Anti-MSLN monoclonal internalization antibody, its preparation method and application

Abstract

This invention discloses an anti-MSLN monoclonal internalization antibody, its preparation method, and its applications. The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain variable region CDR1 as shown in SEQ ID NO.1; heavy chain variable region CDR2 as shown in SEQ ID NO.3; and heavy chain variable region CDR3 as shown in SEQ ID NO.5. The light chain variable region includes light chain variable region CDR1 as shown in SEQ ID NO.7; light chain variable region CDR2 as shown in SEQ ID NO.9; and light chain variable region CDR3 as shown in SEQ ID NO.11. The antibody of this invention has good specificity and high affinity, can bind to cell surface expression, and can be efficiently transported into the cell by cells, carrying toxin molecules to simultaneously enter the cell and kill the cell. This antibody can be widely used in drugs for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody, its preparation method and application, and more particularly to an anti-MSLN monoclonal internalization antibody, its preparation method and application. Background Technology

[0002] Mesothelin (MSLN) is a 40 kDa glycoprotein located on the cell surface, anchored to the cell membrane via glycosylphosphatidylinositol. The mesothelin gene encodes a 69 kDa precursor protein, which is hydrolyzed into two chains by furin-like convertases. The approximately 40 kDa membrane-bound protein at the C-terminus is the mature mesothelin, and the approximately 30 kDa fragment at the N-terminus, called megakaryocyte-promoting factor (MPF), is detached and released extracellularly. Both MPF and the membrane-anchored MSLN are N-glycosylated. MPF can promote the formation of megakaryocyte clones in vitro, and the membrane-anchored MSLN can interact with MUC16 (also known as CA125) and plays an important role in cell adhesion. Therefore, the membrane-anchored MSLN is currently selected as a target in targeted therapy. Thus, MSLN currently specifically refers to the 40 kDa C-terminal fragment of MSLN, i.e., the membrane-anchored MSLN.

[0003] Under normal circumstances, MSLN is expressed at low levels only in mesothelial tissues such as the pleura, peritoneum, and pericardium, and is not expressed in other tissues, exhibiting a very narrow expression spectrum. However, strong expression of MSLN is extremely common in cancerous tissues. High expression levels have been detected in various solid tumors, including mesothelioma, non-small cell lung cancer, ovarian cancer, endometrial cancer, cervical cancer, and pancreatic cancer. MSLN expression has even been found in esophageal cancer, metastatic triple-negative breast cancer, and renal cell carcinoma. Regarding cancer cell lines, MSLN is known to be highly expressed in three ovarian cancer cell lines (HO-8910, HEY-T30, and OVCAR3, especially HO-8910), the metastatic pancreatic cancer cell line AsPC1, and the cervical cancer cell line HeLa. It is expressed at very low levels in two ovarian cancer cell lines (SKOV3 and 3AO) and the lung adenocarcinoma cell line A549, and is not expressed in the human hepatocellular carcinoma cell line Huh7.

[0004] At the cellular level, MSLN participates in cell biological processes such as (cancer) cell proliferation, apoptosis, and adhesion, and adversely affects the efficacy of chemotherapy. For example, SKOV3 cells with downregulated MSLN expression showed decreased tumorigenicity when used for mouse xenograft construction, while also increasing apoptosis and weakening tumor metastasis. Blocking the binding of MSLN to MUC16 decreased cell adhesion. Downregulation of MSLN expression increased the sensitivity of cancer tissue to cisplatin and paclitaxel. Furthermore, MSLN overexpression was observed to induce overexpression of metalloproteinase 9, thereby promoting tumor cell migration and invasion. Therefore, it can be inferred that MSLN interacts with MUC16 to induce the expression of metalloproteinase 9 and other proteins, thereby causing corresponding changes in cell proliferation, adhesion, and apoptosis, playing an important role in the invasion of tumor peritoneal metastases, and promoting tumor cell anti-apoptosis.

[0005] MSLN is almost not expressed in normal cells (only lowly expressed in the pleura, peritoneum, and pericardium) but is strongly expressed in various cancer tissues, making it a good target for targeted therapy. This inherently gives MSLN the potential advantages of low off-target effects and broad indications for targeted therapy. For example, MSLN is expressed in 60-90% of lung cancer tissues, with 20% showing high expression levels, but not in normal lung tissue. Furthermore, lung adenocarcinoma with high MSLN expression has a poor prognosis, suggesting that MSLN is a potential target for lung cancer treatment. Experiments have shown that CAR-T therapy targeting MSLN, administered via tail vein reinfusion to tumor-bearing mice, significantly inhibited tumor growth induced by subcutaneous inoculation with lung adenocarcinoma tissue highly expressing MSLN.

