CN110698562B - Anti-human MSLN monoclonal antibody - Google Patents
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Abstract
The invention discloses an anti-human MSLN monoclonal antibody, which is a specific and high-affinity nano antibody developed by taking human MSLN as a target spot based on a phage display technology. Compared with the prior art, the invention successfully prepares the anti-human MSLN monoclonal antibody which has good specificity and higher affinity and can be combined with the human MSLN expressed on the surface of the cell, and the anti-human MSLN monoclonal antibody is a potential drug for tumor immunotherapy.
Description
Technical Field
The invention relates to an anti-human MSLN monoclonal antibody, belonging to the technical field of monoclonal antibodies.
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, the C-terminal membrane-bound protein of about 40kD is mature mesothelin, and the N-terminal fragment of about 30kD, called megakaryocyte-promoting factor (MPF), 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 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 referred to 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 levels have been detected in various solid tumor tissues such as mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, esophageal cancer, and metastatic triple negative breast cancer. As for the cancer cell lines, it is known that MSLN is highly expressed in three ovarian cancer cell lines HO-8910, HEY-T30, OVCAR3 (particularly HO-8910), the metastatic pancreatic cancer cell line AsPC1 and the cervical cancer cell line Hela, and that it is extremely low expressed in two ovarian cancer cell lines SKOV3, 3AO and the lung adenocarcinoma cell line A549, but not expressed in the human liver cancer cell line Huh 7.
The biological functions of MSLN have not been fully understood so far, and their relationship to cancer is unknown, whether the increase in MSLN expression following carcinogenesis or the increase in MSLN promotes carcinogenesis. It was reported that overexpression of MSLN leads to overexpression of metalloproteinase 9, thereby promoting migration and infiltration of tumor cells. MSLN also mediates cell adhesion through interaction with CA125, plays an important role in the invasion process of tumor peritoneal metastasis, and can promote anti-death of tumor cells. In clinical practice, serum MSLN has been used as an index for the diagnosis of ovarian cancer and mesothelioma.
The MSLN is hardly expressed in normal cells (only low expression in pleura, peritoneum and pericardium) and is 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 advantage of low off-target effect. 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 obvious effect and low possible off-target effect exhibited by the MSLN-targeted therapy, the MSLN-targeted therapy scheme is developed in multiple directions, including recombinant monoclonal antibody drugs, antibody-conjugated drugs, vaccines, adoptive immune cell therapy and the like, wherein the Amatuximab developed by the national cancer institute has completed the second-phase clinical trial for treating malignant mesothelioma.
As for the antibodies currently under development, although many known mesothelin monoclonal antibodies are available, none can exhibit Complement Dependent Cytotoxicity (CDC) against tumor cells. It follows that 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, high transportation and storage requirements and high development and use costs. In conclusion, although the existing traditional antibodies play an extremely important role in the treatment/detection of diseases, the defects thereof are also obvious.
Aiming at MSLN target points, 14 clinical medicines exist at present, and the fastest Amatuximab monoclonal antibody (MORAB-009) is developed to complete the second-phase clinical test. Among the 14 antibodies, some were human-mouse chimeric antibodies and some were human antibodies, all of which were derived from mice, as for their antibody types, and obtained by hybridoma technology. For MSLN-targeting antibodies, there is no public report on the development of nanobodies, and there is no public report on MSLN-targeting antibody screening schemes based on phage display technology.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention discloses an anti-human MSLN monoclonal antibody.
The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the following technical scheme:
an anti-human MSLN monoclonal antibody, which is a single-chain structure and comprises an antigen binding part with an amino acid sequence shown as SEQ ID NO. 1.
As an embodiment of the invention, the anti-human MSLN monoclonal antibody comprises a signal peptide and an antigen binding part, and the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 2.
Or, the anti-human MSLN monoclonal antibody comprises a signal peptide, an antigen binding part and a humanized Fc region, and the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 3.
