CN113788896A

CN113788896A - anti-MSLN monoclonal internalization antibody and preparation method and application thereof

Info

Publication number: CN113788896A
Application number: CN202110981467.9A
Authority: CN
Inventors: 张军锋; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-12-14
Also published as: WO2023024444A1; CN115819597A

Abstract

The invention discloses an anti-MSLN monoclonal internalization antibody, a preparation method and application thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises a heavy chain variable region sequence shown in SEQ ID NO: 1, CDR1 of the heavy chain variable region; as shown in SEQ ID NO: 3, CDR2 of the heavy chain variable region; as shown in SEQ ID NO: 5, CDR3 of the heavy chain variable region; the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 7, light chain variable region CDR 1; as shown in SEQ ID NO: 9, light chain variable region CDR 2; and as shown in SEQ ID NO: 11, CDR3 of the light chain variable region shown in fig. The antibody of the invention has good specificity and higher affinity, can be combined with cell surface expression, can be efficiently transported to cells by the cells, carries toxin molecules to synchronously enter the cells so as to kill the cells, and can be widely applied to the drugs for tumor immunotherapy.

Description

anti-MSLN monoclonal internalization antibody and preparation method and application thereof

Technical Field

The invention relates to a monoclonal antibody, a preparation method and application thereof, in particular to an anti-MSLN monoclonal internalization antibody, and a preparation method and application thereof.

Background

Mesothelin (MSLN) is a 40KD glycoprotein located on the cell surface and anchored to the cell membrane by glycosylphosphatidylinositol. The mesothelin gene encodes a 69kDa precursor protein that is hydrolyzed by furin-like converting enzyme into two chains, a membrane-bound protein with about 40kD at the C-terminus is mature mesothelin, and a fragment called megakaryocyte-promoting factor (MPF) with about 30kD at the N-terminus is shed and released extracellularly. MPF and membrane anchor MSLN are both N-glycosylated, MPF can promote the formation of megakaryocyte clone in vitro, and membrane anchor MSLN can interact with MUC16 (also called CA125) and play an important role in the process of cell adhesion, so that membrane anchor MSLN is selected as a target point in the current targeted therapy, and the current MSLN is exclusively the C-terminal 40KD fragment of MSLN, namely membrane anchor MSLN.

Under normal conditions, MSLN is only expressed in the mesothelial tissues such as pleura, peritoneum, pericardium and the like, but is not expressed in other tissues, and the expression spectrum is extremely narrow. However, strong expression of MSLN is very common in cancerous tissues, and high expression level has been detected in various solid tumor tissues such as mesothelioma, non-small cell lung cancer, ovarian cancer, endometrial cancer, cervical cancer, pancreatic cancer, etc., and MSLN is expressed even in esophageal cancer, metastatic triple negative breast cancer and renal cancer. As for the cancer cell lines, it is known that MSLN is highly expressed in three ovarian cancer cell lines of HO-8910, HEY-T30, OVCAR3 (particularly HO-8910), metastatic pancreatic cancer cell line AsPC1 and cervical cancer cell line Hela, and that it is extremely low expressed in two ovarian cancer cell lines of SKOV3, 3AO and lung adenocarcinoma cell line A549, while it is not expressed in human liver cancer cell line Huh 7.

At the cellular level, MSLN is involved in cellular biological processes such as (cancer) cell proliferation, apoptosis, adhesion, etc., and adversely affects the effects of chemotherapy. For example, SKOV3 cells with downregulated MSLN expression have reduced tumorigenicity when used in mouse transplantation tumor construction, and may also result in increased apoptosis and reduced metastasis of cancer tissues. Blocking the binding of MSLN to MUC16 results in a decrease in cell adhesion. Following downregulation of MSLN expression, cancer tissues have increased sensitivity to cisplatin and paclitaxel. It was further observed that overexpression of MSLN caused overexpression of metalloproteinase 9, thereby promoting migration and infiltration of tumor cells. Therefore, it is presumed that MSLN interacts with MUC16 to cause expression of metalloproteinase 9 and other proteins, and further cause corresponding changes in processes such as cell proliferation, adhesion, apoptosis, etc., and plays an important role in invasion of tumor peritoneal metastasis, and can promote anti-apoptosis of tumor cells.