[0006] Given the broad indications and low potential off-target effects of targeted therapies targeting MSLN, research and development of MSLN-targeted treatments is proceeding in multiple directions. This includes 12 drugs undergoing clinical trials (5 of which are antibody-based), such as recombinant monoclonal monospecific antibodies, bispecific antibodies, synthetic proteins, antibody-drug conjugates (toxins or radioisotopes), vaccines, and adoptive immunotherapy. Among these, Amatuximab monoclonal antibody (MORAb-009), developed by the National Cancer Institute, has completed a phase II clinical trial for the treatment of malignant mesothelioma.

[0007] Regarding antibodies currently under development, while many known mesothelin monoclonal antibodies are available, none exhibit complement-dependent cytotoxicity (CDC) against tumor cells. Antibodies specifically bind to antigens on the cell surface, and their Fc region recruits C1q cells, thereby activating the classical complement activation pathway. This ultimately leads to the formation of a membrane attack complex on the surface of antigen-expressing cells, causing target cell lysis. This mechanism is therefore beneficial for cancer treatment. Obtaining antibodies targeting MSLNs with strong CDC effects using traditional hybridoma technology is challenging. For example, antibodies obtained through traditional methods have poor tissue penetration and insufficient therapeutic efficacy, require humanization due to high immunogenicity, and exhibit poor stability with stringent requirements for transportation and storage. In conclusion, while existing traditional antibodies play a crucial role in disease treatment and detection, the shortcomings of current MSLN-targeting antibodies are also evident.

[0008] Currently, there are 12 MSLN-targeting drugs in clinical trials, including 5 antibody drugs, such as Amatuximab. These antibodies are human-mouse chimeric antibodies or humanized antibodies, all derived from mice and obtained using hybridoma technology. Current therapies targeting MSLN include cell therapy and naked antibodies, but their clinical efficacy is limited, especially for solid tumors, where these two methods are almost ineffective. Researchers generally believe that antibody-drug conjugates (ADCs) will be one of the future hopes for cancer treatment targeting this target. However, these antibodies are not fully human antibodies, and compared to fully human antibodies, they have issues such as higher immunogenicity, poorer safety profile, and potentially lower in vivo stability. Summary of the Invention

[0009] Objectives of the invention: The objective of this invention is to provide an anti-MSLN monoclonal internalization antibody with cell internalization function and CDC effect; another objective of this invention is to provide a method for preparing an anti-MSLN monoclonal internalization antibody; yet another objective of this invention is to provide an application of the anti-MSLN monoclonal internalization antibody.

[0010] Technical solution: The anti-MSLN monoclonal internalization antibody of the present invention includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a heavy chain variable region CDR1 as shown in SEQ ID NO.1; a heavy chain variable region CDR2 as shown in SEQ ID NO.3; and a heavy chain variable region CDR3 as shown in SEQ ID NO.5.

[0011] The SEQ ID NO.1 is GYTFTSYY; the SEQ ID NO.3 is INPSGGST; and the SEQ ID NO.5 is ARDRGTYYYGSGDLGY.

[0012] Furthermore, the light chain variable region includes light chain variable region CDR1 as shown in SEQ ID NO.7; light chain variable region CDR2 as shown in SEQ ID NO.9; and light chain variable region CDR3 as shown in SEQ ID NO.11.

[0013] SEQ ID NO.7 is QGISTW; SEQ ID NO.9 is AAS; SEQ ID NO.11 is QQANSFPLT.

[0014] Furthermore, the antibody is a fully human antibody.

[0015] Furthermore, the antibody specifically binds to the MSLN antigen on the surface of cancer cells.

[0016] On the other hand, the present invention provides a nucleic acid molecule that encodes the above-mentioned monoclonal internalization antibody.

[0017] On the other hand, the present invention provides an expression vector comprising the above-described nucleic acid molecules.

[0018] On the other hand, the present invention provides a host cell comprising the expression vector described above.