Or the anti-human MSLN monoclonal antibody comprises a signal peptide, an antigen binding part, a flexible connecting peptide and a human Fc region, and the amino acid sequence of the anti-human MSLN monoclonal antibody is shown in SEQ ID NO. 4.
The preparation method of the anti-human MSLN monoclonal antibody adopts a method of screening phage display peptide library. The specific method comprises the following steps:
commercial recombinant human MSLN His label protein and phage display natural nano antibody library are used as starting materials. Coating the MSLN His-tag protein on an immune tube, incubating the immune tube with a nano-antibody library, washing off the phage which is not combined/weakly combined, and eluting the phage combined with the MSLN His-tag protein. Repeating the steps for 3 times, changing the coating amount and washing conditions of the MSLN His tag protein each time, gradually eliminating the phage with weak binding and keeping more phage with strong binding as much as possible. And (3) coating a solid medium plate after infecting host bacteria, performing monoclonality on the obtained phage with strong binding capacity, performing ELISA screening by using the monoclonal culture supernatant, and performing nucleic acid sequence determination on the monoclonal. Establishing a coding sequence (in one embodiment of the invention, connecting a signal peptide of the secreted luciferase, a nano antibody, namely an antigen binding site, a human IgG2 Fc and a His tag coding sequence), reading in frame, and constructing a mammalian expression vector. CHO-K1 cells were transfected and the supernatant was taken for flow cytometry detection. And (3) a large number of transfection flow assay positive expression vectors, purifying the fusion protein from the supernatant by using a Ni-NTA affinity chromatography column, and identifying the binding property of the fusion protein and MSLN.
A nucleic acid molecule comprising a sequence encoding said anti-human MSLN monoclonal antibody.
A vector comprising said nucleic acid molecule.
A host cell comprising said nucleic acid molecule or said vector.
A kit comprising said anti-human MSLN monoclonal antibody.
The use of said anti-human MSLN monoclonal antibody in the preparation of a kit for detecting the presence or level of MSLN in a sample.
The anti-human MSLN monoclonal antibody can be applied to the preparation of anti-cancer or cancer detection or drugs.
The invention takes human MSLN as a target spot to develop a specific and high-affinity nano antibody. The nanometer antibody is an antibody which is found in camels and adult cartilaginous fishes (such as sharks) and only contains a heavy chain variable region, and a single heavy chain variable region of the antibody has complete antigen binding capacity. Compared with the traditional antibody, the antibody has the characteristics of small volume, strong tissue penetrating power, high water solubility, high tolerance and stability, low immunogenicity, simple structure, easiness in modification (multivalence and multispecific) and the like, so that the antibody has special significance for treating the target treatment of solid tumors which are hindered by blood brain barriers and have poor CART effect and are difficult for effector molecules to enter and the like. Its smaller size and larger CDR3 region is prone to bind to specific epitopes and thus to specific biological effects. Meanwhile, the antibody screening technology adopted in the invention is phage surface display, and the constructed universal nano antibody library is used as a starting material, so that the screening process is greatly shortened, and the time is saved by 90% compared with the traditional hybridoma method. Based on the antibody of the present invention, a cancer therapeutic drug such as a monoclonal antibody drug, an antibody conjugate drug, and a CART immunotherapy, or a detection agent for cancer diagnosis can be developed. Compared with the researched MSLN-targeted antibody drug, the sequence and the structure of the antibody are completely different because the antibody is a nano antibody, and the antibody is an antibody with a brand new structure different from the existing antibody drug.
Has the beneficial effects that: compared with the prior art, the invention successfully prepares the anti-human MSLN monoclonal antibody which has good specificity and higher affinity and can be combined with the human MSLN expressed on the cell surface, and the anti-human MSLN monoclonal antibody is a potential drug for tumor immunotherapy.