The MSLN is hardly expressed in normal cells (only low expression in pleura, peritoneum and pericardium) and strongly expressed in various cancer tissues, so that the MSLN becomes a good target of targeted therapy, and the MSLN determines that the targeted therapy taking the MSLN as the target naturally has the potential advantages of low off-target effect and wide adaptation diseases. For example, MSLN is expressed in 60-90% of lung cancer tissues, 20% of the tissues are high in expression level but not expressed in normal lung tissues, and the prognosis of lung adenocarcinoma with high expression of MSLN is poor, which indicates that MSLN is a potential target for treating lung cancer. Experiments prove that after CART taking MSLN as a target spot is returned to tumor-bearing mice through tail veins, the tumor volume increase caused by subcutaneous inoculation of lung adenocarcinoma tissues with high expression of MSLN is obviously inhibited.

In view of the characteristics of wide indication and low possible off-target effect of the MSLN-targeted therapy, the MSLN-targeted therapy scheme is developed in multiple directions, and includes 12 drugs such as recombinant monoclonal monospecific antibody drugs, bispecific antibodies, synthetic proteins, antibody-conjugated drugs (toxins or radioisotopes), vaccines, and adoptive immune cell therapy (antibody-based drugs 5). Among them, Amatuximab (MORAB-009), developed by the national cancer institute, has completed a second phase clinical trial for the treatment of malignant mesothelioma.

As for the antibodies currently under development, although many known monoclonal antibodies to mesothelin are available, none can exhibit Complement Dependent Cytotoxicity (CDC) against tumor cells. The antibody specifically binds to an antigen on the cell surface, and the Fc region recruits C1q, which activates the classical pathway of complement activation, eventually forming a membrane-tapping complex on the surface of the antigen-expressing cell, causing lysis of the target cell. This effect is seen to be helpful in cancer treatment. It is a difficult matter to obtain MSLN-targeted antibodies with strong CDC effects using traditional hybridoma technology. For example, the antibody obtained by the traditional method has poor tissue penetration, insufficient treatment effect, strong immunogenicity, requirement of humanized modification, poor stability, and high transportation and storage requirements. In conclusion, although the existing traditional antibodies play an extremely important role in the treatment/detection of diseases, the existing antibodies targeting MSLN have obvious defects.

The number of MSLN target drugs currently in clinical stage is 12, and the number of antibody drugs including Amatuximab is 5. These antibodies are human-mouse chimeric antibodies or humanized antibodies, both derived from mice, and obtained by hybridoma technology. Current therapies targeting MSLN have cell therapy and naked antibodies with limited clinical efficacy, and both approaches are nearly ineffective, particularly for solid tumors. It is widely recognized by developers that antibody-drug conjugates (ADCs) will be one of the hopes for future cancer therapy against this target. In addition, these antibodies are not fully human antibodies, and have problems of higher immunogenicity, lower safety, and possibly lower in vivo stability, compared to fully human antibodies.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide an anti-MSLN monoclonal internalization antibody with cell internalization function and CDC function; another objective of the invention is to provide a method for preparing an anti-MSLN monoclonal internalizing antibody; another object of the invention is to provide the use of an anti-MSLN monoclonal internalizing antibody.

The technical scheme is as follows: the anti-MSLN monoclonal internalizing antibody of the invention comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, CDR1 of the heavy chain variable region; as shown in SEQ ID NO: 3, CDR2 of the heavy chain variable region; and as shown in SEQ ID NO: 5, CDR3 of the heavy chain variable region.

The nucleotide sequence of SEQ ID NO: 1 is GYTFTSYY; the nucleotide sequence of SEQ ID NO: 3 is INPSGGST; the nucleotide sequence of SEQ ID NO: and 5 is ARDRGTYYYGSGDLGY.

Further, the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 7, light chain variable region CDR 1; as shown in SEQ ID NO: 9, light chain variable region CDR 2; and as shown in SEQ ID NO: 11, CDR3 of the light chain variable region shown in fig.