[0019] On the other hand, the present invention provides a method for preparing an anti-MSLN monoclonal internalization antibody, the method comprising culturing host cells comprising the above-described host cells. The specific method is as follows:

[0020] Commercially available recombinant human MSLN biotinylated protein and a phage display antibody library were used as starting materials. MSLN biotinylated protein was co-incubated with streptavidin-coated magnetic beads. MSLN was immobilized on the beads through the interaction between streptavidin and biotin. The antibody library was then incubated with the beads, and unbound / weakly bound phages were washed away, while phages bound to MSLN protein were eluted. This process was repeated three times, each time with varying amounts of MSLN biotinylated protein and washing conditions to progressively eliminate weakly bound phages and retain as many strongly bound phages as possible. Host bacteria were infected with strongly bound phages, plated, and cultured overnight to obtain monoclonal colonies. This process involved monocloning the phage antibodies that could bind MSLN. The supernatant from the monoclonal culture was used for ELISA screening, and the nucleic acid sequences of the monoclonal colonies were determined.

[0021] The coding sequences of the signal peptide, anti-MSLN antibody, and human IgG1 Fc were ligated and read in the same frame to construct a mammalian expression vector. This vector was transfected into 293F cells, and after 5 days of shaking culture, the supernatant was harvested. The fusion protein was purified from the supernatant using protein A magnetic bead affinity chromatography, and its binding characteristics to MSLN were identified. Target cells were cultured, and the antibody was co-incubated with the cells at 4°C in two parallel groups. One group was incubated at 4°C and then transferred to 37°C for further incubation, while the other group was incubated at 4°C. After incubation, fluorescently labeled detection antibodies were added, and the cells were incubated at 4°C. The fluorescent antibodies were detected by flow cytometry, thus identifying the amount of antibody captured by MSLN on the cell surface.

[0022] MSLN antibody. Target cells were seeded in 96-well plates and cultured overnight to allow for full cell adhesion. After adding antibody and / or toxin reagents, cell culture was performed and cell growth characteristics were observed; cell viability was assessed after culture.

[0023] On the other hand, the present invention provides a kit comprising the above-described monoclonal internalizing antibody.

[0024] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned monoclonal internalizing antibody.

[0025] The application of the above-mentioned monoclonal internalized antibodies in the preparation of antibody-drug conjugates.

[0026] The above-mentioned monoclonal internalizing antibodies are used in the preparation of anticancer or cancer detection reagents, products or drugs.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The antibody of the present invention has good specificity and high affinity, can bind to cell surface expression, and can be efficiently transported into the cell by the cell, carrying toxin molecules into the cell simultaneously to kill the cell. This antibody can be widely used in drugs for tumor immunotherapy. Attached Figure Description

[0028] Figure 1 The results of ELISA detection of the binding of phage crude culture medium and human MSLN in Example 2;

[0029] Figure 2 The results of FACS detection of the binding of phage crude culture medium and human MSLN in Example 4;

[0030] Figure 3 This is a schematic diagram of the MSLN antibody expression vector structure in Example 3;

[0031] Figure 4 SDS-PAGE electrophoresis of the purified antibody in Example 3;

[0032] Figure 5The binding performance of the purified antibody in Example 4 to MSLN overexpressed on the cell surface was tested.

[0033] Figure 6 The binding performance of the purified antibody in Example 4 to MSLN on the surface of HeLa cells was tested.

[0034] Figure 7 This is an off-target detection of the purified antibody in Example 4 binding to Huh7 and HEK293 cells;

[0035] Figure 8 EC50 detection of antibody binding to MSLN molecules on the solid-phase support surface in Example 5;

[0036] Figure 9 For the detection of the internalization performance of the purified antibody in Example 6;

[0037] Figure 10 The relative internalization properties of the purified antibody in Example 6;

[0038] Figure 11 This demonstrates the cell-killing efficacy of the purified antibody internalized toxin in Example 7. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Example 1: Phage Display Fully Human Antibody Library Screening

[0041] 1) Activation of host bacteria TG1: Prepare mini agar medium plates [1×M9 salt, 2% glucose, 2mM MgSO4, 0.1mM CaCl2, 1mM vitamin B1], and incubate TG1 overnight at 37℃ using the streak method.

[0042] 2) Magnetic bead washing and blocking: Take 50 μL of magnetic beads (purchased from Invitrogen), place them on a magnetic rack, remove the liquid after adsorption, resuspend in 1 ml of PBS, wash twice, block with 1 ml of blocking agent of 1.5% skim milk powder + 1.5% BSA (the concentration of blocking agent is gradually increased in the second and third rounds of washing) for 1 hour, and remove the liquid.

