A method for preparing an antibody-drug conjugate

Abstract

This invention relates to a method for preparing antibody-drug conjugates, comprising the following steps: ultrafiltration and concentration of antibody-containing cell culture medium to the desired concentration or without further treatment to quantify the antibody concentration; addition of a drug linker, and adjustment of the pH to 7.4-8.5 with sodium hydroxide aqueous solution, reacting at room temperature or 37°C for 2-4 hours; and purification after the reaction to obtain the antibody-drug conjugate. This invention allows for the direct preparation of K248-targeted ADCs in the cell culture medium of an antibody expression system, avoiding the antibody pre-purification step in traditional ADC preparation and simplifying the traditional ADC preparation process. The resulting ADC has similar physicochemical properties to traditional K248-targeted ADCs; exhibits similar killing activity on tumor cells with different antigen expression levels; and this method is compatible with diverse substrate structures and antibody types.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for preparing antibody-drug conjugates (ADCs). Specifically, this invention relates to a method for directly preparing antibody-drug conjugates (ADCs) in a cell expression system and its applications. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of complexes formed by linking antibodies and potent cytotoxic payloads through linkers. These drugs combine strong killing effects with low toxicity and side effects, demonstrating great efficacy and potential in the field of cancer treatment, and have become a focus of cancer drug development in recent years.

[0003] However, due to the complex structure of ADCs (Advanced Dietitian Combinations) consisting of both macromolecules and small molecules, the production process is complex, limiting the rapid development of ADC drugs. Currently, mainstream ADC production technologies rely on multi-step separation and purification processes, including purification after antibody expression, synthesis of drug linkers, and purification of the final product after conjugation. This entire process is time-consuming and costly. Furthermore, host cell proteins, nucleic acid residues, and culture medium components in the cell culture medium can severely interfere with conjugation efficiency, necessitating time-consuming antibody pre-purification, further exacerbating the process complexity.

[0004] Because antibodies contain 80-90 lysine residues on their surface, with approximately half located in the solvent-accessible region, the high abundance of lysine leads to high heterogeneity in lysine-conjugated ADCs, meaning the number and location of drug conjugates on the antibody vary. This heterogeneity presents a series of potential problems for the resulting ADCs in terms of process stability, site reproducibility, efficacy, and safety. Although a number of methods have been developed in recent years, such as Thiomab technology, non-natural amino acid insertion technology, and enzyme-mediated site-specific conjugation technology, to address the issue of ADC uniformity, these technologies largely require genetic engineering of the antibody. This increases production costs, and the introduced new structures may also exhibit immunogenicity.

[0005] Fc-binding peptide (FcBP) is a cyclic peptide derived from Protein A that has a high affinity for the Fc domain of antibodies and has been developed for precise covalent modification of antibodies. Summary of the Invention

[0006] This invention, based on an Fc-binding peptide-guided site-specific modification strategy for antibody K248, develops a method for directly preparing ADCs in a cell expression system. This innovative strategy eliminates the need for antibody pre-purification, enabling high-precision site-specific conjugation of antibodies in unpurified culture medium. By directly preparing site-specific ADCs in a cell expression system, this invention simplifies the ADC production process and opens new pathways for the industrial production and clinical application of ADC drugs.

[0007] Therefore, the purpose of this invention is to provide a method for directly preparing site-specific ADCs in a cell expression system, providing a new approach for optimizing the process of site-specific ADCs.

[0008] According to one aspect of the present invention, a method for preparing an antibody-drug conjugate is provided, characterized by comprising the following steps:

[0009] (1) The cell culture medium containing antibodies is concentrated to the required concentration by ultrafiltration or left untreated to quantify the antibody concentration;

[0010] (2) Add the drug linker and adjust the pH to 7.4-8.5 with sodium hydroxide aqueous solution. React at room temperature or 37°C for 2-4 hours.

[0011] (3) After the reaction is completed, the antibody-drug conjugate is obtained by purification.

[0012] According to one embodiment of the present invention, wherein,

[0013] The antibody-containing cell culture medium in step (1) is obtained from cell expression systems selected from CHO, NSO, Sp2 / O, HEK293 and PER.C6, and can be used for reaction without treatment.

[0014] According to one embodiment of the present invention, wherein,

[0015] In step (2), the amount of drug linker is 5-20 eq of antibody molar concentration; the drug linker is dissolved in DMF or DMSO, and the content of DMF or DMSO in the reaction system is 0.5%-20%; the concentration of sodium hydroxide used to adjust pH is 0.1-2M.