The nucleotide sequence of SEQ ID NO: 7 is QGISTW; the nucleotide sequence of SEQ ID NO: 9 is AAS; the nucleotide sequence of SEQ ID NO: 11 is QQANSFPLT.

Further, the antibody is a fully human antibody.

Further, the antibody specifically binds to a cancer cell surface MSLN antigen.

In another aspect, the invention provides a nucleic acid molecule encoding a monoclonal internalizing antibody as described above.

In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule described above.

In another aspect, the present invention provides a host cell comprising the expression vector described above.

In another aspect, the invention provides a method of making an anti-MSLN monoclonal internalizing antibody, comprising culturing a host cell comprising the above. The specific method comprises the following steps:

taking a commercial recombinant human MSLN biotin label protein and a phage display antibody library as starting materials; incubating MSLN biotin-labeled protein and streptavidin-coated magnetic beads together, fixing the MSLN on the magnetic beads through interaction between the streptavidin and the biotin, incubating the magnetic beads with an antibody library, washing off unbound/weakly bound phage, and eluting phage bound with the MSLN protein; repeating the steps for 3 times, changing the use amount and washing conditions of the MSLN biotin tag protein each time, gradually eliminating the phage with weak binding and keeping more phage with strong binding as much as possible; infecting host bacteria by using strongly combined phage, coating a plate and culturing overnight to obtain a monoclonal colony, namely performing monoclonality on the phage antibody capable of being combined with MSLN; performing ELISA screening by using the monoclonal culture supernatant, and performing nucleic acid sequence determination on the monoclonal;

connecting the coding sequences of a signal peptide, an anti-MSLN antibody and human IgG1 Fc, reading in frame, and constructing a mammalian expression vector; transfecting 293F cells, performing shake culture for 5 days, then harvesting supernatant, purifying the supernatant by using protein A magnetic bead affinity chromatography to obtain fusion protein, and identifying the binding property of the fusion protein and MSLN; culturing target cells, co-incubating the antibodies with the target cells at 4 ℃, and performing two groups in parallel; one group was transferred to 37 ℃ after incubation at 4 ℃ and incubation continued at 4 ℃ for the other group. And after the incubation is finished, adding a fluorescence labeling detection antibody, incubating at 4 ℃ and detecting the fluorescence antibody by using flow cytometry, namely obtaining the anti-MSLN antibody of which the cell surface is captured by the MSLN. Target cells were plated in 96-well plates and cultured overnight to allow for adequate adherence. Adding antibody and/or toxin reagent, culturing cells and observing the growth characteristics of the cells; after the culture was completed, the cell viability was measured.

In another aspect, the invention provides a kit comprising a monoclonal internalizing antibody as described above.

In another aspect, the invention provides a pharmaceutical composition comprising the monoclonal internalizing antibody of claim.

The monoclonal internalization antibody is applied to preparation of an antibody drug conjugate.

The monoclonal internalization antibody is applied to preparation of an anti-cancer or cancer detection reagent, product or medicament.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the antibody of the invention has good specificity and higher affinity, can be combined with cell surface expression, can be efficiently transported to cells by the cells, carries toxin molecules to synchronously enter the cells so as to kill the cells, and can be widely applied to the drugs for tumor immunotherapy.

Drawings

FIG. 1 shows the results of ELISA detection of the combination of the coarse culture solution of phase and human MSLN in example 2;

FIG. 2 shows the results of FACS detection of the combination of the coarse culture solution of phage and human MSLN in example 4;

FIG. 3 is a schematic diagram of the structure of the MSLN antibody expression vector in example 3;

FIG. 4 is SDS-PAGE electrophoresis of the purified antibody of example 3;

FIG. 5 is a graph showing the binding performance of the purified antibody of example 4 to MSLN overexpressed on the cell surface;

FIG. 6 is a graph showing the MSLN binding performance of the purified antibody of example 4 to the HeLa cell surface;

FIG. 7 is an off-target assay for binding of the purified antibody of example 4 to Huh7, HEK293 cells;

FIG. 8 is the EC50 detection of the binding of the antibody of example 5 to MSLN molecules on the surface of a solid support;

FIG. 9 shows the internalization performance of the purified antibody of example 6;

FIG. 10 is the relative internalization performance of the purified antibody of example 6;

FIG. 11 is a graph of the killing potency of purified antibody internalization-mediated toxins on cells in example 7.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

Example 1: panning of phage display fully human antibody library

1) Activation of host bacterium TG 1: preparation of mini agar Medium plate [1 XM 9 salt, 2% glucose, 2mM MgSO₄，0.1mM CaCl₂1mM vitamin B1]The TG1 was streaked overnight in a 37 ℃ incubator.