[0043] 3) Antigen binding: Human MSLN protein (purchased from Acro Biosystems, with the antigen concentration gradually decreasing in subsequent rounds of panning) was diluted to 1 ml in PBS (pH 7.2-7.4) at a concentration of 16 ug / ml, the magnetic beads were resuspended, and the mixture was incubated by rotation for 1 hour.

[0044] 4) Library blocking: Synchronize the binding of antigen to magnetic beads, take 10 11PFU phage virus particles (from the original antibody library or panning amplification products) were incubated with 1 ml of 1.0% skim milk powder + 1.0% BSA blocking agent by rotation for 1 hour.

[0045] 5) Phage binding: Place the magnetic beads on a magnetic rack and remove the liquid. Add the sealed library to the magnetic beads, resuspend and incubate by rotation for 1 hour, then remove the liquid.

[0046] 6) Washing: Wash with 1 ml PBST [0.01 M PBS (pH 7.4), 0.1% Tween-20 (the concentration of Tween-20 for the second and third rounds is 0.2% and 0.3% respectively)], and then wash with 0.01 M PBS (pH 7.4).

[0047] 7) Elution: Aspirate the liquid, elute with 300 μL of 0.2 M glycine-hydrochloric acid (pH 2.2) for 10 minutes, add 20 μL of neutralization buffer [1 M Tris-Cl (pH 9.0)] and mix well. Store temporarily at 4 °C.

[0048] 8) Titration: Take 2 μL, 0.2 μL (2 μL of the stock solution diluted 10 times with 2×YT medium), and 0.02 μL (2 μL of the stock solution diluted 100 times with 2×YT medium) of elution buffer, mix with 0.2 mL of TG1 at mid-log (OD600 = 0.5), incubate at room temperature for 30 minutes, spread evenly on 2×YT-GA100 plates [containing 2% glucose and 100 μg / mL ampicillin], incubate overnight at 37°C, count the number of clones on plates with approximately 50 clones, and calculate the titer based on the dilution factor.

[0049] 9) Phage amplification: While panning, pick TG1 single clones from mini agar plates and inoculate them into 10 ml of 2×YT medium. Incubate at 37°C with shaking at 250 rpm until mid-log phase (OD600 = 0.5). Add 200 μL of the eluted product obtained from panning and incubate at 37°C for 30 minutes. Add helper phage M13KO7 and incubate at 37°C for another 30 minutes. Incubate at 37°C with shaking at 250 rpm for 1 hour. Centrifuge to remove the supernatant, resuspend in 20 ml of 2×YT medium containing working concentrations of 100 μg / ml ampicillin and 50 μg / ml kanamycin, and incubate overnight at 30°C with shaking at 220 rpm.

[0050] 10) Phage precipitation: Centrifuge at 10,000 rpm for 15 minutes to remove bacterial cells. Add 1 / 5 volume of 2.5M NaCl / 20% PEG8000 to the supernatant and incubate on ice for 2 hours. Centrifuge at 10,000 rpm for 10 minutes to obtain phage precipitation. Remove the residual liquid and resuspend the precipitate in 0.2 ml of 0.01M PBS (pH 7.4). Measure the titer as described above.

[0051] 11) Repeat steps 2)-10) two or three times to obtain phage display antibodies with strong binding affinity.

[0052] Example 2: Monoclonal ELISA

[0053] 1) Coat an ELISA plate with 0.3 ug / ml streptavidin overnight at 4°C, treat with 2% BSA / PBS blocking buffer for 2 h, and wash 3 times with PBS.

[0054] 2) Pick single colonies from 2×YT-GA100 plates, culture with shaking until mid-log phase, add helper phage M13KO7, and incubate at 37°C for 30 minutes. Culture at 37°C with shaking at 220 rpm for 1 hour, then centrifuge at 4000 rpm for 15 minutes. Resuspend in 400 μL of 2×YT containing working concentrations of 100 μg / ml ampicillin and 50 μg / ml kanamycin, and culture overnight at 30°C with shaking at 220 rpm. Centrifuge at 4000 rpm for 15 minutes to precipitate the bacterial cells.

[0055] 3) Add 50 μL of 4% BSA / PBS to the microplate, and at the same time add 50 μL of phage supernatant. Mix well and incubate for 1 hour.

[0056] 4) Remove the liquid: wash 5 times with 0.1% PBST, then wash 3 times with PBS to remove the liquid.