[0016] According to one embodiment of the present invention, wherein,

[0017] The drug linker structure in step (2) comprises three parts: an Fc-binding peptide, a cleavable fragment, and a functional molecule.

[0018] The Fc-binding peptide refers to a peptide that has affinity for the Fc domain of an antibody.

[0019] The cleavable fragment refers to a fragment that can break under the attack of a nucleophile to form a new chemical bond and attach a functional molecule to a specific site on the protein. At the same time, the fragment breaks and releases the Fc-binding peptide.

[0020] In some embodiments, the Fc-binding peptide includes, but is not limited to, a cyclic peptide with the sequence SEQ ID No. 1: RGNCAYHRGKLVWCTYH (two cysteine ​​residues are linked by a disulfide bond to form a cyclic peptide chain).

[0021] In some embodiments, the cleavable fragment may be a thioester fragment, a maleimide ester fragment, an ester fragment, etc., preferably a thioester fragment. Preferably, the drug linker has the following structure:

[0022]

[0023] More preferably, the functional molecule may be a bioorthogonal group, a fluorescent molecule, or a drug, etc. Most preferably, the functional molecule is selected from one or more of biotin, the fluorescent molecule FITC, and the cytotoxic MMAE.

[0024] Beneficial effects

[0025] This invention enables the direct preparation of K248 site-directed ADCs in cell culture medium of antibody expression systems, avoiding the antibody pre-purification step in the traditional ADC preparation process and simplifying the traditional ADC preparation process.

[0026] The resulting ADC has similar physicochemical properties to the traditional K248 site-directed ADC, such as comparable hydrophilicity, aggregation stability, and thermal stability; in addition, it exhibits similar killing activity on tumor cells with different antigen expression levels; furthermore, this method is compatible with diverse substrate structures and antibody types, and has universality. Attached Figure Description

[0027] Figure 1 This document outlines a procedure for directly preparing K248 site-directed ADCs in cell culture medium for antibody expression systems.

[0028] Figure 2 Mass spectrometry data before and after Zilovertamab coupling with VC-PAB-MMAE in Example 6.

[0029] Figure 3 Mass spectrometry data for Zilovertamab before and after biotin conjugation in Example 7.

[0030] Figure 4 The mass spectrometry data for Zilovertamab before and after coupling with the fluorescent molecule FITC in Example 8 are shown.

[0031] Figure 5Zilovertamab coupled with Lys(mPEG) in Example 9 24 Mass spectrometry data before and after VC-PAB-MMAE.

[0032] Figure 6 Mass spectrometry data before and after Sacituzumab coupling with VC-PAB-MMAE in Example 10.

[0033] Figure 7 Mass spectrometry data of Sacituzumab before and after biotin conjugation in Example 11.

[0034] Figure 8 The mass spectrometry data for Sacituzumab before and after FITC coupling with the fluorescent molecule in Example 12 are shown.

[0035] Figure 9 Sacituzumab coupled with Lys(mPEG) in Example 13 24 Mass spectrometry data before and after VC-PAB-MMAE.

[0036] Figure 10 Mass spectrometry data before and after Mirvetuximab coupling with VC-PAB-MMAE in Example 14.

[0037] Figure 11 The hydrophobic chromatograms are a comparison of the ADC-1 and ADC-2 prepared in Example 6 and Comparative Example 1.

[0038] Figure 12 The thermal stability comparison diagram shows the ADC-1 and ADC-2 prepared in Example 6 and Comparative Example 1.

[0039] Figure 13 The graph shows a comparison of cell activity and toxicity of ADC-1 and ADC-2 prepared in Example 6 and Comparative Example 1. Specific implementation methods

[0040] definition

[0041] To better understand this invention, the following provides definitions and explanations of relevant terms.

[0042] Unless otherwise stated, all abbreviations herein have meanings well known to those skilled in the art.

[0043] As used herein, the term "antibody" refers to a heterotetraglycoprotein of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0044] As used herein, the term "Fc" refers to the following portion of an antibody, which contains the second (CH2) and third constant regions (CH3) of the first heavy chain, and is bound to the second and third constant regions of the second heavy chain via disulfide bonds and optionally hinge regions. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0045] As used herein, the term "K248" refers to lysine 248 of the Fc domain of an antibody, using the EU antibody sequence numbering system.