Description of the drawings:
FIG. 1: combining the phase crude culture solution with human MSLN to detect the result by ELISA. In the phage panning procedure, duplicate validation was performed with both 50ul and 5ul 2 XYT medium containing the phage virus particles.
FIG. 2: the structure of a vector used for transfection of a CHO-K1 cell line over-expressed by the human MSLN antibody is shown schematically, MCS is a multiple cloning site, and a target gene is inserted into the position.
FIG. 3: and (3) detecting the CHO-K1 overexpression human MSLN antibody fusion protein by SDS-PAGE. In the figure, the left, middle and right lanes are 25ul loading of untransfected CHO-K1 cells, 5ul loading of transfected CHO-K1 cells and 25ul loading of transfected CHO-K1 cells, respectively, and the expressed antibody is shown in the boxed position.
FIG. 4: combining human MSLN and monoclonal antibody to flow test result. The left and right histograms in the figure represent the binding signals to SKOV3 cell line (very weakly expressed MSLN ovarian cancer cell line) and HO-8910 cell line (highly expressed MSLN ovarian cancer cell line), respectively.
FIG. 5: and (3) carrying out ELISA detection on the titer of the purified anti-human MSLN monoclonal antibody fusion protein. Combining the purified antibody with human MSLN, and reacting with HRP-labeled anti-human Fc antibody.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Examples
(1) Phage display antibody library panning
1) Activation of host bacterium TG 1: preparation of mini ear Medium plates [1 XM 9 salt, 2% glucose, 2mM MgSO 4 ，0.1mM CaCl 2 1mM vitamin B1]The TG1 was streaked overnight in a 37 ℃ incubator.
2) Coating: human MSLN protein (from Acro Biosystem) was diluted at 50ug/ml in 0.1M NaHCO 3 (pH8.6), 1.5ml of the solution was added to an immunization tube and the tube was coated overnight at 4 ℃. Removing coating liquid, and adding blocking solution [0.01M PBS (pH7.4), 2% skimmed milk powder]After 1 hour of incubation, the cells were washed 4 times with 0.01M PBS (pH7.4).
3) Combining: get 10 11 pfu phage virions were added to 1.5ml MPBS [0.01M PBS (pH 7.4), 2% skim milk powder in an immune tube]And incubated at room temperature for 1 hour. After the liquid was washed clean, the content of Tween-20 was 0.1% in PBST [0.01M PBS (pH7.4) ]]Washed 8 times and then 8 times with 0.01M PBS (pH7.4).
4) And (3) elution: the tube was repeatedly washed with 1ml of 100mM triethylamine for 10 minutes, mixed with 0.5ml of a neutralizing solution [1M Tris-Cl (pH 7.6) ] and temporarily stored at 4 ℃.
5) And (3) measuring the titer: 2ul, 0.2ul (2 ul after stock solution was diluted 10 times with 2 XYT medium) and 0.02ul (2 ul after stock solution was diluted 100 times with 2 XYT medium) of eluates were mixed with 0.2ml of TG1 in the middle logarithmic phase (OD 600= 0.5), incubated at room temperature for 30min, 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.
6) Phage amplification: an overnight-cultured TG1 strain was inoculated into 20ml of 2 XYT medium at a ratio of 1. The helper phage was added, incubated at room temperature or 37 ℃ for an additional 30 minutes, and ampicillin was added to a working concentration of 100ug/ml and kanamycin was added to a working concentration of 50ug/ml. The cells were cultured overnight at 37 ℃ with shaking at 220 rpm. The cells were removed by centrifugation at 6000rpm for 10 minutes, and 1/5 volume of 2.5M NaCl/20% was added to the supernatant to prepare PEG8000, which was then cooled in ice for 2 hours. The phage pellet was obtained by centrifugation at 10000rpm for 10min, the residue was removed and 0.2ml of 0.01M PBS (pH 7.4) was added to resuspend the pellet and the titer was determined as above.
