An antibody conjugate drug against esophageal cancer and a preparation method and application thereof

Abstract

This invention discloses an EGFR-targeting anti-esophageal cancer antibody-drug conjugate, its preparation, and its application. The drug comprises a fusion protein Fv-LDP-D3 and an isoematilin derivative conjugated site-specifically via a linker. This invention is the first to synthesize an isoematilin-linker-maleimide active intermediate, overcoming the technical barrier of direct conjugation of natural flavonoids, achieving highly efficient, site-specific conjugation (DAR 4.0, activity retention >90%). Equimolar comparison experiments confirmed that this conjugate drug exhibits significantly higher anti-esophageal cancer activity than free isoematilin and its additive form both in vitro and in vivo, demonstrating significant synergistic effects. This drug selectively inhibits EGFR-positive esophageal cancer cells, induces apoptosis, inhibits migration, and is safe for normal cells. This invention provides a novel strategy for the targeted delivery of isoematilin and the treatment of esophageal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and particularly relates to an antibody-drug conjugate for esophageal cancer, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of drugs that combine the specificity of an antibody with the cytotoxic effects of a small molecule drug. Structurally, an ADC consists of a targeting antibody moiety, a chemical linker moiety, and a small molecule drug moiety. This drug design strategy combines the targeting ability of an antibody with the cytotoxicity of a cytotoxic drug, aiming to improve the therapeutic index—that is, to increase efficacy while reducing side effects. In ADC design, in addition to the selection of the antibody and small molecule drug moieties, the design of the linker moiety connecting the antibody and the small molecule drug is also crucial.

[0003] Esophageal cancer is one of the most common malignant tumors worldwide, with esophageal squamous cell carcinoma (ESCC) having a relatively high incidence in China. Epidermal growth factor receptor (EGFR) is one of the ideal targets for cancer therapy. The EGFR receptor protein consists of 1186 amino acids, which can be divided into three regions: an extracellular ligand-binding region of amino acids 1-620, a transmembrane region of amino acids 621-643, and an intracellular region containing a tyrosine kinase region. Overexpression or mutation of the EGFR receptor leads to dysregulation of downstream signaling pathways. EGFR is overexpressed in most esophageal tumors, making the development of antibody-drug conjugates (ADCs) for esophageal cancer of great significance. Currently, several ADCs are used clinically.

[0004] Eupatilin (CAS: 22368-21-4, 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxybenzopyran-4-one) is a natural flavonoid isolated from plants in the Asteraceae family. It possesses effective anti-inflammatory activity, but its poor water solubility and lack of targeting limit its clinical application. Currently, there are no reports on the site-directed chemical conjugation of eupatilin with anti-EGFR antibodies to form a stable antibody-drug conjugate (ADC). The main technical obstacle lies in the fact that the eupatilin molecule itself lacks active groups that can react with the natural amino acids on the antibody, and its strong hydrophobicity easily leads to protein aggregation and inactivation during the conjugation process. Therefore, developing a method to achieve efficient, site-directed, and activity-preserving conjugation of eupatilin with anti-EGFR antibodies, with a synergistic effect against esophageal cancer, is a pressing technical challenge in this field. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an antibody-drug conjugate for esophageal cancer, its preparation method, and its application.

[0006] In a first aspect, the present invention provides an antibody-drug conjugate targeting EGFR, the drug molecule comprising: The fusion protein Fv-LDP-D3 is composed of an anti-EGFR single-chain antibody scFv, a lidamycin peptide chain olefinic peptide (LDP), and the third domain (D3) of human serum albumin linked sequentially via a peptide linker; the amino acid sequence of the fusion protein Fv-LDP-D3 is shown in SEQ ID No. 6; An iso-zellin active intermediate, wherein the iso-zellin active intermediate is covalently coupled to the interchain cysteine ​​residues of the fusion protein Fv-LDP-D3.

[0007] Preferably, the iso-zellin active intermediate is formed by an ester bond between the linker molecule and the 7-position hydroxyl group in the iso-zellin molecule.

[0008] Preferably, the linker molecule is a C5-C6 straight-chain alkyl linker, which may be selected from maleimide valerate (5-maleimide valerate, DMVA) or maleimide hexanoic acid (6-maleimide hexanoic acid, EMCA).

[0009] Preferably, the drug-antibody molar ratio (DAR) of the antibody-drug conjugate is 2 to 6:1, more preferably 4:1.