2) Magnetic bead washing and sealing: 50ul of magnetic beads (purchased from Invitrogen) were pipetted and placed on a magnetic rack, the liquid was pipetted after adsorption, 1ml of PBS was resuspended and washed twice, and 1ml of a blocking agent consisting of 1.5% nonfat dry milk and 1.5% BSA (the concentration of blocking agent gradually increased during the second and third rounds of panning) was used for 1 hour to remove the liquid.

3) Antigen binding: human MSLN protein (purchased from Acro biosystems, and the antigen concentration was gradually decreased in each subsequent round of panning) was diluted to 1ml in PBS (pH7.2-7.4) at a concentration of 16ug/ml, the beads were resuspended, and the cells were incubated for 1 hour with rotation.

4) And (4) closing the library: taking 10 simultaneous with antigen binding to magnetic beads¹¹pfu phage virus particles (from the original antibody library or panning amplification products), with 1ml of 1.0% skim milk powder + 1.0% BSA blocking agent spin incubation for 1 hours.

5) Phage binding: and (5) placing the magnetic beads on a magnetic frame, and removing the liquid. The sealed pool was added with magnetic beads, resuspended and incubated for 1 hour with rotation, and the liquid removed.

6) Washing: washed with 1ml of PBST [0.01M PBS (pH7.4), 0.1% Tween-20 (second and third rounds of Tween-20 concentrations 0.2%, 0.3%, respectively) ], and then washed with 0.01M PBS (pH7.4).

7) And (3) elution: the liquid was aspirated, eluted with 300ul of 0.2M glycine-hydrochloric acid (pH2.2) for 10 minutes, mixed with 20ul of a neutralizing solution [1M Tris-Cl (pH9.0) ] and stored temporarily at 4 ℃.

8) And (3) measuring the titer: 2ul and 0.2ul (2 ul after the stock solution was diluted 10 times with 2 XYT medium) and 0.02ul (2 ul after the stock solution was diluted 100 times with 2 XYT medium) were mixed with 0.2ml of TG1 in the middle logarithmic phase (OD 600. about.0.5), incubated at room temperature for 30 minutes, spread on 2 XYT-GA 100 (containing 2% glucose and 100ug/ml ampicillin) plates, cultured overnight at 37 ℃ and the number of clones on about 50 plates was counted, and the titer was calculated from the dilution factor.

9) Phage amplification: while panning was performed, TG1 single clones on mini agar plates were picked and inoculated into 10ml of 2 XYT medium, and cultured at 37 ℃ with shaking at 250rpm until mid-log phase (OD 600. RTM. 0.5). 200ul of eluted product from panning was added and incubated at 37 ℃ for 30 minutes. The helper phage M13KO7 was added and incubated at 37 ℃ for an additional 30 minutes and incubated at 37 ℃ for 1 hour with shaking at 250 rpm. The supernatant was centrifuged off and resuspended in 20ml of 2 XYT containing 100ug/ml ampicillin and 50ug/ml kanamycin at 30 ℃ overnight with shaking at 220 rpm.

10) Phage precipitation: the cells were removed by centrifugation at 10000rpm for 15 minutes, and 1/5 volumes of 2.5M NaCl/20% PEG8000 were added to the supernatant, which was then cooled in ice for 2 hours. The phage pellet was centrifuged at 10000rpm for 10 minutes, the residue was removed and 0.2ml of 0.01M PBS (pH7.4) was added to resuspend the pellet, and the titer was determined as above.

11) Repeating the steps 2) -10) for two or three times to obtain the phage display antibody with strong binding force.