7) Repeating the steps 2) -6) twice, adjusting the coating concentration of the MSLN protein to 30ug/ml for the first time, adjusting the coating concentration to 15ug/ml for the second time, increasing the washing times and keeping the rest constant.
(2) Monoclonal ELISA
1) 0.5ug/ml human MSLN protein 4 ℃ overnight coated enzyme label plate, blocking solution treatment for 2h, and PBS washing 4 times.
2) Single colonies picked from 2 XYT-GA 100 plates were cultured with shaking to the middle logarithmic phase, helper phage was added, incubated at room temperature or 37 ℃ for 30 minutes, ampicillin was added to a working concentration of 100ug/ml, and kanamycin was added to a working concentration of 50ug/ml. The cells were cultured overnight at 37 ℃ with shaking at 220 rpm. The cells were centrifuged at 6000rpm for 10 minutes to remove the cells.
3) Taking 50ul or 5ul of supernatant, taking MPBS as a medium, supplementing 100ul, adding into an enzyme label plate, incubating for 1 hour, removing liquid, and washing with PBS for 4 times.
4) The HRP-labeled anti-M13 phage antibody was diluted with 1% skim milk powder in PBS, 100ul of the diluted HRP-labeled anti-M13 phage antibody was added to the microplate, incubated for 1 hour, the liquid was removed, and the microplate was washed 4 times with PBS.
5) 100ul of TMB developer was added, incubation was carried out at 37 ℃ for 10min or until the yellow color was fully developed, 100ul 1M sulfuric acid was added to stop the reaction, and OD450 was read on a microplate reader. The results are shown in FIG. 1, the monoclonal ELISA reading values are obviously higher than the control, the result is positive, and the monoclonal ELISA reading values have the function of binding the human MSLN protein at the ELISA level.
(3) Preparation and flow detection 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) The chemical synthesis nanometer antibody fusion gene sequence is characterized in that a signal peptide of the secreted luciferase, a nanometer antibody, a flexible connecting peptide, human IgG2 Fc and a His tag are connected and read in frame, the nucleic acid sequence is shown as SEQ ID NO.5, and the amino acid sequence of the coded antibody is shown as SEQ ID NO. 6.
3) The above nucleic acid fragments are inserted into MCS region of eukaryotic expression vector pLOE-purOR (figure 2) in sequence to synthesize fusion protein with N end as signal peptide, middle section as nano antibody, C end as Fc and His label.
4) Preparing sterile non-toxic plasmid, transfecting CHO-K1 cell according to the requirement of transfection reagent, and collecting supernatant.
5) SDS-PAGE detects the expression of the antibody, and as shown in figure 3, the fused antibody protein is successfully expressed.
6) Flow assay for cell binding capacity
(1) Fully digesting the SKOV3 cell of the extremely weak expression MSLN ovarian cancer cell strain and the HO-8910 cell of the ovarian cancer cell strain strongly expressing the humanized MSLN by using 0.25 percent of pancreatin, stopping digestion by serum, centrifugally collecting the cells, and lightly blowing and beating by PBS to prepare single cell suspension.
(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 10 5 The cells were collected by centrifugation in a 1.5ml centrifuge tube.
(4) Adding 200 μ l of CHO-K1 cell culture supernatant obtained in step 4), mixing, and incubating at room temperature for 30min.
(5) Cells were harvested by centrifugation and washed 1 time with 1ml PBS.
(6) Add 50. Mu.l of 20-fold diluted secondary antibody of APC-labeled goat anti-human IgG Fc fragment (2.5. Mu.l/2.5X 10) 5 Optionally binding with human IgG), and reacting at room temperature in dark for 20min.
(7) Cells were washed 1 time with 1ml PBS, centrifuged at 1000rpm for 8min and the supernatant removed.
(8) Add 200. Mu.l PBS to re-suspend into single cell suspension, and detect on machine with flow cytometer. As shown in FIG. 4, compared with SKOV3 cell line not expressing MSLN, HO-8910 signal of the cell line highly expressing MSLN is obviously enhanced, the bar chart is shifted to the right, and the antibody can be combined with MSLN on the cell surface.
