CN103819559A - Anti-mesothelin nanometer antibody as well as coding gene and application thereof - Google Patents
Anti-mesothelin nanometer antibody as well as coding gene and application thereof Download PDFInfo
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- CN103819559A CN103819559A CN201310667427.2A CN201310667427A CN103819559A CN 103819559 A CN103819559 A CN 103819559A CN 201310667427 A CN201310667427 A CN 201310667427A CN 103819559 A CN103819559 A CN 103819559A
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Abstract
The invention discloses an anti-mesothelin nanometer antibody, as well as a coding gene and application thereof, and relates to an anti-tumor differentiation antigen nanometer antibody as well as a coding gene and application thereof. The amino acid sequence of the anti-mesothelin nanometer antibody provided by the invention is as shown in SEQ ID NO.2. The nucleotide sequence of the coding gene of the anti-mesothelin nanometer antibody is shown in SEQ ID NO.1. The anti-mesothelin nanometer antibody is used for preparing a probe for highly-expressed mesothelin cancer cells or preparing a medicine for inhibiting the highly-expressed mesothelin cancer cells. The nanometer antibody mesnab1 provided by the invention can specifically bind to mesothelin expressed on the surfaces of tumor cells, and can be used for treating and diagnosing mesothelin highly-expressing, tumor and researching the effect of the mesothelin in tumorigenesis. The anti-mesothelin nanometer antibody can be applied to the fields of biology and medicine.
Description
Technical field
The present invention relates to a kind of purposes of nano antibody and encoding gene and this nano antibody of antitumor differentiation antigen.
Background technology
Mesothelin (mesothelin, MSLN) is newfound a kind of Tumor Differentiation antigen in recent years.Its precursor is glycophospholipin acyl-glycero (GPI) grappling, glycosylated cells surface protein; the N-end secreted protein that can be 31kD by furin protease hydrolysis and the C-terminal protein of 40kD; the latter mainly exists with the form of membrane-bound GPI grappling; limitedly be distributed in normal mesothelial cell and superficial epithelium cell, therefore called after mesothelin.
Mesothelin is limited expression in healthy tissues, and in some tumour high expression level, as carcinoma of the pancreas (100% case), adenocarcinoma ovaries (70% case), adenocarcinoma of lung (50% case) and malignant mesothe (100% case) etc.The mechanism that mesothelin induced tumor occurs may comprise following three aspects:
The first, mesothelin can by with Mucin1 6(be CA125) interacting promotes peritoneal seeding and the transfer of tumour cell;
The second, mesothelin can promote by NF-κ B signal path survival and the propagation of tumour cell;
The 3rd, the expression of mesothelin can strengthen the tolerance of tumour cell to some specific medicine (as: TNF-α, taxol, platinum-endoxan mixture).
As can be seen here, mesothelin is a desirable target for oncotherapy.
Summary of the invention
Mesothelin, as a kind of desirable target for oncotherapy, the invention provides a kind of purposes of nano antibody and encoding gene and this nano antibody of antitumor differentiation antigen.
The amino acid of the nano antibody of the anti-mesothelin of the present invention is as shown in SEQ ID NO:2.
The nucleotide sequence of the nano antibody encoding gene of above-mentioned anti-mesothelin is as shown in SEQ ID NO:1.
Prepare the probe of high expression level mesothelin cancer cells with the nano antibody of above-mentioned anti-mesothelin.
Suppress the medicine of high expression level mesothelin cancer cells with the nano antibody preparation of above-mentioned anti-mesothelin.
The nano antibody of the anti-mesothelin of the present invention is named as nano antibody mesnab1.Nano antibody mesnab1 can specific binding tumor cell surface expression mesothelin, by restraining effect such as the migrations to tumour cell for the treatment of high expression level mesothelin cancer.
Nano antibody mesnab1 of the present invention can be used for the tumour (as carcinoma of the pancreas, adenocarcinoma ovaries, adenocarcinoma of lung and malignant mesothe) for the treatment of and diagnosis mesothelin high expression level, and the effect of research mesothelin in tumour occurs.
Nano antibody mesnab1 molecular weight of the present invention, about 15kD, therefore has better tissue permeability, also stronger in conjunction with the ability of the epitope of presence bit inhibition effect on space.So, can be used for the treatment of using nano antibody mesnab1 of the present invention as oral medicine the tumour of mesothelin high expression level.
Nano antibody mesnab1 of the present invention has the binding ability with FcRn, reaches 10h plasma half-life.
Nano antibody mesnab1 of the present invention can express at prokaryotic expression system, has advantages of that production cost is low, the cycle is short.