[0057] 5) Dilute the HRP-labeled anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) 3000 times with 2% BSA, add 100ul to the ELISA plate, incubate for 1 hour, remove the liquid, wash 3 times with 0.1% PBST, and pat dry the residual liquid.

[0058] 6) Add 100 μL of TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL of 1M sulfuric acid to terminate the reaction. Read the OD450 on a microplate reader. (Data can be found in...) Figure 1 As shown in Table 1 and Figure 1, during the phage panning process, 50 μL of 2×YT medium containing phage virus particles was used for detection. The raw data are shown in Table 1, where the values ​​are the absorbance values ​​at 450 nm and their ratios.

[0059] Table 1. ELISA detection of Phage crude culture medium and human MSLN.

[0060]

[0061] 7) Select positive monoclonal clones for sequencing and perform nucleic acid sequencing on the V region gene of monoclonal internalization antibody No. ID 1 (G11). The CDR region was defined based on the calculation results from the Vbase2 website.

[0062] The heavy and light chain sequences of monoclonal internalizing antibody No. ID 1 (G11) are as follows:

[0063] The amino acid sequence of the antibody heavy chain variable region CDR1, SEQ ID NO.1, is GYTFTSYY;

[0064] The nucleotide sequence of the antibody heavy chain variable region CDR1, SEQ ID NO.2, is GGATAC ACC TTC ACC AGC TACTAT;

[0065] The amino acid sequence of the CDR2 variable region of the antibody heavy chain, SEQ ID NO.3, is INPSGGST;

[0066] The nucleotide sequence of the variable region CDR2 of the antibody heavy chain, SEQ ID NO.4, is ATC AAC CCT AGT GGT GGT AGCACA;

[0067] The amino acid sequence of the variable region CDR3 of the antibody heavy chain, SEQ ID NO. 5, is ARDRGTYYYGSGDLGY;

[0068] The nucleotide sequence of the variable region CDR3 of the antibody heavy chain, SEQ ID NO.6, is GCG AGAGAT CGG GGAACG TATTAC TAT GGT TCG GGG GAC TTG GGC TAC;

[0069] The amino acid sequence of the heavy chain variable region of G11, SEQ ID NO.13, is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTS YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGTYYYGSGDLGYWGQGTLV TVSS;

[0070] Heavy chain variable region nucleic acid sequence of G11 SEQ ID NO.14 is: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACG CACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCGGGGAACGTATTACTATGGTTCGGGGGACTTGGGCTACTGGGGCCAGGGGGACCCTGGTCACCGTCTCCTCA;

[0071] The amino acid sequence of the CDR1 variable region of the antibody light chain, SEQ ID NO.7, is QGISTW;

[0072] The nucleotide sequence of the antibody light chain variable region CDR1, SEQ ID NO.8, is CAG GGT ATT AGC ACC TGG;

[0073] The amino acid sequence of the CDR2 variable region of the antibody light chain, SEQ ID NO. 9, is AAS;

[0074] The nucleotide sequence of the variable region CDR2 of the antibody light chain, SEQ ID NO.10, is GCT GCA TCC;

[0075] The amino acid sequence of the CDR3 variable region of the antibody light chain, SEQ ID NO.11, is QQANSFPLT;

[0076] The nucleotide sequence of the variable region CDR3 of the antibody light chain, SEQ ID NO.12, is CAACAG GCC AAC AGT TTC CCGCTC ACC;

[0077] The amino acid sequence of the light chain variable region of G11, SEQ ID NO.15, is: DIVMTQTPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPKPLISAASSLQSGVPSR FSGSGSGTDFILTISSLQPEDSATYYCQQANSFPLTFGGGTKVEIK;

[0078] The nucleic acid sequence of the light chain variable region of G11 SEQ ID NO.16 is: GATATTGTGATGACCCAGACTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAACAGAAGCCAGGGAAAGCCCCTAAGCCCTGATCTCTGCTGCATCCA GTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCATTCTCACCATCAGTAGTCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGGCCAACAGTTTCCCGCTCACCTTCGGCGGAGGGACCAAGGTGGAAATCAAA.

[0079] Example 3: Preparation of Human MSLN Monoclonal Antibody

[0080] 1) Single colonies picked from 2×YT-GA100 plates were cultured overnight with liquid shaking, and phagemids were extracted using the plasmid extraction method.

[0081] 2) Synthesize primers and amplify the antibody gene coding region displayed by the bacteriophage using PCR.