[0010] Secondly, the present invention provides a method for preparing an antibody-drug conjugate targeting EGFR, comprising the following steps: S1: Synthesis of the active intermediate of iso-zellin To address the issue of the inability to directly couple natural isoematilin, this invention first prepares an isoematilin-linker through chemical synthesis. Specifically, utilizing the nucleophilic activity of the C-7 phenolic hydroxyl group of isoematilin under alkaline conditions, it undergoes an esterification reaction with the carboxyl group of 5-maleiminovalerate (DMVA) or 6-maleiminohexanoate (EMCA) catalyzed by N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), forming an isoematilin-7-O-ester-linked Eupatilin-DMVA or Eupatilin-EMCA active intermediate. The maleimide group at the end of this intermediate can undergo a specific Michael addition reaction with the partially reduced and exposed free thiol groups on the fusion protein in subsequent coupling reactions, forming a stable thioether bond. S2: Preparation of fusion protein Fv-LDP-D3 The purified Fv-LDP-D3 protein was obtained through genetic engineering and HEK293E cell expression system, and the concentration was adjusted to 5-7 mg / mL. S3: Selective reduction of fusion proteins Fv-LDP-D3 solution (PBS, pH 7.4, containing 1 mM EDTA) was mixed with tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) to a final TCEP concentration of 0.8-1.2 mM (preferably 1.0 mM), and reacted at 16-30°C (preferably 25°C) for 1-3 hours (preferably 2 hours). Under these conditions, selective reduction of interchain disulfide bonds can be achieved, exposing a limited number of free thiol groups, while the disulfide bonds within scFv remain intact. S4: Coupling reaction The reduced protein solution obtained in S3 is mixed with DMSO and the iso-euphorbia flavonoid active intermediate solution obtained in S1, controlling the final volume ratio of DMSO to be 5%-15% (preferably 10%), and the molar ratio of iso-euphorbia flavonoid active intermediate to protein to be 4:1 to 8:1 (preferably 6:1). The mixture is reacted at 16-30°C (preferably 25°C) in the dark for 30-120 minutes (preferably 60 minutes). S5: Termination of Reaction and Purification Cysteine ​​was added to the reaction solution at a final concentration of 5-15 mM (preferably 10 mM) to quench unreacted maleimide groups. The molar ratio of cysteine ​​to the initial isozepine active intermediate was 1:3 to 1:5 (preferably 1:4). The reaction solution was washed five times with PBS buffer using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to remove small molecules and organic solvents, yielding the purified antibody-drug conjugate. S6: Product Characterization Concentration: BCA method; DAR value: Ultraviolet spectrophotometry (dual wavelengths of 280 nm and 360 nm); Coupling site confirmation: trypsin digestion-LC-MS / MS analysis; Activity retention: ELISA was used to determine the binding activity with EGFR before and after coupling; The order of steps S1 and S2 is not important.

[0011] Thirdly, the present invention also provides the use of the antibody-drug conjugate described in the first aspect in the preparation of an anti-esophageal cancer drug, characterized in that the in vitro concentration of the antibody-drug conjugate is 2.5 to 80 μg / mL, and the in vivo dose is 0.1 to 1.0 mg / kg (calculated as fusion protein).

[0012] Beneficial effects: Compared with the prior art, the present invention has the following advantages: Overcoming the technical obstacle of site-specific conjugation of flavonoid drugs, site-specific, efficient, and controllable conjugation of iso-eupatorin with antibodies was achieved for the first time; Rigorous equimolar comparison experiments have demonstrated that this conjugate drug exhibits anti-esophageal cancer activity far exceeding that of free isozygoxin and its additive form both in vitro and in vivo, showing significant synergistic effects. The drug has a clear mechanism of action, good safety profile, and a favorable therapeutic window. Attached Figure Description

[0013] Figure 1 Synthetic route and structural formula of Eupatilin-EMCA, an active intermediate of iso-eupatilin.

[0014] Figure 2 Quantitative analysis results of selective reduction conditions of fusion protein Fv-LDP-D3, including (A) content of free thiol groups in the protein after treatment with different TCEP concentrations (Ellman reagent method, n=3); (B) monomer retention rate by SEC-HPLC analysis after treatment with different TCEP concentrations; (C) detection rate of degradation fragments after treatment with different TCEP concentrations (n=3); (D) retention rate of antigen binding activity after treatment with different TCEP concentrations (ELISA method, n=3).

[0015] Figure 3 Comparison of in vitro cell activity at equimolar concentrations of conjugated and free drugs.

[0016] Figure 4 Comparison of in vivo antitumor activity (equivalent to isoesphine).

[0017] Figure 5 The binding activity of the fusion protein Fv-LDP-D3 to the EGFR antigen.

[0018] Figure 6 Effects of different concentrations of antibody-drug conjugates on the viability of KYSE450, KYSE30, TE1, and HEEC cells.

[0019] Figure 7 Effects of antibody-drug conjugates on KYSE30 cell migration.

[0020] Figure 8 The apoptotic effect of antibody-drug conjugates on KYSE30 cells.

[0021] Figure 9 : The expression of apoptosis-related proteins in KYSE30 cells by antibody-drug conjugates. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] Example 1: Construction and expression of fusion protein Fv-LDP-D3 (1) Construction and cloning of Fv-LDP-D3 gene Overlap extension PCR was used to assemble a complete Fv-LDP-D3 fusion gene by modifying and splicing the gene fragments encoding the anti-EGFR single-chain antibody scFv (containing the VH sequence shown in GenBank: AFF61391.1, with IgV-H mutated to aspartic acid (D) at position 52, tyrosine (Y) at position 99, and methionine (M) at position 104 in the framework region), the gene fragment encoding the ledamycin peptide chain endolyne peptide (LDP), and the gene fragment encoding the third domain of human serum albumin (D3, Accession: 1BJ5_A, amino acids 372-538). The above mutations increased the affinity of scFv for EGFR by 3-fold compared to the parental sequence (scFv derived from cetuximab), improved the monomer detection rate at 40°C from 70% to 90%, and increased the soluble expression yield by 50%.