[0046] The "antibody-drug conjugate" or ADC mentioned in this article refers to a conjugate formed by covalently linking an antibody to a toxic drug via a linker. ADCs typically possess a ternary structure of antibody-linker-drug, combining the properties of antibodies with the characteristics of drugs.

[0047] The functional molecules mentioned in this article refer to structures with certain functions, such as those with anti-tumor, anti-infection, or anti-inflammatory effects, which can kill or inhibit cell (such as cancer cells) growth and are used to treat or alleviate certain diseases (such as cancer); for example, bioorthogonal groups that can be used in bioorthogonal reactions; and molecules that can emit light and are used for diagnosis and tracing.

[0048] As used herein, the term "Fc-binding peptide" refers to a linear or cyclic peptide that has the ability to reversibly bind to the Fc region of a target protein, particularly an antibody.

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, all reagents and materials used in this invention are commercially available. For example, the Fc-binding peptide was purchased from Genscript Biotech, Ltd., MMAE from Shanghai Ruishan Pharmaceutical Co., Ltd., China. Other chemical reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai Bid Pharmaceutical, Jier Biochemical, and Maclean's, etc. Chromatographic grade acetonitrile, trifluoroacetic acid, and deuterated reagents were purchased from Bailingwei. Unless otherwise specified, commercially available reagents and solvents were used directly without further purification. Trans5α competent cells were purchased from Beijing TransGen Biotech Co., Ltd., OPM-293CD05 Medium was purchased from Shanghai OPMI Biotechnology Co., Ltd., Invitrogen HEK293F cells, FBS was purchased from Thermo Fisher Scientific, and FaDu, NCI-N87, BxPC-3, NCI-H441, HCC-1806, and Calu-6 cells were all purchased from the Institute of Life Sciences, Chinese Academy of Sciences. The instruments used in this invention include: an analytical high-performance liquid chromatograph (HPLC) of Thermo Ultimate 3000, a preparative HPLC of Beijing Innovation Tongheng LC3000, a reversed-phase C18 column (Agilent Poroshell 120EC-C18, 4.6×150mm, 4μm) and a reversed-phase C18 semi-preparative column (Waters, 19×250mm, 5μm) and HIC column (TSKgel, 4.6×10cm, 2.5μm)) and a freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.). The centrifuge (Thermo Sorvall ST 8R), the sterile laminar flow hood (Thermo 1300 Series A2 1384), and the CO2 incubator (Thermo 3111 Forma) were all purchased from Thermo. High-resolution mass spectrometry data were determined using Waters Xevo G2-XS QTOF.

[0051] The abbreviations used in this article are as follows:

[0052]

[0053]

[0054] Example 1

[0055] Preparation of Fc-binding peptide-thioester-VC-PAB-MMAE complex P1

[0056]

[0057] a) Dissolve 12.8 mg (38.04 μmol) of 2-(triphenylthio)acetic acid in 500 μL of N,N-dimethylformamide (DMF), then add 7.2 mg (19.0 μmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 4.9 μL (28.53 μmol) of DIPEA, and 20 mg (9.5 μmol) of Fc-binding peptide (sequence Ac-RGNCAYHRGKLVWCTYH-NH2, with two cysteine ​​residues linked by disulfide bonds to form a cyclic peptide chain). React at room temperature for 1 h, monitored by LC-MS. After the reaction is complete, add diethyl ether; a white solid precipitates. Filter, collect the precipitate, and obtain 22.6 mg of white powder, i.e., S1 (yield 94%).

[0058] b) S1 was dissolved in 500 μL of dichloromethane, and 450 μL of trifluoroacetic acid (TFA) and 50 μL of triisopropylsilane (TIPS) were added under ice bath conditions. The reaction was carried out at 4 °C for 1 h, and monitored by LC-MS. After the reaction was completed, the mixture was dried under a nitrogen stream, purified by semi-preparative reversed-phase chromatography, and the target component was collected and lyophilized to obtain a white powder, S2. HRMS (ESI) analysis calculated value: C 95 H 137 N 31 O 23 S3:[M+H] + 2176.9744, [M+2H] 2+ 1088.9911, [M+3H] 3+ 726.3300, Measured values: m / z = 1088.9951, 726.3297.

