% humidity.

[0027] HeLa cells were cultured in Dulbecco modified Eagle medium containing L-alanylglutamine, supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate.

[0028] SKOV3 cells were cultured in DMEM containing L-alanylglutamine and supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate.

[0029] During passage, adherent cells were detached by adding 1×TrypLE Express, then diluted with PBS and transferred to new culture flasks.

[0030] To evaluate Amanitin-based O-3-ADCs and P-3-ADCs, 2 μL of live cell suspension was seeded into 96-well plates, with 5000 live HeLa cells per well and 5000 live SKOV3 cells per well, and then cultured overnight in a humidified incubator at 37°C.

[0031] ADC-1 and ADC-2 were prepared at a starting concentration of 10x and then serially diluted as follows: Figure 1 The x-axis is shown in the figure. Test compounds were prepared at a 10-fold starting concentration, serially diluted at a 1:2 volume ratio, and 12 μL was added to the cells. The experiment was performed using CellTiter-Glo reagent according to the manufacturer's instructions. Cell viability was determined after 72 hours.

[0032] The contents were mixed on an orbital oscillator for 2 minutes to induce cell lysis, followed by incubation at room temperature for 10 minutes to stabilize the luminescence signal. Luminescence values ​​were measured using a TECAN Spark® multimode microplate reader and normalized to the luminescence values ​​of untreated control cells. Dose-response curves were fitted to a variable-slope four-parameter function of the logarithm (inhibitor) of the GraphPad Prism. Data are expressed as relative cell viability percentages against compound molar concentrations. IC50 values ​​were calculated from multiple experiments. 50 Geometric mean. The luminescence value of the control group was set as 100% survival rate. The relative cell viability (%) of the treatment group was calculated using the following formula: (luminescence value of treatment group / luminescence value of control group) × 100%.

[0033] Cell viability results as follows Figure 1 As shown. From Figure 1 The results showed that the antitumor activities of O-3-ADC and P-3-ADC were evaluated in HeLa and SKOV3 cell lines. Both ADCs exhibited selective cell-killing activity in the double-digit nanomolar range, and their activity was higher in HeLa cells than in SKOV3 cells. In both HeLa and SKOV3 cell lines, the IC50 of O-3-ADC was significantly higher than that in SKOV3 cells. 50 The values ​​are highest at concentrations of 8.5 nm and 20 nm, respectively, while the IC50 of the P-3-ADC is... 50 The values ​​were highest at concentrations of 7.5 nm and 27 nm, respectively. The ADC compounds that performed well in both cell lines had the following structural features: (i) maleimide chemistry for binding to antibody thiol groups, (ii) a bicyclic octapeptide structure characteristic of Amanitin, and (iii) the use of asparagine residues as linker attachment sites.

[0034] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cyclic peptide toxin, Amanitin-type ADC, characterized in that, The specific structure is any one of the following compounds: ； In the formula, Me represents methyl and antibody trastuzuma represents antibody.

2. A method for preparing the amanitin-type cyclic peptide toxin ADCs according to claim 1, characterized in that, The specific preparation method of compound 1 is as follows: The antibody is reduced. Drug-linker-1 was coupled with cysteine ​​to obtain compound 1; The structure of drug-linker-1 is shown below: 。 3. The method for preparing amanitin-type cyclic peptide toxin ADCs according to claim 2, characterized in that, The specific process by which drug-linker-1 undergoes cysteine ​​coupling to obtain compound 1 is as follows: The drug-linker-1 stock solution was mixed with the reduced antibody solution, wherein the molar ratio of drug-linker-1 to antibody was 6-8:1; the coupling reaction was carried out at room temperature in the dark for 1-2 hours, and the mixture was purified to obtain compound 1.

4. The method for preparing amanitin-type cyclic peptide toxin ADCs according to claim 2, characterized in that, The specific preparation method of compound 2 is as follows: The antibody is reduced. Drug-linker-2 was coupled with cysteine ​​to obtain compound 2; The structure of drug-linker-2 is shown below: 。 5. The method for preparing amanitin-type cyclic peptide toxin ADCs according to claim 4, characterized in that, The specific process for compound 2 is as follows: The drug-linker-2 stock solution was mixed with the reduced antibody solution, wherein the molar ratio of drug-linker-2 to antibody was 6-8:1; the coupling reaction was carried out at room temperature in the dark for 1-2 hours, and the mixture was purified to obtain compound 2.

6. The method for preparing amanitin-type cyclic peptide toxin ADCs according to claim 4, characterized in that, The specific process of the antibody reduction reaction is as follows: Trastuzumab and reaction buffer were mixed at a mass-volume ratio of 2 mg to 5 mg to 1 L to obtain a mixture. Tris(2-carboxyethyl)phosphonic acid hydrochloride was added to the mixture at a mass-volume ratio of 8 times the molar amount of trastuzumab, and the mixture was incubated for 2 to 4 hours.

7. The method for preparing amanitin-type cyclic peptide toxin ADCs according to claim 6, characterized in that, The reaction buffer contains the following components at final concentrations: 25 mM L-histidine, 1×PBS, 0.15 M sodium chloride and 2.5 mM ethylenediaminetetraacetic acid.

8. The use of the amanitin-type cyclic peptide toxin ADCs according to claim 1 in the preparation of antitumor drugs, characterized in that, The anti-tumor drug is an anti-cervical cancer drug or an anti-ovarian cancer drug.

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