[0024] The wild-type scFv-VH sequence (GenBank: AFF61391.1) is SEQ ID No. 1: QLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTRLKHQWGQG TLVTV.

[0025] After the mutation, CDR-H2 has a D at position 52, CDR-H3 has a Y at position 99, and the frame region has an M at position 104; the mutated scFv-VH sequence is SEQ ID No. 2. QLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPDFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTRYKHQWMQGTLVTV.

[0026] The scFv-VL sequence is SEQ ID No. 3: ELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIGKNNRPSGIPDRFSGSSSSGNTASLTITGAQAEDEADYYCNSRDSSGPVFGGGTKLTV.

[0027] The core sequence of LDP is SEQ ID No. 4: AQIEGDVVVKPAVVAANQGVTAVTQVLGAPK.

[0028] The HSA D3: 372-538 sequence is SEQ ID No. 5: KVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPK.

[0029] The overall sequence structure of the constructed fusion protein Fv-LDP-D3 is SEQ ID No. 6: (mutated scFv-VH)-(GGGGS)3-(scFv-VL)-(GGGGS)2-(LDP core sequence)-(GSTSGSGKPGSGEGSTKG)-(HSA D3:372-538).

[0030] Mutant primer design: Based on the wild-type scFv-VH gene sequence (GenBank: AFF61391.1), complementary forward and reverse primers were designed for the three mutation sites. The primers were 30-40 bp in length, with the mutation sites located in the middle region of the primers. The mutant codons were designed as aspartic acid (GAC), tyrosine (TAC), and methionine (ATG), respectively. The primers were synthesized by Qingke Biotechnology Co., Ltd. Primer information is shown in Table 1.

[0031]

[0032] PCR amplification of mutant plasmids: Using pUC57 plasmid containing the wild-type scFv gene sequence as a template (50 ng), add upstream and downstream mutant primers (125 ng each), dNTP mixture (10 mM, 1 μL each), PfuUltra high-fidelity DNA polymerase (2.5 U), and matching buffer, and bring the volume to 50 μL with ddH2O. The PCR amplification program is as follows: 95°C pre-denaturation for 30 seconds; 95°C denaturation for 10 seconds, 60°C annealing for 30 seconds, 68°C extension for 2 minutes, for a total of 30 cycles; final extension at 68°C for 10 minutes.

[0033] DpnI restriction enzyme digestion to remove wild-type template: After PCR amplification, add 1 μL of DpnI restriction endonuclease (20 U / μL) to the reaction system, mix gently, and incubate at 37°C for 1 hour. DpnI specifically cleaves methylated wild-type template DNA, while mutant plasmid DNA synthesized in vitro is not cleaved. After digestion, inactivate the enzyme by placing the reaction system in an 80°C water bath for 10 minutes.

[0034] Transformation and screening: 5 μL of the PCR product digested with DpnI was added to 100 μL of ice-cold *E. coli* DH5α competent cells and incubated on ice for 30 minutes. The cells were then heat-shocked in a 42°C water bath for 60 seconds, and immediately placed in an ice bath for 2 minutes to cool. 800 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 200 rpm for 1 hour. 200 μL of the bacterial culture was then evenly spread onto LB solid medium containing ampicillin (100 μg / mL) and incubated upside down at 37°C for 12–16 hours.

[0035] Sequencing validation: Five single colonies were picked from the plate and inoculated into 5 mL of LB broth (containing 100 μg / mL ampicillin), and cultured overnight at 37°C with shaking at 220 rpm. The plasmid was extracted and sent to a sequencing company for DNA sequencing. The sequencing primers were the universal primers M13F and M13R for the pUC57 vector. The sequencing results were compared with the designed sequence to confirm the correct mutation site and the absence of other non-specific mutations. The correctly sequenced mutant plasmid was named pUC57-scFv(VH-mut)-VL.

[0036] The introduction of the combined mutations was carried out in a stepwise manner. First, the mutation at position 52 (I52D) was introduced to screen for a single mutant plasmid that was correctly sequenced. Then, using this single mutant plasmid as a template, the mutation at position 99 (L99Y) was introduced to obtain a double mutant plasmid. Finally, using the double mutant plasmid as a template, the mutation at position 104 (G104M) in the framework region was introduced to obtain a triple mutant plasmid. Each mutated plasmid was confirmed by sequencing. The final scFv-VH sequence containing the three mutations was obtained, with the following amino acid sequence: QLVQSGAEVKKPG SSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPDFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTRYKHQWMQG TLVTV. Compared to the original sequence (GenBank: AFF61391.1), the 52nd position changed from I to D, the 99th position changed from L to Y, and the 104th position changed from G to M.

[0037] Evaluation of the technical effects of the above mutations: As determined by the SPR method (Table 2), the dissociation constant (KD) of the scFv and EGFR antigen after the three mutation combinations decreased by one-third compared to the original parent wild-type sequence (parental KD = 5.31 × 10⁻⁶). -8 M, mutant KD=1.7×10 -8 The affinity was increased by approximately 3 times. Accelerated stability testing at 40°C (4 weeks) showed that the monomer detection rate of the mutant scFv was 90%, compared to 70% for the parental sequence. E. coli expression system assays (Table 3) showed that the soluble expression yield of the mutant scFv was 45.1 mg / L, compared to 28.8 mg / L for the parental sequence, representing an increase of 56.6%.