[0059] c) Dissolve NH2-VC-PAB-MMAE (10 mg, 8.9 μmol) in 200 μL DMF, add disuccinimide glutarate (DSG) (14.5 mg, 44.5 μmol) and triethylamine (3.7 μL, 26.7 μmol), and react at room temperature for 2 h, monitored by LC-MS. After the reaction is complete, separate and purify by semi-preparative reversed-phase chromatography, collect the target component, and lyophilize to obtain 10.8 mg of white powder, which is S3 (yield 91%). HRMS (ESI): Calculated value: C 67 H 103 N 11 O 17 [M+H] + 1334.7612, measured value: 1334.7655.

[0060] d) S3 (6.2 mg, 4.6 μmol) was dissolved in 200 μL of DMF, followed by the addition of S2 (10 mg, 4.6 μmol) and triethylamine (1.9 μL, 13.8 μmol). The reaction was carried out at room temperature for 1 h, monitored by LC-MS. After the reaction was complete, the target component was purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain a white powder, which was P1 (yield 76%). HRMS (ESI) analysis calculated: C 158 H 235 N 41 O 37 S3:[M+H] + 3395.7007, [M+3H] 3+ 1132.5721, [M+4H] 4+ 849.6810, Measured values: m / z = 1133.5587, 849.6596.

[0061] Example 2

[0062] Preparation of Fc-binding peptide-thioester-biotin complex P2

[0063]

[0064] Biotin-NHS ester (1.9 mg, 5.5 μmol) was dissolved in 200 μL of DMF, followed by the addition of S2 (10 mg, 4.6 μmol) and triethylamine (1.9 μL, 13.8 μmol). The reaction was carried out at room temperature for 30 min, monitored by LC-MS. After the reaction was completed, the target component was purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain a white powder, which was P2 (yield 75%). HRMS (ESI) analysis calculated: C 105 H 151 N 33 O 25 S4:[M+H] + 2403.052, [M+3H] 3+ 801.6892, [M+4H] 4+ 601.5188, Measured values: m / z = 801.6801, 601.5139.

[0065] Example 3

[0066] Preparation of Fc-binding peptide-thioester-fluorescent molecule complex P3

[0067]

[0068] a) Dissolve 4,7,10,13,16-pentaenodecane-1,19-diacid (27 mg, 80 μmol) in 700 μL LMF, then add HATU (24.3 mg, 64 μmol), DIPEA (16.5 μL, 96 μmol), and FITC (11.1 mg, 32 μmol) sequentially. React at room temperature in the dark for 2 h, monitored by LC-MS. After the reaction is complete, the target component is purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain 14.5 mg of a yellow powder, which is S4 (yield 68%). HRMS (ESI) analysis calculated: C 34 H 37 NO 13 [M+H] + 668.2343, measured value: 668.2415.

[0069] b) and c) FITC-PEG5-COOH(S4) (14.5 mg, 21.7 μmol) was dissolved in 200 μL DMF, and NHS (5 mg, 43.4 μmol) and EDCI (7.67 μL, 43.4 μmol) were added. The reaction was carried out overnight at room temperature in the dark, and monitored by LC-MS. After the reaction was complete, thioglycolic acid (4.52 μL, 65.1 μmol), tris(2-carbonylethyl)phosphohydrochloride (9.3 mg, 32.55 μmol) and 200 μL of 0.2 M NaH2PO4-Na2HPO4 buffer solution (pH = 7.4) were added to the reaction solution, and the reaction was continued at room temperature in the dark for 2 h, monitored by LC-MS. After the reaction was complete, the target component was separated and purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain 11.5 mg of yellow powder, which was S5 (yield 72%). HRMS (ESI) Anal. Calcd for C 36 H 39 NO 14 S[M+H] + 742.2169, found 742.2214.

[0070] d) S5 (2 mg, 2.7 μmol) was dissolved in 200 μL of DMF, and HATU (1 mg, 2.7 μmol), DIPEA (0.7 μL, 4.05 μmol), and Fc-binding peptide (8.5 mg, 4.05 μmol) were added sequentially. The reaction was carried out at room temperature in the dark for 2 h, and monitored by LC-MS. After the reaction was complete, the target component was separated and purified by semi-preparative reversed-phase chromatography. The target component was collected, lyophilized to obtain 6.1 mg of yellow powder, which was P3 (yield 80%). HRMS (ESI) analysis calculated: C 129 H 172 N 32 O 35 S3[M+H] +2826.1903, [M+2H] 2+ 1413.5991, [M+3H] 3+ 942.7353, measured values: 1413.6048, 942.7214.