Example 2: monoclonal ELISA

1)0.3ug/ml streptavidin was coated onto the elisa plate overnight at 4 ℃, treated with 2% BSA/PBS blocking solution for 2h, and washed 3 times with PBS.

2) Single colonies were picked from 2 XYT-GA 100 plates, cultured with shaking to the middle logarithmic phase, supplemented with the helper phage M13KO7, and incubated at 37 ℃ for 30 minutes. The cells were cultured at 37 ℃ for 1 hour with shaking at 220rpm, and centrifuged at 4000rpm for 15 minutes. 400ul containing working concentration 100ug/ml ampicillin and working concentration 50ug/ml kanamycin 2 XYT heavy suspension, 30 degrees C, 220rpm shaking culture overnight. The cells were centrifuged at 4000rpm for 15 minutes to precipitate the cells.

3) 50ul of 4% BSA/PBS was added to the ELISA plate, and 50ul of phage supernatant was added, mixed well and incubated for 1 hour.

4) Remove liquid, wash 5 times with 0.1% PBST, wash 3 times with PBS, remove liquid.

5) An HRP-labeled anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) is diluted 3000 times with 2% BSA, 100ul of the diluted antibody is added into an enzyme label plate, the plate is incubated for 1 hour, the liquid is removed, the plate is washed 3 times with 0.1% PBST, and the residual liquid is drained.

6) 100ul of TMB developing solution is added, the mixture is incubated at 37 ℃ for 10min or until blue color is fully developed, 100ul of 1M sulfuric acid is added to stop the reaction, and OD450 is read on a microplate reader. The data are shown in FIG. 1 and Table 1, and as shown, 50ul of 2 XYT medium containing phage virus particles was used for detection during phage panning. The raw data are shown in Table 1, where the values are the absorbance at 450nm and its ratio.

TABLE 1 phase crude culture solution and humanized MSLN combined ELISA detection

7) And (3) selecting positive monoclonal for sequencing, and performing nucleic acid sequence determination on the V region gene of the monoclonal internalization antibody No. ID 1.

The sequences of the heavy and light chains of monoclonal internalizing antibody No. id1 are as follows:

antibody heavy chain variable region CDR1 amino acid sequence SEQ ID NO: 1 is GYTFTSYY;

the nucleotide sequence of CDR1 in heavy chain variable region of antibody is shown in SEQ ID NO: 2 is GGA TAC ACC TTC ACC AGC TAC TAT;

antibody heavy chain variable region CDR2 amino acid sequence SEQ ID NO: 3 is INPSGGST;

the nucleotide sequence of CDR2 in heavy chain variable region of antibody is shown in SEQ ID NO: 4 is ATC AAC CCT AGT GGT GGT AGC ACA;

antibody heavy chain variable region CDR3 amino acid sequence SEQ ID NO: 5 is ARDRGTYYYGSGDLGY;

the nucleotide sequence of CDR3 in heavy chain variable region of antibody is shown in SEQ ID NO: 6 is GCG AGA GAT CGG GGA ACG TAT TAC TAT GGT TCG GGG GAC TTG GGC TAC;

antibody light chain variable region CDR1 amino acid sequence SEQ ID NO: 7 is QGISTW;

antibody light chain variable region CDR1 nucleotide sequence SEQ ID NO: 8 is CAG GGT ATT AGC ACC TGG;

antibody light chain variable region CDR2 amino acid sequence SEQ ID NO: 9 is AAS;

antibody light chain variable region CDR2 nucleotide sequence SEQ ID NO: 10 is GCT GCA TCC;

antibody light chain variable region CDR3 amino acid sequence SEQ ID NO: 11 is QQANSFPLT;

antibody light chain variable region CDR3 nucleotide sequence SEQ ID NO: 12 is CAA CAG GCC AAC AGT TTC CCG CTC ACC.

Example 3: preparation of human MSLN monoclonal antibody

1) Single colonies picked from 2 XYT-GA 100 plates were cultured overnight with shaking in liquid, and phagemids (phagemid) were extracted by the plasmid extraction method.