[0038] ;

[0039]

[0040] Assembly of fusion genes Overlap extension PCR was used to modify and splice the above-mentioned mutant anti-EGFR single-chain antibody scFv encoding gene fragment, the lidamycin peptide chain endoyne peptide (LDP) encoding gene fragment, and the human serum albumin domain 3 (D3, Accession: 1BJ5_A, amino acids 372-538) encoding gene fragment to assemble a complete Fv-LDP-D3 fusion gene. The domains are linked by DNA sequences encoding flexible linker peptides. The overall sequence structure is (scFv-VH)-(GGGGS)3-(scFv-VL)-(GGGGS)2-(LDP core sequence)-(GSTSGSGKPGSGEGSTKG)-(HSA D3: 372-538).

[0041] Construction of recombinant expression plasmids The fusion gene fragment obtained by the above PCR amplification was ligated with the pTT5 mammalian cell expression vector that had been double-digested with restriction endonucleases Nde I and Xho I using the ClonExpress® II One Step cloning kit to construct the recombinant expression plasmid pTT5-Fv-LDP-D3.

[0042] (2) Expression of the fusion protein in HEK293E cells The correctly sequenced recombinant plasmid pTT5-Fv-LDP-D3 was subjected to high-purity plasmid DNA extraction (concentration ≥1 μg / μL, A) to obtain high-purity plasmid DNA. 260 / A 280=1.8-2.0). HEK293E cells were passaged in DMEM medium containing 10% fetal bovine serum after resuscitation. 24 hours before transfection, the cells were replaced with serum-free CDM4HEK293™ medium, and the cell density was adjusted to 2.0 × 10⁻⁶ cells / year. 6 Cells / mL. Polyethyleneimine (PEI) transfection was performed using a PEI ratio of 150 μg plasmid and 450 μg PEI (1:3 mass). After standing at room temperature for 15 minutes, the mixture was added to the cell suspension in a shake flask. High-efficiency feed was added on day 4 post-transfection, and cell culture was collected on day 7. The cells were centrifuged at 4000×g for 30 minutes at 4°C, and the supernatant was filtered through a 0.22 μm filter and stored at 4°C for later use.

[0043] (3) Purification of fusion protein Purification was performed using the AKTA Pure protein purification system and a HiTrap Protein A HP affinity column. The affinity column was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). The filtered cell culture supernatant was loaded at a flow rate of 0.5 mL / min, with a loading volume of approximately 120 mL. After loading, the sample was washed with 20 column volumes of binding buffer, followed by elution with 5 column volumes of elution buffer (0.1 M glycine-HCl, pH 3.0). The elution peaks were collected (1 mL per tube, pre-added with 100 μL of 1 M Tris-HCl neutralization buffer, pH 9.0). The elution peaks were combined and ultrafiltered using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa. The final buffer was replaced with PBS buffer (pH 7.4, containing 1 mM EDTA). The protein concentration was determined to be 6.2 mg / mL using the BCA method, with a total protein content of approximately 4.5 mg.

[0044] (4) Identification of purity and activity of fusion protein Purity was analyzed by SEC-HPLC under the following chromatographic conditions: TSKgel G3000SWXL column, mobile phase PBS (pH 7.4, containing 150 mM NaCl), flow rate 0.5 mL / min, and detection wavelength 280 nm. The results showed that the purity of the fusion protein monomer was 95.2%, and no degradation fragments were detected.

[0045] Antigen binding activity was detected using ELISA. Human recombinant EGFR protein was coated onto 96-well microplates, and serially diluted Fv-LDP-D3 fusion protein (0.1-10 μg / mL) was added. After incubation, anti-His-tag primary antibody and HRP-labeled secondary antibody were added. OD was measured after TMB color development. 450 The concentration-response curve was fitted using a four-parameter Logistic model, and EC was calculated.50 The value was 0.52 μg / mL (95% confidence interval 0.48–0.56 μg / mL), compared with the control antibody cetuximab (EC). 50 =0.48 μg / mL) is equivalent to, see Figure 5 Surface plasmon resonance (SPR) and BCA assays were used to detect the affinity and expression levels of the mutants, respectively. Individual mutations (D52, Y99, M104) only increased affinity by 1.2-1.8 times, but the combination of the three increased affinity by 3.0 times and expression levels by 50%. The purified Fv-LDP-D3 fusion protein was aliquoted and stored at -80°C for later use.

[0046] Example 2: Synthesis of Eupatilin-EMCA, an active intermediate of iso-eupatilin Under nitrogen protection, isoematilin (100 mg, 0.29 mmol), EMCA (80 mg, 0.38 mmol, 1.30 eq), DCC (90 mg, 0.44 mmol, 1.50 eq), DMAP (7 mg, 0.06 mmol, 0.20 eq), and anhydrous DMF (8 mL) were added sequentially to a 50 mL two-necked round-bottom flask. The reaction system was placed on a magnetic stirrer and stirred at room temperature (25 ± 2 °C) for 24 hours. During the reaction, DCC reacted with the carboxyl group of EMCA to form an active O-acylurea intermediate, which then underwent nucleophilic substitution with the C-7 phenolic hydroxyl group of isoematilin to form an ester bond, simultaneously generating dicyclohexylurea (DCU) as a byproduct. The Eupatilin-EMCA reaction route is as follows: Figure 1 As shown.