(4) Preparation of humanized MSLN monoclonal antibody from CHO-K1 cell
1) Large amount of positive plasmid for flow detection is prepared, and three T25 bottles of CHO-K1 cells are transfected.
2) 72 hours after transfection, the supernatant (total 12 ml) was collected while cells were trypsinized and collected by centrifugation.
3) Cells were suspended in 5ml cold PBS. The ultrasonic power is set to 25% of the maximum power, the work is carried out for 2s, the pause is carried out for 6s, and the total time is 3min. Cells were disrupted by sonication in an ice bath and combined with cell culture supernatant after completion. A cocktail protease inhibitor was added.
4) Taking 4ml of Ni-NTA agarose gel, adding the gel into a screening plate of a purification column, and filling a gravity purification column.
5) The column was equilibrated with 1ml of 20mM Tris-HCl buffer (pH 8.0)/500 mM NaCl.
6) The combined solution was applied to a purification column at low temperature, and the flow rate was controlled at 3 s/drop.
7) The column was washed with 10ml of 20mM Tris-HCl buffer (pH 8.0)/500 mM NaCl solution containing 30mM imidazole.
8) The antigen-binding peptide fusion protein was eluted with 4ml of 20mM Tris-HCl buffer (pH 8.0)/500 mM NaCl solution containing 300mM imidazole.
9) Adding into deionized water wet ultrafiltration tube with molecular weight cut-off of 10KD, adding cold PBS to 10ml, centrifuging at 4 deg.C and 3000rpm for 10min until the liquid level is at the upper edge of V-shaped ultrafiltration membrane. The filtrate was aspirated off, 10ml of cold PBS was added to the inner column, and the column was centrifuged until the liquid surface was on the upper edge of the V-shaped ultrafiltration membrane.
10 The liquid in the upper column was aspirated, the ultrafiltration membrane was washed with 0.4ml of cold PBS, and the liquids were combined for a total of about 1.5ml.
(5) Titer detection of purified human MSLN monoclonal antibody
1) Concentration of fusion protein by Bradford method
Using Quick Start from Bio-Rad TM Bradford 1x Dye Reagent (cat # 5000205) kit was used for the assay, and the test samples were tested at four concentrations, 2X, 5X, 10X, and 20X. According to the kit operation, 595n is carried out after the completionAnd reading the absorbance at m.
2) The plate was coated with 100ul of 2ug/ml human MSLN under 4 ℃ overnight conditions.
3) 0.05% PBST wash plate 3 times, closed with 200ul 2% skim milk powder for 1 hour at room temperature, 0.05% PBST wash plate 3 times.
4) The purified protein was diluted to 1ng/ul, 5ng/ul, 25ng/ul, 125ng/ul, 625ng/ul with 1% nonfat dry milk in PBS, 100ul of the diluted purified protein was added to the ELISA plate, and 1% nonfat dry milk in PBS was added to the control wells.
5) Incubate at room temperature for 2 hours, shaking several times during this period. Remove liquid, 0.05% PBST wash 3 times.
6) Secondary antibody of HRP-labeled rabbit anti-human IgG Fc fragment was added and incubated at room temperature for 1 hour. Remove liquid, 0.05% PBST washing 3 times.
7) 100ul of TMB substrate was added and incubated at 37 ℃ for about 10 minutes until the yellow color fully developed.
8) The reaction was stopped by adding 100ul 1M sulfuric acid and the OD450 read on a microplate reader. The results are shown in FIG. 5, where the purified antibody bound to the antigen in vitro, showing a typical antigen-antibody binding concentration-dependent curve.