[0082] 3) Insert the above nucleic acid fragments sequentially into the MCS region of the eukaryotic expression vector Abexp-uIgG1. Figure 3 ), which encodes a fusion protein with an N-terminal signal peptide, a mid-terminal antibody, and a C-terminal Fc tag.

[0083] 4) Shake culture 15ml of bacterial culture and use a plasmid extraction kit (purchased from Kangwei Century) to prepare sterile, endotoxin-free plasmids.

[0084] 5) Preparation of transfection complex: Take 23 μg and dilute with 0.75 mL of diluent (e.g., OPM-293CD05 medium). Simultaneously, add 70 μL of transfection reagent (e.g., PEI solution) to 0.75 mL of diluent (e.g., OPM-293CD05 medium) and mix gently. Add the PEI diluent to the plasmid diluent and immediately mix gently with a pipette. Let stand at room temperature for 15 min, avoiding disturbance.

[0085] 6) Add to 25 ml of 293F cells and their culture medium, and incubate at 80 rpm, 37°C, and 5% CO2 for 24 hours. Then add 25 ml of fresh growth medium (such as OPM-293CD05), and continue incubating at 80 rpm, 37°C, and 5% CO2 for 72 hours.

[0086] 7) Centrifuge at 10,000 rpm for 10 minutes and collect the supernatant. Incubate with equilibrated protein A affinity beads by rotation for 1 hour, then place on a magnetic rack and remove the supernatant.

[0087] 8) Wash three times with 30 ml PBS, add 5 ml 0.1 M glycine (pH 3.0) and elute for 10 minutes. Place on a magnetic rack, aspirate the supernatant and immediately neutralize with 1 M Tris-HCl buffer (pH 8.5) to obtain the purified antibody.

[0088] 9) SDS-PAGE analysis of purified antibodies, along with concentration determination, is performed. Figure 4 As shown, using reduction electrophoresis, the disulfide bonds in the antibody molecule are broken, and the molecule migrates electrophoretically in an extended single peptide chain state.

[0089] The amino acid sequence of scFv constructed by G11 SEQ ID NO.17 is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGTYYYGSGDLGYWGQGTLV TVSSGGGGSGGGGSGGGGSDIVMTQTPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPKPLISAASSLQSGVPSRFSGSGSGTDFILTISSLQPEDSATYYCQQANSFPLTFGGGTKVEIK;

[0090] The scFv nucleic acid sequence SEQ ID NO.18 constructed by G11 is as follows: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCGGGGAACGTATTACTATGGTTCGGGGGACTTGGGCTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCAGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGATATTGTGATGACCCAGACTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAACAGAAGCCAGGGAAAGCCCCTAAGCCCCTGATCTCTGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCATTCTCACCATCAGTAGTCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGGCCAACAGTTTCCCGCTCACCTTCGGCGGAGGGACCAAGGTGGAAATCAAA;

[0091] Protein sequence SEQ ID NO.19 (G11scFv-Fc) for constructing expression vector in the preparation of G11 scFv: MHSSALLCCLVLLTGVRAQVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGTYYYGSGDLGYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPKPLISAASSLQSGVPSRFSGSGSGTDFILTISSLQPEDSATYYCQQANSFPLTFGGGTKVEIKEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0092]

[0093] Example 4: Flow cytometry (FACS) detection of phage or antibody cell binding ability

[0094] 1) Cells expressing MSLN (cell binding assay, such as HeLa, CHOK1 overexpressing MSLN) or not expressing MSLN (off-target assay, such as Huh7 overexpressing HEK293) are thoroughly digested with 0.25% trypsin, digestion is terminated with serum, cells are collected by centrifugation, and single-cell suspension is prepared by gently pipetting with PBS.

[0095] 2) Wash the cells once with 10ml PBS, centrifuge at 1000rpm for 5min, then resuspend the cells in 1ml PBS and count the cells.

[0096] 3) Take 2.5 × 10 5 Cells were collected by centrifugation in 96-well cell culture plates.

[0097] 4) Add 100 μl of phage supernatant from step 2) of Example 2 or 100 μl of purified antibody from step 8) of Example 3 (10 ug / ml), mix well, and incubate at room temperature for 30 minutes to 1 hour.

[0098] 5) Collect cells by centrifugation and wash them once with 300ul PBS.