Accompanying drawing explanation
Fig. 1 is that in embodiment 3, nano antibody mesnab1 and mesothelin are in conjunction with ELISA measurement result figure, and in Fig. 1, " ■ " curve is nano antibody mesnab1, the negative control antibodies m01s of "●" curve in Fig. 1;
Fig. 2 is the Proliferation of Human Ovarian Cell SK-OV-3 cell light field observation figure of hatching altogether with nano antibody mesnab1 in embodiment 4;
Fig. 3 is the Proliferation of Human Ovarian Cell SK-OV-3 cell Texas Red fluoroscopic examination figure of hatching altogether with nano antibody mesnab1 in embodiment 4;
Fig. 4 is the Proliferation of Human Ovarian Cell SK-OV-3 cell DAPI staining examine figure of hatching altogether with nano antibody mesnab1 in embodiment 4;
Fig. 5 is the Proliferation of Human Ovarian Cell SK-OV-3 cell light field observation figure of hatching altogether with antibody m01s in embodiment 4;
Fig. 6 is the Proliferation of Human Ovarian Cell SK-OV-3 cell Texas Red fluoroscopic examination figure of hatching altogether with antibody m01s in embodiment 4;
Fig. 7 is the Proliferation of Human Ovarian Cell SK-OV-3 cell DAPI staining examine figure of hatching altogether with antibody m01s in embodiment 4;
Fig. 8 is embodiment 5 flow cytometry detection figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is illustrated, technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one:
The amino acid of the nano antibody of the anti-mesothelin of present embodiment is as shown in SEQ ID NO:2.
The nano antibody of the anti-mesothelin of present embodiment can be used for the treatment of high expression level mesothelin cancer, comprises carcinoma of the pancreas, adenocarcinoma ovaries, adenocarcinoma of lung and malignant mesothe etc.
The nano antibody of the anti-mesothelin of present embodiment can be used as probe, for detection of the tumour of high expression level mesothelin, as carcinoma of the pancreas, adenocarcinoma ovaries, adenocarcinoma of lung and malignant mesothe etc.
The nano antibody of the anti-mesothelin of present embodiment can also merge with self or other polypeptide, for treatment and the detection of high expression level mesothelin cancer.
The nano antibody of the anti-mesothelin of present embodiment and other molecule coupling, include but are not limited to radio isotope and toxin, forms treatment and the detection of coupling antibody for high expression level mesothelin cancer.
Present embodiment nano antibody mesnab1 has higher anti-ability of aggregation.
Present embodiment nano antibody mesnab1 introduces a pair of disulfide linkage, and therefore stability is high, and its Tm value is not less than 80 ℃.Present embodiment nano antibody mesnab1 has more excellent solubility expression and protease resistant.
Embodiment two:
The nucleotide sequence of the nano antibody of the anti-mesothelin of present embodiment is as shown in SEQ ID NO:1.
The nucleotide sequence of the nano antibody of the anti-mesothelin of present embodiment is synthesized by bio-engineering corporation.
Embodiment 1.
The gene that obtains nano antibody mesnab1 by SEQ ID NO:1 synthetic, is cloned in prokaryotic expression carrier pComb3, is transformed in E.coli HB2151; Inoculate bacterial classification and (in 1L substratum, contain 30g Tryptones, 20g yeast extract and 10g MOPS in the SB substratum containing 100 μ g/ml ammonia benzyls, pH value is adjusted to 7.0 with NaOH) in, until OD600 reach at 0.7～1.0 o'clock and add IPTG to final concentration be 200 μ g/ml, under 37 ℃, the condition of 220r/min, carry out abduction delivering 14～16h; Then the centrifugal collection thalline of 15min under 4 ℃, 6000r/min condition, abandon substratum, precipitation is resuspended in Buffer A(50mM Tris-HCl, 450mM NaCl, pH8.0) in, then process centrifugal collection supernatant after 1 hour through PXB (polymyxin B); With Ni-NTA resin (QIAGEN company) purifying nano antibody mesnab1.
Embodiment 2.
The purity of verifying nano antibody mesnab1 through SDS-PAGE is more than 98%.The ultra-filtration centrifuge tube that is 3kD with molecular weight cut-off subsequently (Merck Millipore company) ultrafiltration and concentration nano antibody mesnab1.The C-end that is the nano antibody mesnab1 that vector expression was obtained with pComb is containing 6 × His label and FLAG label.
Embodiment 3.
Mesothelin (2 μ g/mL) is coated on elisa plate, after 4 ℃ of overnight incubation, seals 1h with PBS+3%milk in 37 ℃.Add the nano antibody mesnab1 of different weaker concns, hatch after 2 hours for 37 ℃ and wash four times with PBST (PBS+0.05%Tween20), the mouse-anti FLAG monoclonal antibody that adds again horseradish peroxidase (HRP) mark is hatched after 1 hour in 37 ℃, wash four times with PBST, then add ABTS to detect.And with antibody m01s(Gong R, et al., J Biol Chem., 2011) as negative control.
The EC50 of nano antibody mesnab1 and mesothelin combination is 273nM, and antibody m01s can not be with mesothelin in conjunction with (as shown in Figure 1).ELISA experimental results show that nano antibody mesnab1 can combine with antigen mesothelin.
Embodiment 4.