Sequence listing
<110> Zhejiang Landun pharmaceuticals Co., ltd
<120> anti-human MSLN monoclonal antibody
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<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr Ser
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Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Ser Gly Gly Gly Ser Tyr Tyr Ala Asp Ser Val
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Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
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Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
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Ala Ala Glu Arg Trp Cys Tyr Gly Leu Val Thr Arg Gln Ala Asp Phe
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Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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<211> 142
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<213> Artificial Sequence (Artificial Sequence)
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Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
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Ser Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln
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Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr
35 40 45
Ser Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
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Val Ala Ala Ile Tyr Thr Ser Gly Gly Gly Ser Tyr Tyr Ala Asp Ser
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Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
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Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
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Cys Ala Ala Glu Arg Trp Cys Tyr Gly Leu Val Thr Arg Gln Ala Asp
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Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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<213> Artificial Sequence (Artificial Sequence)
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Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
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Ser Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln
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Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr
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Ser Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
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Val Ala Ala Ile Tyr Thr Ser Gly Gly Gly Ser Tyr Tyr Ala Asp Ser
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Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
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Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
100 105 110
Cys Ala Ala Glu Arg Trp Cys Tyr Gly Leu Val Thr Arg Gln Ala Asp
115 120 125
Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Pro Ala
130 135 140
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
195 200 205
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 4
<211> 374
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
1 5 10 15
Ser Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln
20 25 30
Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr
35 40 45
Ser Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
50 55 60
Val Ala Ala Ile Tyr Thr Ser Gly Gly Gly Ser Tyr Tyr Ala Asp Ser
65 70 75 80
Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
85 90 95
Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
100 105 110
Cys Ala Ala Glu Arg Trp Cys Tyr Gly Leu Val Thr Arg Gln Ala Asp
115 120 125
Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Ala Pro
145 150 155 160
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
165 170 175
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
180 185 190
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
195 200 205
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
210 215 220
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
225 230 235 240
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
245 250 255
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
260 265 270
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
275 280 285
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
290 295 300
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
305 310 315 320
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
325 330 335
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
340 345 350
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
355 360 365
Ser Leu Ser Pro Gly Lys
370
<210> 5
<211> 1143
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgagag catggcccag 60
gtaaagctgg aggagtctgg gggaggctcg gtgcaggctg gagggtctct gagactctcc 120
tgtgtagcct ctggatacac ctacagtacc atgggctggt tccgccaggc tccagggaag 180
gagcgcgagg gggtcgcagc tatttatact agtggtggtg gctcatacta tgccgactcc 240
gtgaagggcc gattcaccat ctcccaagac aatgccaaga acacggtgta tctgcaaatg 300
aacggcttga aacctgagga cactgccatg tactactgtg cggcagagag atggtgctat 360
gggttggtca cgaggcaggc tgactttggt tactggggcc aggggaccca ggtcaccgtc 420
tcctcaggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc gccagcacca 480
cctgtggcag gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 540
tcccggaccc ctgaggtcac gtgcgtggtg gtggacgtga gccacgaaga ccccgaggtc 600
cagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccacgggag 660
gagcagttca acagcacgtt ccgtgtggtc agcgtcctca ccgttgtgca ccaggactgg 720
ctgaacggca aggagtacaa gtgcaaggtc tccaacaaag gcctcccagc ccccatcgag 780
aaaaccatct ccaaaaccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 840
tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac 900
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 960
acacctccca tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1020
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1080
aaccactaca cgcagaagag cctctccctg tctccgggta aacaccacca tcaccaccat 1140
tga 1143
<210> 6
<211> 380
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
1 5 10 15
Ser Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln
20 25 30
Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Tyr
35 40 45
Ser Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
50 55 60
Val Ala Ala Ile Tyr Thr Ser Gly Gly Gly Ser Tyr Tyr Ala Asp Ser
65 70 75 80
Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
85 90 95
Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
100 105 110
Cys Ala Ala Glu Arg Trp Cys Tyr Gly Leu Val Thr Arg Gln Ala Asp
115 120 125
Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Ala Pro
145 150 155 160
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
165 170 175
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
180 185 190
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
195 200 205
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
210 215 220
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
225 230 235 240
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
245 250 255
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
260 265 270
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
275 280 285
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
290 295 300
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
305 310 315 320
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
325 330 335
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
340 345 350
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
355 360 365
Ser Leu Ser Pro Gly Lys His His His His His His
370 375 380
Claims (9)
1. An anti-human MSLN monoclonal antibody is characterized in that the antibody is a nano antibody, and the amino acid sequence is shown as SEQ ID NO. 1.