[0099] 6) For phage detection, add 100 μL of anti-M13 phage antibody diluted 1000-fold with PBS (purchased from Beijing Yiqiao Shenzhou), incubate for 30 min, wash once with PBS, add 100 μL of fluorescently labeled (e.g., FITC, APC) antibody diluted 100-fold with PBS, react at room temperature in the dark for 20 min, and detect by flow cytometry. The results are shown in Table 2 and [Table data would be inserted here]. Figure 2 The above;

[0100] Table 2: FACS detection results of Phage crude culture medium combined with human MSLN

[0101]

[0102] like Figure 2 As shown in Table 2, the left and right bars in the figure represent the binding signals of wild-type CHO-K1 and MSLN-overexpressing CHO-K1 cell lines, respectively. The values ​​in the table are the median fluorescence intensity (MFI) and its ratio.

[0103] For antibody purification detection, add 100 μl of fluorescently labeled (e.g., FITC, APC) antibody diluted 200-fold with PBS, react at room temperature in the dark for 20 min, and then detect by flow cytometry. Data are shown in Table 3. Figure 5 As shown.

[0104] Table 3: Detection of the binding performance of purified antibody to MSLN overexpressed on the cell surface

[0105]

[0106] like Figure 5 As shown in Table 3, flow cytometry was used to detect the binding of antibodies to CHO-K1 cells overexpressing MSLN. Amatuximab was used as a positive control and hIgG1 as a negative control. Each antibody was detected using wild-type cells (gray curve) and MSLN-overexpressing cells (black curve). The values ​​in the table are median fluorescence intensities (MFI).

[0107] Table 4: Detection of the binding performance of purified antibodies to MSLN on the surface of HeLa cells

[0108]

[0109] like Figure 6 As shown in Table 4, flow cytometry was used to purify and detect MSLN binding to the surface of cancer cells. Amatuximab was used as a positive control, and hIgG1 as a negative control. The three antibodies bound to HeLa cells respectively. The values ​​in the table are median fluorescence intensity (MFI), or the ratio of the MFI of the antibody to the MFI of hIgG1.

[0110] Table 5: Off-target detection of purified antibody binding to Huh7 (left) and HEK293 (right) cells

[0111]

[0112] like Figure 7 As shown in Table 5, flow cytometry was used for off-target detection, with hIgG1 serving as the negative control (CK). Both the test antibody and hIgG1 were measured twice. The values ​​in the table represent the median fluorescence intensity (MFI), or the ratio of the antibody's MFI to the hIgG1's MFI.

[0113] Example 5: Detection of purified antibody levels using ELISA (enzyme-linked immunosorbent assay).

[0114] 1) Dilute the antigen with coating buffer and coat overnight at 4°C.

[0115] 2) Remove the coating solution the next day and wash with PBS.

[0116] 3) Add blocking buffer and block at 37°C for 1 hour, then wash with PBS. Simultaneously, serially dilute the antibody starting at 10 μg / ml to obtain 16 antibody solutions with different concentrations.

[0117] 4) Add it to the blocked ELISA plate, bind at 30°C for 30 min, and wash with PBST.

[0118] 5) Add HRP-labeled anti-human IgG secondary antibody (purchased from Biolegend) at a dilution ratio of 1:10000, bind at 30℃ for 30 min, and wash with PBST.

[0119] 6) Add 100 μL of TMB chromogenic solution, incubate at 37°C for 10 min or until the blue color is fully developed, then add 100 μL of 1M sulfuric acid to stop the reaction. Read the OD450 on a microplate reader. Data as follows: Figure 8 As shown in Table 6, the antibodies were serially diluted 2-fold. hIgG1 was used as a negative control and set up for parallel detection. Since it does not bind, there is no linear relationship and therefore no EC50 value. The values ​​in the table are absorbance values ​​at 450 nm.

[0120] Table 6: EC50 detection of antibody binding to MSLN molecules on solid-phase support surface

[0121]

[0122] Example 6: Antibody internalization function detection

[0123] 1) Digest OVCAR3 cells with 0.25% Typsin-EDTA, centrifuge and discard the supernatant, then resuspend in complete culture medium.

[0124] 2) Add 75 μL of cell suspension to a 96-well plate, ensuring each well contains 1.5 × 10⁶ cells. 5 Each cell.

[0125] 3) Add 25 μL of antibody at the EC80 working concentration (i.e., 80% of the saturation concentration) and mix well. Divide each antibody into two plates, and prepare one well for each plate. Incubate at 4°C for 1 hour.

[0126] 4) Add PBS to a final volume of 250 μL, centrifuge at 500 g for 5 min at room temperature, discard the supernatant and retain the cell pellet, blot away any remaining liquid with absorbent paper, gently tap the experimental plate to disperse the cells, wash again with PBS to disperse the cells. This step removes excess unbound antibodies.