[0071] Example 4

[0072] Fc-binding peptide-thioester-Lys(mPEG) 24 Preparation of )-VC-PAB-MMAE P4

[0073]

[0074] Pre-a) Diethylene glycol (26.8 mg, 0.2 mmol) was dissolved in 400 μL of DMF, and p-nitrophenol (66.7 mg, 0.48 mmol) and EDCI (84.8 μL, 0.48 mmol) were added. The mixture was reacted overnight at room temperature under TLC monitoring. After the reaction was complete, diethyl ether was added, and the mixture was stirred to precipitate a large amount of pale yellow solid. The precipitate was collected by centrifugation and washed three times with diethyl ether to obtain 52 mg of pale yellow solid, which was S6 (yield 69%). 1 H NMR (500MHz, DMSO-d6) δ8.37-8.32(m,4H),7.56-7.49(m,4H),4.69(s,4H). 13 CNMR(126MHz,DMSO-d6)δ168.13,154.85,145.21,126.21,125.40,123.15,115.81,67.48.

[0075] a) and b) CH3O-PEG 24 -COOH (58 mg, 0.05 mmol) was dissolved in 1 mL of acetonitrile, and N-hydroxysuccinimide (NHS) (6.8 mg, 0.06 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) (7.9 μL, 0.06 mmol) were added. The reaction was carried out overnight at room temperature. After the reaction was completed, Fmoc-Lys(NH2)-OH (18.4 mg, 0.05 mmol) was added, and the reaction was carried out for 2 h at room temperature, monitored by LC-MS. After the reaction was complete, the target component was separated and purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain 57.9 mg of a colorless oil, which was S7 (yield 78%). HRMS (ESI) analysis calculated value: C 73 H 126 N2O 30 [M+H] + 1511.8474, [M+2H] 2+ 756.4275, measured value: 756.4218.

[0076] c) and d) S7 (13.5 mg, 8.9 μmol) was dissolved in 400 μL DMF, and HATU (6.8 mg, 17.8 μmol), DIPEA (4.6 μL, 27.6 μmol), and NH2-VC-PAB-MMAE (10 mg, 8.9 μmol) were added. The reaction was carried out at room temperature for 2 h, and monitored by LC-MS. After the reaction was complete, 100 μL piperidine was added, and the reaction was carried out at room temperature for 15 min. After the reaction was completed, the product was purified by semi-preparative reversed-phase chromatography. The target component was collected, lyophilized, and 18.3 mg of white powder was obtained, which was S8 (yield 86%). HRMS (ESI) analysis calculated value: C 116 H 208 N 12 O 39 [M+H] + 2394.4741, [M+3H] 3+ 798.8299, measured value: 798.8312.

[0077] e) and f) S8 (14 mg, 5 μmol) was dissolved in 200 μL DMF, and S6 (5.6 mg, 15 μmol) and triethylamine (3.36 μL, 25 μmol) were added. The reaction was carried out at room temperature for 20 min, and monitored by LC-MS. After the reaction was complete, 1.05 μL, 15 μmol, mercaptoacetic acid was added, and the reaction was carried out at room temperature for 1 h, and monitored by LC-MS. After the reaction was completed, the target component was purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain 10.5 mg of white powder, which was S9 (yield 81%). HRMS (ESI) analysis calculated value: C 122 H 214 N 12 O 44 S[M+H] + 2584.4676, [M+3H] 3+ 862.1611, [M+4H] 4+ 646.8728, Measured values: 862.1596, 646.8756.

[0078] g) S9 (6.15 mg, 2.38 μmol) was dissolved in 200 μL of DMF, followed by the sequential addition of HATU (1.35 mg, 4.76 μmol), DIPEA (1.23 μL, 7.14 μmol), and Fc-binding peptide (5 mg, 2.38 μmol). The reaction was carried out at room temperature for 2 h, and monitored by LC-MS. After the reaction, the target component was purified by semi-preparative reversed-phase chromatography, collected, and lyophilized to obtain 6.2 mg of white powder, which was P4 (yield 56%). HRMS (ESI) analysis calculated: C 215 H 347 N43 O 65 S3[M+H] + 4668.4409, [M+5H] 5+ 934.4944, [M+6H] 6+ 778.9133, Measured values: 934.5015, 778.9215.