2) Synthesizing primer, PCR amplifying the antibody gene coding region displayed by phage.

3) The above nucleic acid fragments were inserted into the MCS region of the eukaryotic expression vector Abexp-uIgG1 in the order (FIG. 3) to encode a fusion protein having a signal peptide at the N-terminus, an antibody at the middle, and an Fc tag at the C-terminus.

4) 15ml of the bacterial solution was cultured by shaking, and a plasmid extraction kit (purchased from Kong, century) was used to prepare endotoxin-free sterile plasmids.

5) Preparation of transfection complex: 23ug was diluted with 0.75mL of a diluent (e.g., OPM-293CD05 medium) and 70. mu.L of a transfection reagent (e.g., PEI solution) was added to the 0.75mL of the diluent (e.g., OPM-293CD05 medium) and gently mixed. The PEI diluent was added to the plasmid diluent, immediately mixed gently with a gun, and allowed to stand at room temperature for 15min to avoid disturbance.

6) Adding into 25ml 293F cell and its culture solution at 80rpm, 37 deg.C, and 5% CO₂Culturing under the conditions of 24 hr, adding 25mL of fresh growth medium (such as OPM-293CD05), 80rpm, 37 deg.C, and 5% CO₂The culture was continued for 72 hours.

7) Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was collected. And rotationally incubating the mixture with equilibrated proteinA affinity magnetic beads for 1 hour, placing the mixture on a magnetic frame, and removing supernatant.

8) The mixture was washed 3 times with 30ml of PBS, eluted with 5ml of 0.1M glycine (pH 3.0) for 10 minutes, placed on a magnetic frame, and the supernatant was immediately aspirated to neutrality with 1M Tris-HCl buffer (pH 8.5) to obtain a purified antibody.

9) The purified antibody was examined by SDS-PAGE and the concentration was measured, and as shown in FIG. 4, by reduction electrophoresis, disulfide bonds in the antibody molecules were opened and the molecules were electrophoretically migrated in an extended single-peptide chain state.

Example 4: flow-through detection of phase or antibody cell binding Capacity (FACS)

1) MSLN expression (cell binding assays, e.g., Hela, CHOK1 overexpressing MSLN) or no expression (off-target assays, e.g., overexpressing Huh7, HEK293) was digested extensively with 0.25% pancreatin, serum was stopped, cells were harvested by centrifugation, and single cell suspensions were prepared by gentle pipetting with PBS.

2) Cells were washed 1 time with 10ml PBS, centrifuged at 1000rpm for 5min, then suspended in 1ml PBS and counted.

3) Take 2.5X 10⁵The cells were collected by centrifugation in 96-well cell culture plates.

4) Add 100. mu.l of the phage supernatant from step 2) of example 2 or 100. mu.l of 10ug/ml of the purified antibody from step 8) of example 3, mix well and incubate at room temperature for 30min to 1 hour.

5) Cells were harvested by centrifugation and washed 1 time with 300ul PBS.

6) If the detection is for phase detection, 100ul of anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) diluted by 1000 times with PBS is added, the incubation is carried out for 30min, the PBS is washed once, 100ul of fluorescence labeling (such as FITC and APC) antibody diluted by 100 times with PBS is added, the reaction is carried out for 20min in a dark place at room temperature, and the detection is carried out by a flow cytometer, wherein the result data are shown in Table 2 and FIG. 2;

table 2: FACS detection result combining phage rough culture solution and human source MSLN

As shown in FIG. 2 and Table 2, the left and right histograms in the graph represent the binding signals to wild-type CHO-K1 and MSLN overexpressing CHO-K1 cell lines, respectively, and the values in the tables are the Median Fluorescence Intensity (MFI) and its ratio.

For the detection of purified antibody, 100 μ l of fluorescence labeled (e.g., FITC, APC) antibody diluted 200 times with PBS was added, and the mixture was subjected to light shielding reaction at room temperature for 20min and detected by flow cytometry, and the data are shown in Table 3 and FIG. 5.