[0047] The reaction progress was monitored using thin-layer chromatography (TLC) with dichloromethane:methanol (10:1, v / v) as the developing solvent. Observation was performed under 254 nm UV light, and confirmation was achieved after vanillin-sulfuric acid color development. The Rf value of the isoematilin starting material was approximately 0.55, and the Rf value of the target product, Eupatilin-EMCA, was approximately 0.35. After 24 hours of reaction, TLC showed that the isoematilin starting material spot had essentially disappeared, indicating the reaction was stopped.

[0048] After the reaction was complete, the reaction solution was cooled to 4°C in an ice-water bath to allow the byproduct dicyclohexylurea (DCU) to fully precipitate. The reaction solution was filtered to remove the DCU precipitate, and the filter cake was washed with a small amount of cold dichloromethane (DCM) (3 mL × 2 times). The filtrate was poured into 50 mL of ice water, and the pH was adjusted to 5.0-6.0 with 1M HCl. The solution was extracted with ethyl acetate (25 mL × 3 times), and the organic phases were combined and washed successively with deionized water (25 mL × 2 times) and saturated sodium chloride solution (25 mL × 1 time). The organic phase was dried over anhydrous sodium sulfate for 1 hour, filtered, and concentrated under reduced pressure (40°C) to obtain a yellow crude product.

[0049] The crude product was purified by silica gel column chromatography. 15 g of silica gel (200-300 mesh) was used, and the column was packed wet-packed with dichloromethane as the initial solvent for equilibration. The crude product was dissolved in a small amount of dichloromethane:methanol (1:1) and then loaded onto the column using a wet-packing method. The elution gradient was as follows: dichloromethane:methanol (20:1, 100 mL) to wash nonpolar impurities, dichloromethane:methanol (15:1, 100 mL) as a transition, dichloromethane:methanol (10:1, 200 mL) to elute the target product, and finally dichloromethane:methanol (8:1, 100 mL) to elute the product tailings. Each 8 mL tube was collected, and the fractions containing the target product were combined by TLC. The combined solution was concentrated under reduced pressure at 40°C to obtain 85 mg of a pale yellow solid product, with a yield of 52%. Multiple independent experiments validated that the yield range of this method is 50%-65%.

[0050] Thin-layer chromatography analysis showed that the product had an Rf value of 0.33-0.36 in the developing solvent dichloromethane:methanol (10:1), and appeared as a single spot. The vanillin-sulfuric acid color development was yellow-brown.

[0051] High-performance liquid chromatography (HPLC) purity analysis was performed using a C18 reversed-phase column (4.6 × 250 mm, 5 μm). The mobile phase was acetonitrile:water (60:40, v / v, containing 0.1% trifluoroacetic acid), the flow rate was 1.0 mL / min, the detection wavelengths were 280 nm and 350 nm, and the column temperature was 30°C. The product retention time was 9.8 min, and the purity was determined to be 91.5% by area normalization.

[0052] High-resolution mass spectrometry analysis was performed using electrospray ionization (ESI) in positive ion mode. The product mass spectrum shows [M+H]. + The peak value is m / z 565.18, which is closer to the theoretical value of 565.1817 (C). 29 H 28 N2O 10 The relative error was 0.0005%, confirming the molecular weight of the target product.

[0053] The proton nuclear magnetic resonance (NMR) spectrometry analysis was performed using a 400 MHz NMR spectrometer with DMSO-d6 as the solvent. The chemical shifts (δ, ppm), splitting modes, coupling constants (J values), and relative integral values ​​of each proton are as follows: δ 12.93 (s, 1H, 5-OH), 7.55 (d, J = 2.0 Hz, 1H, H-2'), 7.48 (dd, J = 8.4, 2.0 Hz, 1H, H-6'), 7.02 (s, 2H, maleimide CH=CH), 6.99 (d, J = 8.4 Hz, 1H, H-5'), 6.85 (s, 1H, H-3), 6.80 (s, 1H, H-8), 6.73 (s, 1H, H-6), 3.99 (s, 3H, 6-OCH3), 3.88 (s, 3H, 3'-OCH3), 3.86 (s, 3H, 4'-OCH3), 2.62 (t, J = 7.4 ppm). The δ values ​​were 2.42 (t, J = 7.2 Hz, 2H, -CH2-CO-), 1.78-1.65 (m, 4H, -CH2-CH2-), and 1.55-1.48 (m, 2H, -CH2-). The signal of the C-7 hydroxyl group (δ approximately 10.8-11.0) of the starting material isoenstatin completely disappeared in the product spectrum, while the characteristic proton signal of the methylene group in the linker (δ 2.62, 2.42, 1.78-1.65, 1.55-1.48) appeared, proving that the C-7 hydroxyl group had been successfully esterified and the EMCA linker had been attached to the isoenstatin core. The Eupatilin-EMCA product was stored at -20°C in a light-protected, sealed container under nitrogen protection. Stability tests showed that after 6 months of storage under these conditions, the HPLC purity remained above 90%, indicating good product stability. When using, dissolve the solution in anhydrous DMSO to prepare a 10 mM stock solution and store at -20°C for later use.