[0079] Example 5

[0080] Zilovertamab expression

[0081] 50 μL of Trans5α competent cells were slowly thawed on ice, and Zilovertamab heavy and light chain plasmids (constructed by Nanjing Genscript Biotech Co., Ltd.) were slowly and gently added separately. The mixture was then incubated on ice for 30 min. Afterward, the cells were heat-shocked in a 42°C water bath for 90 s. The cells were then transferred to ice for 5 min. Finally, 500 μL of antibiotic-free LB medium was added. The cells were incubated at 37°C with shaking at 200 rpm for 1 h. The culture solution was then evenly spread onto LB agar plates (with added ampicillin). After absorption, the plates were inverted and incubated overnight at 37°C. Single colonies from the plates were picked and placed in shake flasks containing LB liquid medium (with ampicillin) and incubated at 37°C with shaking at 200 rpm for 16 h. The culture was collected, and plasmid extraction was performed according to the HiPure Plasmid EFMaxi Kit instructions. The plasmid concentration was determined using NanoDrop after extraction. OPM-293CD05 Medium (purchased from Shanghai Aopumai Biotechnology Co., Ltd.) was pre-treated in a 37°C water bath. Cells stored in liquid nitrogen were removed and thawed in a 37°C water bath. They were then transferred to centrifuge tubes pre-filled with medium, centrifuged at 800 rpm for 5 min, and the liquid was discarded. When resuspending the cells, 1 mL of fresh medium was used, and the cells were seeded into 125 mL triangular culture flasks pre-filled with 20 mL of medium. The flasks were gently shaken and incubated at 37°C, 120 rpm, and 5% CO2. Cell viability and density were checked one day in advance, and the cell density was adjusted to 0.5 × 10^6 cells / mL. After approximately 24 hours of culture, the cell count and viability were recalculated. Cells with a viability ≥ 95% at a concentration of 1 × 10^6 cells / mL were ready for plasmid transfection. Total plasmid: Total cell count: PEI transfection reagent = 1 mg: 10^6 cells: 3 mL, where light chain plasmid: heavy chain plasmid = 3:1. Plasmids and transfection reagents were diluted separately with 10% of the total cell solution volume of culture medium. After gentle mixing, the PEI dilution was added to the plasmid DNA dilution, gently mixed, and allowed to stand for 15 min. Afterward, the mixture was added dropwise to the cell solution, gently agitating during the addition. Cells were cultured at 37℃, 120 rpm, 5% CO2 for 7 days. Cell viability was checked and found to be less than 60%. Cells were collected, centrifuged, and filtered through a 0.45 μm filter to obtain the Zilovertamab expression system supernatant. Antibody concentration was quantified by reducing SDS-PAGE electrophoresis and image analysis using Image Lab software, and stored at -20℃.

[0082] Experimental Example 1

[0083] Purification of antibodies and antibody conjugates

[0084] Pre-fill the chromatography column with Protein A packing material, then flush and equilibrate the column with 5 column volumes of equilibration buffer / wash buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0). In a 4°C incubator, connect a peristaltic pump to allow cell fluid to flow through the packing material for sample loading, controlling the flow rate at 1 mL / min. After loading, use 10-15 column volumes of equilibration buffer / wash buffer to remove contaminating proteins. Finally, elute the proteins with 5-10 column volumes of elution buffer (0.1M glycine, pH 3.0).

[0085] The collected cell supernatant, washing buffer, and elution buffer were analyzed by SDS-PAGE. Samples containing protein bands of the target molecular weight were collected, centrifuged, concentrated, and replaced with 1×PBS preservation solution. The protein concentration of the collected ADCs was determined using NanoDrop, aliquoted, and stored at -20°C.

[0086] Example 6

[0087] Method for direct preparation of Zilovertamab antibody conjugates in cell expression systems

[0088] The supernatant of the Zilovertamab expression system was diluted with OPM-293CD05 Medium to a concentration of 2 mg / mL. Then, 20 eq (0.1336 mM) of the Fc-binding peptide-thioester-VC-PAB-MMAE complex P1 was added, and the pH was adjusted to 7.5 with sodium hydroxide aqueous solution. The reaction was carried out at 37°C for 2 h. After the reaction, ADC-1 was purified using the method described in Experimental Example 1.