By SK-OV-3(5 × 10 of 500 μ L
5～6 × 10
5individual/ml) cell inoculates 24 orifice plates, adds nano antibody mesnab1 after cultivating 14～16h, hatch 2 hours for 37 ℃; Use afterwards PBSA(PBS+0.5%BSA), PBS cleans respectively and after 3 times, adds fluorescence two anti-(sheep anti-mouse igg of Texas Red coupling) to hatch after 1h in 37 ℃, clean respectively and wash after 3 times the dyeing situation of observation of cell under fluorescent microscope with PBSA, PBS again, and using antibody m01s as negative control.
DAPI dyeing is in order to the existence of indicator cells core.According to Fig. 2～Fig. 7, the Proliferation of Human Ovarian Cell SK-OV-3 cell of hatching altogether with nano antibody mesnab1 is by red-dyed, and SK-OV-3 has been proved to be able to high expression level mesothelin, show that nano antibody mesnab1 can be combined with the mesothelin of Proliferation of Human Ovarian Cell SK-OV-3 cell surface expression.As negative control, antibody m01s does not detect the combination with mesothelin.
Embodiment 5.
Get SK-OV-3 cell (approximately 1 × 10
6individual), hatch 2 hours at 37 ℃ with nano antibody mesnab1, add mouse-anti FLAG monoclonal antibody after cleaning respectively 3 times with PBSA, PBS, hatch 1 hour for 37 ℃; Clean respectively and after 3 times, add fluorescence two anti-(sheep anti-mouse igg of Texas Red coupling) with PBSA, PBS again, hatch after 1h in 37 ℃, clean respectively 3 times with PBSA, PBS afterwards after in analyzing on flow cytometer.And using antibody m01s as negative control.
As shown in Figure 8, add the fluorescence of the SK-OV-3 cell of nano antibody mesnab1 to have significant migration, confirmed the conclusion of embodiment 4 immunofluorescent stainings.Illustrate that nano antibody mesnab1 is inhibited to high expression level mesothelin cancer cells simultaneously.
The present invention is not limited to above-mentioned preferred forms, and anyone should learn the structural changes of making under enlightenment of the present invention, and every have identical or close technical scheme with the present invention, within all falling into protection scope of the present invention.
Claims (4)
1. a nano antibody for anti-mesothelin, is characterized in that the amino acid of described nano antibody is as shown in SEQ ID NO:2.
2. the encoding gene of the nano antibody of anti-mesothelin as claimed in claim 1, is characterized in that the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
3. the purposes of the nano antibody of anti-mesothelin as claimed in claim 1, is characterized in that preparing with the nano antibody of anti-mesothelin the probe of high expression level mesothelin cancer cells.
4. the purposes of the nano antibody of anti-mesothelin as claimed in claim 1, is characterized in that suppressing with the nano antibody preparation of anti-mesothelin the medicine of high expression level mesothelin cancer cells.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121941A1 (en) * | 2020-12-09 | 2022-06-16 | 江苏先声药业有限公司 | Anti-human msln antibody and application thereof |
| CN114685667A (en) * | 2020-12-28 | 2022-07-01 | 浙江纳米抗体技术中心有限公司 | Mesothelin binding molecules and uses thereof |
| CN114685666A (en) * | 2020-12-28 | 2022-07-01 | 浙江纳米抗体技术中心有限公司 | Anti-mesothelin nano antibody and application thereof |
| WO2022143550A1 (en) * | 2020-12-28 | 2022-07-07 | 浙江纳米抗体技术中心有限公司 | Mesothelin binding molecule and application thereof |
| WO2023066133A1 (en) * | 2021-10-18 | 2023-04-27 | 普米斯生物技术(珠海)有限公司 | Anti-mesothelin nanobodies and use thereof |
| CN116019939A (en) * | 2022-11-24 | 2023-04-28 | 华中科技大学同济医学院附属协和医院 | Molecular probe targeting MSLN and application thereof |
| CN116063529A (en) * | 2021-12-29 | 2023-05-05 | 华道(上海)生物医药有限公司 | Anti-mesothelin high-affinity nano antibody and application thereof |
| WO2023131276A1 (en) * | 2022-01-06 | 2023-07-13 | 原启生物科技(上海)有限责任公司 | Antigen binding protein targeting msln and use thereof |
| WO2023179740A1 (en) * | 2022-03-25 | 2023-09-28 | Shanghai Henlius Biotech , Inc. | Anti-msln antibodies and methods of use |
| WO2024088371A1 (en) * | 2022-10-28 | 2024-05-02 | 原启生物科技(上海)有限责任公司 | Antigen-binding protein targeting msln |
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| CN114685666B (en) * | 2020-12-28 | 2023-10-03 | 浙江纳米抗体技术中心有限公司 | Anti-mesothelin nanobody and application thereof |
| CN114685667A (en) * | 2020-12-28 | 2022-07-01 | 浙江纳米抗体技术中心有限公司 | Mesothelin binding molecules and uses thereof |
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| WO2023179740A1 (en) * | 2022-03-25 | 2023-09-28 | Shanghai Henlius Biotech , Inc. | Anti-msln antibodies and methods of use |
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| CN116019939A (en) * | 2022-11-24 | 2023-04-28 | 华中科技大学同济医学院附属协和医院 | Molecular probe targeting MSLN and application thereof |
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