[0054] Example 3: Optimization and Verification of Selective Reduction Conditions Five aliquots (1 mg / mL, PBS buffer, pH 7.4, containing 1 mM EDTA) of Fv-LDP-D3 protein solution were prepared, each 1 mL. Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was added to each aliquot to final concentrations of 0 (control), 0.5, 1.0, 2.0, and 5.0 mM, respectively. The aliquots were incubated at 25°C with shaking for 2 hours. After the reaction, the reducing agent was removed using a Zeba desalting column (pre-equilibrated with PBS), and the protein fractions were collected for subsequent analysis. Each experiment was performed in triplicate. The content of free thiol (-SH) groups in the protein under different TCEP treatment conditions was determined using the Ellman reagent (DTNB) method. The absorbance at 412 nm was measured using a microplate reader. The molar concentration of free thiol groups was calculated using a cysteine ​​standard curve (0-100 μM), and then divided by the molar concentration of the protein (calculated based on a molecular weight of 75 kDa) to obtain the number of free thiol groups per protein molecule. The results are shown in the figure. Figure 2 As shown in Figure A, the results of the free thiol group content determination are as follows: 0 mM TCEP: 0.8 ± 0.2 mol / mol 0.5 mM TCEP: 1.5 ± 0.3 mol / mol 1.0 mM TCEP: 3.8 ± 0.4 mol / mol 2.0 mM TCEP: 7.2 ± 0.6 mol / mol 5.0 mM TCEP: 12.5 ± 1.0 mol / mol; Among them, the control group had 0.8 free thiols, which is consistent with the expectation that only a very small number of free thiols are exposed in the native folded state of the fusion protein; the 1.0 mM TCEP group had 3.8 free thiols, which is highly consistent with the theoretical value of selective reduction of interchain disulfide bonds (expected to release 2-4 thiols), and significantly lower than the fully reduced state (14 thiols), indicating that the intrachain disulfide bonds were not destroyed; the ≥2.0 mM TCEP group had 7.2-12.5 free thiols, which exceeds the theoretical range of selective reduction, suggesting that the intrachain disulfide bonds inside the scFv region were non-specifically reduced.

[0055] SEC-HPLC was used to analyze the aggregation state and degradation of the protein before and after reduction to assess the integrity of the protein structure; the results are as follows: Figure 2 As shown in B and 2C, the SEC-HPLC monomer retention results are as follows: 0 mM TCEP: 98.2 ± 0.5% 0.5 mM TCEP: 97.5 ± 0.6% 1.0 mM TCEP: 96.8 ± 0.5% 2.0 mM TCEP: 85.3 ± 1.2% 5.0 mM TCEP: 62.1 ± 1.5%; The results showed that in the 1.0 mM TCEP group, the monomeric protein retention rate was >96%, and no degradation fragments (<20 kDa) were detected, indicating that the intrachain disulfide bonds within the scFv region remained intact and the protein did not degrade. In the ≥2.0 mM TCEP group, significant degradation fragments (4.5% - 22.6%) were observed, indicating that the intrachain disulfide bonds within the scFv region were reduced, leading to protein structural damage.

[0056] The binding activity of the fusion protein to EGFR antigen before and after reduction was determined by ELISA to assess the effect of reduction conditions on the antigen binding site. Results are as follows: Figure 2 As shown in Figure D, the results indicate that in the 1.0 mM TCEP group: EC 50 The value was 0.55 μg / mL, with 95% of the relative binding activity retained, showing no statistically significant difference compared to the control group (0.52 μg / mL) (p>0.05). This indicates that the reduction conditions did not cause significant damage to the antigen binding sites. ≥2.0 mM TCEP group: EC 50 The value increased significantly (p<0.01), and the binding activity decreased to below 67%, indicating that excessive reduction disrupted the conformation of the scFv region.

[0057] The combined results of the three quantitative assays indicate that, under the conditions of 1.0 mM TCEP concentration, 25°C reaction temperature, and 2 hours reaction time, optimized selective reduction of the interchain disulfide bonds in the fusion protein Fv-LDP-D3 was achieved. This provides 2-4 free thiol active sites for subsequent site-directed coupling with the isopropanol active intermediate, while maintaining the integrity of the protein structure and its antigen-binding activity. These results were validated in three independent experiments, demonstrating good reproducibility.

[0058] Example 4: Preparation and characterization of antibody-drug conjugate (Fv-LDP-D3-Eupatilin) 1.0 mL of Fv-LDP-D3 solution (6 mg / mL) was reduced under the optimized conditions determined in Example 3. A DMSO solution (10 mM) of Eupatilin-EMCA prepared in Example 2 was slowly added dropwise to the reduced protein to achieve a molar ratio of 6:1 (Eupatilin-EMCA:protein) and a final DMSO concentration of 10%. The mixture was stirred at 25°C in the dark for 1 hour. Cysteine ​​was added to a final concentration of 10 mM to terminate the reaction. The conjugated drug was obtained after ultrafiltration purification.