[0089] Comparative Example 1

[0090] Methods for purifying antibodies to prepare Zilovertamab antibody conjugates

[0091] The drug linker (Fc-binding peptide-thioester-VC-PAB-MMAE) was dissolved in DMF to prepare a 10 mM stock solution. 20% DMF was added to a 50 mM sodium acetate (pH = 7.2) reaction solution beforehand to aid dissolution, followed by the addition of purified Zilovertamab antibody and Fc-binding peptide-thioester-VC-PAB-MMAE P1. The antibody concentration was 5 mg / mL, and the linker was 0.334 mM. The reaction was incubated at 37°C for 2 h. After the reaction, ADC-2 was purified using the method described in Experimental Example 1.

[0092] Example 7

[0093] The steps in this embodiment are the same as in Example 6, except that the linker P1 is replaced with the Fc-binding peptide-thioester-biotin complex P2 to prepare ADC-3.

[0094] Example 8

[0095] The steps in this embodiment are the same as in Example 6, except that the linker P1 is replaced with the Fc-binding peptide-thioester-fluorescent molecule complex P3 to prepare ADC-4.

[0096] Example 9

[0097] The steps in this embodiment are the same as in Example 6, except that the linker P1 is replaced with Fc-binding peptide-thioester-Lys(mPEG). 24 ADC-5 was prepared by using )-VC-PAB-MMAE P4.

[0098] Example 10

[0099] The steps in this embodiment are the same as in Example 6, except that the Zilovertamab cell expression system is replaced with the Sacituzumab cell expression system to prepare ADC-6.

[0100] Example 11

[0101] The steps in this embodiment are the same as in Example 7, except that the Zilovertamab cell expression system is replaced with the Sacituzumab cell expression system to prepare ADC-7.

[0102] Example 12

[0103] The steps in this embodiment are the same as in Example 8, except that the Zilovertamab cell expression system is replaced with the Sacituzumab cell expression system to prepare ADC-8.

[0104] Example 13

[0105] The steps in this embodiment are the same as in Example 9, except that the Zilovertamab cell expression system is replaced with the Sacituzumab cell expression system to prepare ADC-9.

[0106] Example 14

[0107] The steps in this embodiment are the same as in Example 6, except that the Zilovertamab cell expression system is replaced with the Mirvetuximab cell expression system to prepare ADC-10.

[0108] Experimental Example 2

[0109] LC-MS characterization of ADCs

[0110] All ADCs mass spectrometry samples were deglycosylated using the Endo-S2 enzyme before detection. Data were measured using an LC-MS mass spectrometer (Agilent 6545LC-TOF, Agilent) with a C4 column (AdvanceBio RP-mAb C4 2.5×50mm, 3.5micron), and the molecular weight was obtained by deconvolution processing using Agilent bioconfirmation software.

[0111] Mobile phase A: water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Instrument detection method: Set the mobile phase gradient parameters as follows: 0-2 min, mobile phase B 5-5%, 2-10 min, mobile phase B 5-90%; Flow rate: 0.3 mL / min; Detection absorbance: 280 nm.

[0112] Mass spectrometry data of ADC-1 to ADC-10 prepared in Examples 6 to 14 and Comparative Example 1 were measured according to the method described above. See the results below. Figures 2 to 10 .

[0113] Depend on Figures 2 to 10 It can be seen that, compared with the antibody itself, the molecular weight of all antibody conjugates is increased by the molecular weight of two functional molecules, indicating that they have good uniformity and are all conjugated with two functional molecules.

[0114] Experimental Example 3

[0115] Hydrophilicity analysis of ADCs

[0116] The hydrophilicity of ADCs was determined using an AdvanceBio HIC column (4.6 × 100 mm, 3.5 μm, Agilent) on a high-performance liquid chromatograph (LC-20ADXR, Shimadzu). Mobile phase solutions: Phase A (4.4 g K₂HPO₄, 198.2 g (NH₄)₂SO₄ dissolved in 1 L ddH₂O, 5% volume ratio solution poured out and an equal volume of isopropanol added); Phase B (2.5 g K₂HPO₄, 1.6 g KH₂PO₄ dissolved in 1 L ddH₂O, 20% volume ratio solution poured out and an equal volume added). Instrument detection method: The mobile phase gradient parameters were set to 0-20 min; mobile phase B 40-100%; flow rate: 0.6 mL / min; absorbance: 280 nm. (See [link to instrument description]). Figure 11 .

[0117] Depend on Figure 11 It can be seen that by using the method of the present invention, antibody-drug conjugates with similar hydrophilicity can still be obtained even without omitting the antibody pre-purification step.