Table 3: detection of binding performance of purified antibody and MSLN overexpressed on cell surface

As shown in FIG. 5 and Table 3, flow cytometry was used for antibody binding assays with CHO-K1 overexpressing MSLN, where Amatuximab was the positive control and hIgG1 was the negative control, and each antibody was assayed using wild-type cells (grey curve) and MSLN overexpressing cells (black curve), with the values in the tables being the Median Fluorescence Intensity (MFI).

Table 4: detection of binding performance of purified antibody and Hela cell surface MSLN

As shown in fig. 6 and table 4, flow cytometry was used to purify MSLN binding assays to cancer cell surfaces, where Amatuximab was a positive control, hIgG1 was a negative control, and three antibodies were each bound to Hela cells. The values in the table are the Median Fluorescence Intensity (MFI), or the ratio of MFI of the antibody to the MFI of hIgG 1.

Table 5: purified antibody binding off-target detection with Huh7 (left) and HEK293 (right) cells

As shown in fig. 7 and table 5, flow cytometry was used for off-target detection, in which hIgG1 was the negative control CK, and both the test antibody and hIgG1 were tested in duplicate 2 times. The values in the table are the Median Fluorescence Intensity (MFI), or the ratio of MFI of the antibody to the MFI of hIgG 1.

Example 5: purified antibody ELISA level (enzyme linked immunosorbent assay) binding assay

1) The antigen was diluted with coating buffer and coated overnight at 4 ℃.

2) The next day, the coating solution was removed and washed with PBS.

3) Add blocking solution to block for 1h at 37 ℃ and wash with PBS. Simultaneously, the antibody is diluted from 10ug/ml in a multiple ratio to obtain 16 antibody solutions with gradient concentration.

4) Adding the mixture into a closed enzyme label plate, binding for 30min at 30 ℃, and washing by PBST.

5) HRP-labeled anti-human IgG secondary antibody (purchased from Biolegend) was added at 1:10000 dilution, combined for 30min at 30 ℃ and washed with PBST.

6) 100ul of TMB developing solution is added, the mixture is incubated at 37 ℃ for 10min or until blue color is fully developed, 100ul of 1M sulfuric acid is added to stop the reaction, and OD450 is read on a microplate reader. Data as shown in figure 8 and table 6, antibodies were diluted in a 2-fold gradient. hIgG1 was set as a negative control for parallel detection and had no EC50 value since it did not bind and therefore did not correlate, with the values in the table being absorbance at 450 nm.

Table 6: EC50 detection of antibody binding to MSLN molecule on solid phase carrier surface

Example 6: antibody internalization function detection

1) OVCAR3 cells were digested with 0.25% Typsin-EDTA, centrifuged to discard the supernatant, and resuspended in complete medium.

2) 75ul of cell suspension was added to a 96-well plate and allowed to stand at 1.5X 10 per well⁵And (4) cells.

3) 25ul of antibody was added at the working concentration of EC80 (i.e., 80% of saturation concentration) and mixed well. Each antibody was divided into two plates, one well for each plate. Incubate at 4 ℃ for 1 hour.

4) Adding PBS to the final volume of 250ul, centrifuging at 500g for 5min at normal temperature, discarding the supernatant to retain cell precipitate, completely sucking the residual liquid with absorbent paper, gently patting the test plate to disperse the cells, washing with PBS again to disperse the cells. This step removes excess unbound antibody.

5) The cells were resuspended in 100ul of basal medium, one plate was incubated at 37 ℃ for 2 hours and the other plate was incubated at 4 ℃ for 2 hours. Antibody was transferred to cells during incubation at 37 ℃ and internalization was inhibited at 4 ℃.

6) 100ul of fluorescent secondary antibody diluted with PBS was added (the concentration of the primary antibody was 2-fold of the working concentration), and the mixture was incubated at 4 ℃ for 0.5 hour.

7) Centrifuging at room temperature for 5min at 500g, discarding the supernatant to retain cell precipitate, removing the residual liquid with absorbent paper, gently patting the test plate to disperse cells, washing with PBS once again to disperse cells.

8) Add 100ul PBS heavy suspension, flow cytometry machine detection, Graphpad Prism processing data. The data are shown in fig. 9, table 7, and fig. 10.