[0127] 5) Resuspend the cells in 100 μL of basal culture medium. Incubate one plate at 37°C for 2 hours and the other at 4°C for 2 hours. During incubation at 37°C, antibodies will transfer into the cells, while incubation at 4°C will inhibit internalization.

[0128] 6) Add 100 μL of fluorescent secondary antibody diluted with PBS (the concentration of the secondary antibody before addition should be twice the working concentration), and incubate at 4°C for 0.5 hours.

[0129] 7) Centrifuge at 500g at room temperature for 5 minutes, discard the supernatant and retain the cell pellet, absorb the residual liquid with absorbent paper, gently tap the experimental plate to disperse the cells, wash again with PBS to disperse the cells.

[0130] 8) Resuspend in 100 μL PBS, perform flow cytometry analysis, and process data using Graphpad Prism. Data is as follows: Figure 9 Table 7 Figure 10 As shown.

[0131] Table 7: Detection of the internalization properties of purified antibodies

[0132]

[0133] like Figure 9 As shown in Table 7, after the antibody binds to the cell, the antibody bound to the cell surface is detected by fluorescent secondary antibody. The values ​​in the table are the median fluorescence intensity (MFI).

[0134] like Figure 10 As shown in Table 7, the average MFI of the tested samples (hIgG1, Amatuximab, and seq No. ID1) measured at 37℃ is divided by the average MFI measured at 4℃ for each sample, which is defined as relative internalization. A higher value indicates a weaker internalization ability. The values ​​in the table are median fluorescence intensities (MFI).

[0135] Example 7: Detection of the cell-killing efficacy of antibody internalization-mediated toxins

[0136] 1) Digest 786-0 cells with 0.25% Typsin-EDTA, centrifuge and discard the supernatant, then resuspend in complete culture medium.

[0137] 2) Add 90 μL of cell suspension to a 96-well plate, so that there are 2500 cells in each well.

[0138] 3) Dilute the antibody to a 10-fold EC80 value, add 45 nM FabFc-ZAP human reagent (working concentration 4.5 nM; purchased from Advanced Targeting Systems), take 10 μL of the mixture, add it to the cells in step 2), and mix well.

[0139] 4) Incubate at 37℃ in a 5% CO2 incubator for 72 hours.

[0140] 5) Equilibrate the CCK8 reagent kit to room temperature for 15 minutes. Remove the 96-well cell culture plate, add the CCK8 reagent, and incubate at 37°C in the dark for 1 to 3 hours. Read the OD450 value using a microplate reader. Data as follows: Figure 11 As shown in Table 8.

[0141] Table 8: Cell-killing efficacy of purified antibody internalization-mediated toxins

[0142]

[0143] like Figure 11 As shown in Table 8, after co-incubation with antibody, toxin, and cells, the cell viability measured by the CCK8 assay was lower than that after co-incubation with antibody and cells alone. The figure also shows that the cytotoxic effect of the toxin itself was not significant, while the effect of the ADC was obvious. The values ​​in the table represent the absorbance at 450 nm (A450). Value A is defined as the absorbance at 450 nm measured after simultaneous treatment of cells with antibody Amatuximab or antibody No. ID 1 and toxin, divided by the absorbance at 450 nm measured after treatment with only antibody Amatuximab or antibody No. ID 1. Value B is defined as the absorbance at 450 nm measured after toxin treatment of cells, divided by the absorbance at 450 nm measured in untreated cells. The A / B ratio represents the relative cytotoxicity.

Claims

1. An anti-MSLN monoclonal internalization antibody, said antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO.5; The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO.

11.

2. The monoclonal internalizing antibody according to claim 1, characterized in that, The antibody specifically binds to the MSLN antigen on the surface of cancer cells.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal internalization antibody as described in any one of claims 1-2.

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, The host cell includes the expression vector as described in claim 4.

6. A method for preparing an anti-MSLN monoclonal internalization antibody, characterized in that, The method includes culturing host cells comprising the host cells of claim 5.

7. A reagent kit, characterized in that, The kit includes the monoclonal internalizing antibody as described in claim 1.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal internalizing antibody of claim 1.

9. The use of the monoclonal internalizing antibody of claim 1 in the preparation of anticancer or cancer detection reagents or drugs, wherein the cancer or cancer is ovarian cancer or kidney cancer.

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