[0118] Experimental Example 4

[0119] Thermal stability testing of ADCs

[0120] The thermal stability of the prepared antibodies and ADCs was detected by differential scanning fluorescence (DSF).

[0121] Take 60 μg of ADC sample, 8 mL of 5×PBS, 8 mL of 50×SYPRO Orange dye, and add ddH2O to bring the liquid volume to 40 μL. Perform sample analysis using a real-time PCR instrument with a program of 5-95℃ and a heating rate of 0.3℃ / 6s. Data processing was performed using GraphPad Prism 6. The x-axis was set to T / min, and the y-axis was set to d(RFU) / d(T). The peak value of the spectral line is the Tm value. See [link to graph]. Figure 12 .

[0122] Depend on Figure 12 It can be seen that by using the method of the present invention, antibody-drug conjugates with similar thermal stability can still be obtained even without omitting the antibody pre-purification step.

[0123] Experimental Example 5: Method for Detecting Cytotoxicity

[0124] The cytotoxicity assays used Trop2-positive cells: FaDu, NCI-N87, BxPC-3, NCI-H441, HCC-1806, and Trop2-negative cells Calu-6, all of which were purchased from the Institute of Life Sciences, Chinese Academy of Sciences.

[0125] All cell types were seeded at a rate of 5000 cells per well into 96-well plates and incubated overnight at 37°C with 5% CO2. Cell drug administration was performed at an initial concentration of 0.15 mg / mL (1 μM), with 5-fold dilutions in 9 gradients and 3 replicates. After 144 hours of drug treatment, adherent cells required the removal of the original culture medium, and each well was then filled with a medium solution containing CCK-8, and agitated thoroughly. Suspension cells did not require discarding the medium; 10 μL of CCK-8 was added directly to each well. Afterward, the cells were incubated for 1-4 hours (ideally until the absorbance reading on the microplate reader is 0.8-1.2), and absorbance was measured at 450 nm. Figure 13 .

[0126] Depend on Figure 13 It can be seen that by using the method of the present invention, antibody-drug conjugates with similar cell activity / toxicity (tumor cell killing activity) can be obtained even without omitting the antibody pre-purification step.

Claims

1. A method for preparing an antibody-drug conjugate, characterized in that, Includes the following steps: (1) The cell culture medium containing antibodies is concentrated to the required concentration by ultrafiltration or left untreated to quantify the antibody concentration; (2) Add the drug linker and adjust the pH to 7.4-8.5 with sodium hydroxide aqueous solution. React at room temperature or 37°C for 2-4 hours. (3) After the reaction is completed, the antibody-drug conjugate is obtained by purification.

2. The preparation method according to claim 1, wherein, The antibody-containing cell culture medium in step (1) is obtained from cell expression systems selected from CHO, NSO, Sp2 / O, HEK293 and PER.C6, and can be used for reaction without treatment.

3. The preparation method according to claim 1, wherein, In step (2), the amount of drug linker is 5-20 eq of antibody molar concentration. The drug linker is dissolved in DMF or DMSO. The content of DMF or DMSO in the reaction system is 0.5%-20%. The concentration of sodium hydroxide used to adjust pH is 0.1-2M.

4. The preparation method according to claim 1, wherein, The drug linker structure in step (2) comprises three parts: an Fc-binding peptide, a cleavable fragment, and a functional molecule. The Fc-binding peptide refers to a peptide that has affinity for the Fc domain of an antibody. The cleavable fragment refers to a fragment that can break under the attack of a nucleophile to form a new chemical bond and attach a functional molecule to a specific site on the protein. At the same time, the fragment breaks and releases the Fc-binding peptide.

5. The preparation method according to claim 1, wherein, The Fc-binding peptide comprises the amino acid sequence as described in SEQ ID No.

1.

6. The preparation method according to claim 1, wherein, The cleavable fragment is a thioester fragment, a maleimide ester fragment, or an ester fragment.

7. The preparation method according to claim 6, wherein, The cleavable fragment is a thioester fragment.

8. The preparation method according to claim 1, wherein, The drug linker has the following structure:

9. The preparation method according to claim 1, wherein, The functional molecule is a bioorthogonal group, a fluorescent molecule, or a drug.

10. The preparation method according to claim 1, wherein, The functional molecule is selected from one or more of biotin, fluorescent molecule FITC, and cytotoxic MMAE.

Images