[0059] Characterization results: Protein concentration: 5.2 mg / mL, yield: 87%; DAR value: A 360 / A 280 The calculation result is 4.1; Coupling sites: LC-MS / MS confirmed that they are mainly coupled to the heavy chain Cys-XX; Activity retention: ELISA showed that the affinity for EGFR binding after conjugation was 94% of that before conjugation.

[0060] Example 5: Validation of synergistic effects in vitro (equimolar comparison) KYSE30 cells were collected and the following treatment groups were set up (48h, CCK-8 assay): Group 1: Free iso-euphorbia flavonoids (4 μM, equivalent to iso-euphorbia flavonoids in the conjugated drug) Group 2: Free fusion protein Fv-LDP-D3 (20 μg / mL) Group 3: Conjugated drug (20 μg / mL, iso-euphorbia 4 μM) Group 4: Group 1 + Group 2 (combined administration, concentration as above).

[0061] The results are as follows Figure 3 As shown: Group 1 Inhibition rate: 8%; Group 2 Inhibition rate: 35%; Group 4 (combined administration) inhibition rate: 43.5%; Group 3 (drug conjugate) inhibition rate: 85%.

[0062] It is worth noting that the combined administration of free *Eupatorium fortunei* flavonoids and free Fv-LDP-D3 (group 4) achieved an actual inhibition rate of 43.5%, slightly higher than the arithmetic sum of the inhibition rates of the two drugs acting alone (8% + 35% = 43%), indicating that there was no synergistic effect or the synergistic effect was extremely weak. In contrast, the conjugate drug of this invention (group 3) achieved an inhibition rate of 85% under the condition of an equivalent amount of *Eupatorium fortunei* flavonoids, significantly higher than the combined administration value (43.5%), demonstrating that integrating the two drugs into a single entity through chemical linkage is a necessary technical means to achieve synergistic effects.

[0063] Example 6: In vivo synergistic effect verification (equivalent to isoesphagein) Establishment of nude mouse KYSE150 xenograft model (n=6 / group): Control group: Injected with an equal volume of PBS via tail vein, and measured every three days; Free iso-euphorbia group: administered at the iso-euphorbia equivalent in the conjugate drug (0.02 mg / kg, i.p., measured every three days); Free Fv-LDP-D3 group: 0.2 mg / kg of free Fv-LDP-D3 was injected via tail vein, and the results were measured every three days; Combined administration group: Free isozygoflavin (0.02 mg / kg, intraperitoneal injection) and free Fv-LDP-D3 (0.2 mg / kg, tail vein injection) were administered simultaneously, and the results were measured every three days; Conjugate group: The conjugate drug of the present invention, Fv-LDP-D3-Eupatilin (0.2 mg / kg, of which the iso-eupatilin equivalent is 0.02 mg / kg), was injected via tail vein and tested every three days.

[0064] Results after 30 days of treatment Figure 4 As shown: Control group: tumor volume ~1500 mm³; Free iso-eupatorium flavonoids group: tumor inhibition rate 30.8%; Free Fv-LDP-D3 group: tumor inhibition rate was 52.2%; Combined drug administration group: tumor inhibition rate was 58.0%; The conjugate drug group showed a tumor inhibition rate of 86.9%.

[0065] Under the same absolute dosage of isopropionate (0.02 mg / kg / dose), the tumor inhibition efficiency of the conjugate drug was 2.82 times that of the free drug, proving that the ADC form greatly enhances the antitumor efficacy of isopropionate, and this enhancement exceeds the expectation of simple targeted delivery, reflecting the synergistic effect between antibody and drug.

[0066] Example 7: Effect of antibody-drug conjugates on the activity of esophageal cancer cells The CCK-8 method was used for detection. Figure 6 As shown, the results indicated that after 48 hours of treatment with the conjugated drug, TE-1 and KYSE30 cells underwent morphological changes, with cells shrinking and some cells losing their adherence and exhibiting nuclear fragmentation. HEEC esophageal cells showed no significant morphological changes. The IC50 values ​​of the conjugated drug after 72 hours of treatment with KYSE450, KYSE30, and TE-1 esophageal cancer cells were also discussed. 50 The concentrations were 30 μg / mL, 20 μg / mL, and 40 μg / mL, respectively. These concentrations had no significant effect on HEECs in normal esophageal cells.

[0067] Example 8: Effect of antibody-drug conjugates on the migration ability of esophageal cancer cells The cell scratch assay was used for detection. Figure 7 As shown, the results indicate that the migration rate of KYSE30 esophageal cancer cells treated with the conjugated drug (20 μg / mL) for 24 h and 48 h was 4% and 0%, respectively.

[0068] Example 9: Effect of antibody-drug conjugates on apoptosis of esophageal cancer cells AO-EB staining and Western blot were used for detection. Figure 8-9 As shown, the results indicate that the conjugated drug significantly promotes apoptosis in esophageal cancer cells, increases the expression of the pro-apoptotic protein Bax, inhibits the expression of the anti-apoptotic protein Bcl-2, and increases the Bax / Bcl-2 ratio by 5-fold.