Table 7: purified antibody internalization performance detection

As shown in fig. 9 and table 7, after the antibody binds to the cell, the test antibody bound to the cell surface was detected by a fluorescent secondary antibody, and the numerical value in the table is the Median Fluorescence Intensity (MFI).

As shown in FIG. 10 and Table 7, the relative internalization was defined as the mean MFI values measured at 37 ℃ for the test samples hIgG1, Amatuximab, and seq No. ID1 divided by the mean MFI values measured at 4 ℃ for each, with higher values indicating less internalization. The values in the table are Median Fluorescence Intensity (MFI).

Example 7: detection of cell killing efficacy of antibody internalization mediated toxin

1) 786-0 cells were digested with 0.25% Typsin-EDTA, centrifuged to discard the supernatant, and resuspended in complete medium.

2) 90ul of cell suspension was added to a 96-well plate and 2500 cells per well were allowed.

3) The antibody was diluted to a 10-fold EC80 value, to which was added FabFc-ZAP human reagent at a concentration of 45nM (working concentration of 4.5 nM; purchased from Advanced Targeting Systems), taking 10ul of the mixture, adding to the cells in step 2), and mixing.

4)37℃，5％CO₂The incubator was incubated for 72 hours.

5) The CCK8 kit was equilibrated at room temperature for 15 min. And taking out the 96-well cell culture plate, adding a CCK8 reagent, incubating for 1 to 3 hours in an incubator at 37 ℃ in a dark place, and reading the OD450 value by using an enzyme-labeling instrument. The data are shown in FIG. 11 and Table 8.

Table 8: killing potency of purified antibody internalization-mediated toxins on cells

As shown in fig. 11 and table 8, after the antibody, toxin and cell were incubated, the activity of the cell measured by CCK8 method was lower than that of the antibody and cell alone, and it can also be seen from the figure that the killing of the toxin itself was not significant, and the effect of ADC was significant. The table shows the absorbance at 450nm (A450). The value A is the absorbance value of 450nm measured after the cell is treated by the antibody Amatuximab or antibody No. ID1 and toxin simultaneously, divided by the absorbance value of 450nm measured after the cell is treated by the antibody Amatuximab or antibody No. ID1 only, and the value B is the absorbance value of 450nm measured after the cell is treated by the toxin, divided by the absorbance value of 450nm measured by the untreated cell, and the value A/B is the relative killing.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Arg Asp Arg Gly Thr Tyr Tyr Tyr Gly Ser Gly Asp Leu Gly Tyr

1 5 10 15

<210> 6

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcgagagatc ggggaacgta ttactatggt tcgggggact tgggctac 48

<210> 7

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gln Gly Ile Ser Thr Trp

1 5

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagggtatta gcacctgg 18

<210> 9

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ala Ala Ser

1

<210> 10

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gctgcatcc 9

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gln Gln Ala Asn Ser Phe Pro Leu Thr

1 5

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caacaggcca acagtttccc gctcacc 27

Claims

1. An anti-MSLN monoclonal internalizing antibody, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1, CDR1 of the heavy chain variable region; as shown in SEQ ID NO: 3, CDR2 of the heavy chain variable region; and as shown in SEQ ID NO: 5, CDR3 of the heavy chain variable region.

2. The monoclonal internalizing antibody according to claim 1, wherein said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, light chain variable region CDR 1; as shown in SEQ ID NO: 9, light chain variable region CDR 2; and as shown in SEQ ID NO: 11, CDR3 of the light chain variable region shown in fig.

3. The monoclonal internalizing antibody according to claim 1, wherein said antibody specifically binds to a cancer cell surface MSLN antigen.

4. A nucleic acid molecule encoding the monoclonal internalizing antibody of any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. A method of making an anti-MSLN monoclonal internalizing antibody, comprising culturing a host cell of claim 6.

8. A kit comprising a monoclonal internalizing antibody according to any one of claims 1-3.

9. A pharmaceutical composition comprising a monoclonal internalizing antibody according to any one of claims 1-3.

10. Use of a monoclonal internalizing antibody of any of claims 1-3 to prepare an anti-cancer or cancer detection reagent, product, or medicament.