2. The anti-human MSLN monoclonal antibody of claim 1, comprising a signal peptide and an antigen binding portion, the amino acid sequence of which is shown in SEQ ID No. 2.
3. The anti-human MSLN monoclonal antibody of claim 1, comprising a signal peptide, an antigen binding portion and a human Fc region, the amino acid sequence of which is shown in SEQ ID No. 3.
4. The anti-human MSLN monoclonal antibody of claim 1, comprising a signal peptide, an antigen binding portion, a flexible linker peptide and a humanized Fc region, the amino acid sequence of which is shown in SEQ ID No. 4.
5. A nucleic acid molecule encoding the anti-human MSLN monoclonal antibody of claim 1.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.
8. A kit comprising an anti-human MSLN monoclonal antibody of any one of claims 1-4.
9. Use of the anti-human MSLN monoclonal antibody of any one of claims 1-4 in the preparation of an ovarian cancer detection reagent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911052830.8A CN110698562B (en) | 2019-10-31 | 2019-10-31 | Anti-human MSLN monoclonal antibody |
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| CN111349165B (en) * | 2020-05-25 | 2020-09-01 | 南京蓝盾生物科技有限公司 | Anti-human MSLN monoclonal antibody and application thereof |
| US20240124563A1 (en) * | 2020-12-09 | 2024-04-18 | Nanjing Zaiming Pharmaceutical Co., Ltd. | Anti-Human MSLN Antibody And Application Thereof |
| CN114685667B (en) * | 2020-12-28 | 2024-04-12 | 浙江纳米抗体技术中心有限公司 | Mesothelin binding molecules and uses thereof |
| JP2024505368A (en) * | 2020-12-28 | 2024-02-06 | 浙江納米抗体技術中心有限公司 | Mesothelin binding molecules and their applications |
| CN113061187B (en) * | 2021-04-27 | 2022-07-12 | 艾柏森(江苏)生物科技有限公司 | Anti-human MSLN monoclonal antibody or antigen binding fragment thereof |
| WO2022242703A1 (en) * | 2021-05-19 | 2022-11-24 | 江苏先声药业有限公司 | Anti-msln antibody and application thereof |
| CN117412991A (en) * | 2021-06-18 | 2024-01-16 | 南京再明医药有限公司 | Anti-human MSLN humanized antibody and application thereof |
| CN115991782A (en) * | 2021-10-18 | 2023-04-21 | 普米斯生物技术(珠海)有限公司 | Anti-mesothelin nanobody and use thereof |
| CN118510809A (en) * | 2022-01-06 | 2024-08-16 | 原启生物科技(上海)有限责任公司 | MSLN-targeted antigen binding proteins and uses thereof |
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| CN101012280A (en) * | 2007-02-07 | 2007-08-08 | 吴小华 | Method of preparing monoclonal antibody against human soluble mesothelin related protein |
| AU2008308956B2 (en) * | 2007-10-01 | 2013-03-14 | Bristol-Myers Squibb Company | Human antibodies that bind mesothelin, and uses thereof |
| WO2014004549A2 (en) * | 2012-06-27 | 2014-01-03 | Amgen Inc. | Anti-mesothelin binding proteins |
| CN112321714B (en) * | 2019-01-08 | 2022-06-28 | 上海霖科生物科技有限公司 | Monoclonal antibodies binding to mesothelin and nucleic acid molecules thereof |
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