[0069] Based on the above results, the inventiveness of the present invention can be manifested in the following three aspects: (1) It overcomes the technical bias that flavonoids cannot be site-directedly coupled. Isorhynchonine molecules themselves lack active groups that can react with antibodies, and their strong hydrophobicity easily leads to protein aggregation. Those skilled in the art generally believe that "natural flavonoids are difficult to use in ADC preparation." This invention, by designing and synthesizing an isorhynchonine-linker-maleimide active intermediate and optimizing reduction and coupling conditions, has for the first time achieved highly efficient site-directed coupling of isorhynchonine and antibodies (DAR 4.0, activity retention >90%), overcoming long-standing technical obstacles.

[0070] (2) Combinatorial mutations of the antibody sequence produced an unexpected dual synergistic effect. Individual mutations (D52, Y99, M104) can only increase affinity by 1.2-1.8 times, but the combination of the three mutations increases affinity by 3.0 times and expression level by 50%. This dual synergistic effect far exceeds the expectations of conventional rational design in this field.

[0071] (3) The necessity of coupling and the unpredictability of synergistic effects The combined administration of free isozygoflavin and the free fusion protein did not produce a synergistic effect (the actual inhibition rate was approximately equal to the sum of the expected value of 43.5%), while the conjugate drug of this invention achieved an inhibition rate of 85% (in vitro) and a tumor inhibition rate of 86.9% (in vivo) under the same dosage conditions, significantly better than the sum of the expected value. This result demonstrates that integrating the two into a single entity through chemical linkage is a necessary technical means to achieve synergistic effects, an effect that is not obvious to those skilled in the art.

Claims

1. An antibody-drug conjugate targeting EGFR, characterized in that, The drug molecule comprises: The fusion protein Fv-LDP-D3 has the amino acid sequence shown in SEQ ID No. 6; An iso-zellin active intermediate, wherein the iso-zellin active intermediate is covalently coupled to the interchain cysteine ​​residues of the fusion protein Fv-LDP-D3.

2. The antibody-drug conjugate targeting EGFR according to claim 1, characterized in that, The iso-zellone active intermediate is formed by an ester bond between the linker molecule and the 7-hydroxyl group in the iso-zellone molecule.

3. The antibody-drug conjugate targeting EGFR according to claim 2, characterized in that... The linker molecule is 6-maleimide hexanoic acid or 5-maleimide valerate.

4. The antibody-drug conjugate targeting EGFR according to claim 1, characterized in that, The drug-antibody molar ratio (DAR) of the antibody-drug conjugate is 2–6:

1.

5. The antibody-drug conjugate targeting EGFR according to claim 4, characterized in that, The DAR ratio is 4:

1.

6. A method for preparing an antibody-drug conjugate targeting EGFR, characterized in that, Includes the following steps: S1: Synthesis of the active intermediate of iso-zellin; S2: Preparation of the fusion protein Fv-LDP-D3: The purified Fv-LDP-D3 fusion protein was obtained through genetic engineering and HEK293E cell expression system, and the concentration was adjusted to 5-7 mg / mL. S3: Selective reduction of the fusion protein: Mix Fv-LDP-D3 solution with TCEP to achieve a final TCEP concentration of 0.8-1.2 mM, and react at 16-30°C for 1-3 hours. S4: Coupling reaction The reduced protein solution obtained from S3 was mixed with DMSO and the iso-euphorbia flavonoid active intermediate solution obtained from S1. The final volume ratio of DMSO was controlled at 5%-15%, the molar ratio of iso-euphorbia flavonoid active intermediate to protein was 4:1 to 8:1, the reaction temperature was 16-30°C, and the reaction time was 30-120 minutes in the dark. S5: Termination of Reaction and Purification Add cysteine ​​to the reaction solution at a final concentration of 5-15 mM to quench unreacted maleimide groups; the molar ratio of cysteine ​​to the initial isozygoflavin active intermediate is 1:3 to 1:5; wash the reaction solution 5 times with PBS buffer using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to remove small molecules and organic solvents, and obtain the purified antibody-drug conjugate. The order of steps S1 and S2 is not important.

7. The method for preparing an antibody-drug conjugate targeting EGFR according to claim 6, characterized in that, In step S3, the final concentration of TCEP is 1.0 mM, the reaction temperature is 25°C, and the reaction time is 2 hours.

8. The method for preparing an antibody-drug conjugate targeting EGFR according to claim 6, characterized in that, In step S4, the final volume ratio of DMSO is controlled at 10%, the molar ratio of isozygoflavin active intermediate to protein is 6:1, the reaction temperature is 25°C, and the reaction time is 60 minutes.

9. A method for preparing an antibody-drug conjugate targeting EGFR according to claim 6, characterized in that, In step S5, the final concentration of cysteine ​​added to the reaction solution is 10 mM, and the molar ratio of cysteine ​​to the initial isozelin active intermediate is 1:

4.

10. The use of the antibody-drug conjugate according to any one of claims 1-5 in the preparation of an anti-esophageal cancer drug, characterized in that, The in vitro concentration of the antibody-drug conjugate is 2.5–80 μg / mL, and the in vivo dose is 0.1–1.0 mg / kg, based on the fusion protein.

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