High-affinity C-type single domain antibody of targeting mesothelin as well as preparation method and application thereof

A single-domain antibody and affinity technology, applied in chemical instruments and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the weak stability of CH2 and other problems, to achieve good inhibitory effect, good tissue permeability, and good application prospects

Active Publication Date: 2018-06-08
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] CH2 is less stable

Method used

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  • High-affinity C-type single domain antibody of targeting mesothelin as well as preparation method and application thereof
  • High-affinity C-type single domain antibody of targeting mesothelin as well as preparation method and application thereof
  • High-affinity C-type single domain antibody of targeting mesothelin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning of the gene for mesothelin

[0032] Inoculate approximately 2 × 10 cells in a 6-well plate for cultured cells 6 Personal ovarian cancer cell OVCAR-3 was cultured for 12-16 hours, and the cells grew into a single layer. The medium was removed, and the total cellular RNA was extracted with Trizol Reagent (Invitrogen Company), and the obtained total cellular RNA was dissolved in 50 μL of RNase-free water. Then use M-MLV Reverse Transcriptase Kit (Promega Company) and use random primers to reverse transcribe to obtain cDNA.

[0033] According to the gene sequence of mesothelin (GenBank No.AY743922), its amino acid sequence was analyzed, and the forward and reverse primers of the mesothelin gene were designed to amplify the extracellular domain of the mesothelin polypeptide (amino acid 296-600) (Feng Y, et al. al., MolCancer Ther., 2009): forward primer G GAATTC TG GAAGTGGAGAAGACAGC (horizontal line marked as EcoR I restriction site); reverse primer CG...

Embodiment 2

[0035] Example 2: Expression and purification of mesothelin

[0036] 1 day before transfection, 293F cells (control cell density was 5×10 5 cells / ml) inoculate and culture 40mL of cell fluid at 37°C, 8% CO 2 , Cultivated overnight in a constant temperature incubator at 135r / min. Take 40 μg of plasmid and place it in a 14 mL Falcon tube. Add 4mL LD-PBS, mix gently 3 times, every 6 seconds. Add 60 μL (2mg / mL) PEI, and mix well as above. The mixture was allowed to stand at room temperature for 20-30 minutes, and added drop by drop to the cells, so that the mixture was in contact with the cells as fully as possible. Place the cells transfected with the plasmid at 37°C, 8% CO 2 , Cultured in a constant temperature incubator at 135r / min.

[0037] Observe the cell samples, and collect the cell samples transfected for 24h, 48h, 72h, 96h, and 120h, and use mouse anti-His as the primary antibody (the vector used provides a 6×His Tag tag at the C-terminus of the expressed protein f...

Embodiment 3

[0039] Example 3: Construction of phage display library for screening

[0040] m01s is a mutant of the CH2 domain of human antibody IgG. Compared with CH2, its N-terminal seven amino acids are truncated to enhance anti-aggregation ability, and the pH-dependent binding to FcRn is enhanced; in addition to its own disulfide bond (Nativedisulfide bond), also contains a pair of artificial disulfide bonds (Engineered disulfide bond) to enhance stability. The three loop regions contained in m01s are Loop BC, Loop DE and Loop FG, which can be used to introduce mutations to construct antibody libraries.

[0041] Using m01s as the backbone (its gene sequence is Seq ID No.10 and its amino acid sequence is Seq ID No.9), a phage library was constructed according to the method of existing literature (Gong R, et al., PLoS ONE, 2012), and breastfeeding The mesothelin expressed by animal cells was screened as an antigen, and after 4 rounds of screening, an enriched clone was obtained, which w...

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Abstract

The invention discloses a high-affinity C-type single domain antibody of targeting mesothelin as well as a preparation method and application thereof. The high-affinity C-type single domain antibody is obtained by taking a mutant m01s of a structural domain in a human antibody IgG constant region CH2 as a skeleton, constructing a polypeptide library and then screening by taking the mesothelin as an antigen; firstly, the antibody is smaller in molecular weight, has better tissue permeability and ability to combine with antigenic epitopes with steric effects compared with a full-length single antibody, is used for treating and diagnosing some tumors (such as ovarian adenocarcinoma) with high mesothelin expression and even can be developed into an oral medicine; secondly, the plasma half lifeof the high-affinity C-type single domain antibody can reach 10 hours; finally, the high-affinity C-type single domain antibody can be expressed in a prokaryotic expression system and is low in production cost and short in period. F3 screened from the antibody has a better inhibiting effect on OVCAR-3 with high mesothelin expression and good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-affinity C-type single-domain antibody targeting mesothelin and its preparation method and application. Background technique [0002] Mesothelin (MSLN) is a newly discovered tumor differentiation antigen in recent years. Its precursor is a glycosylphosphatidyl alcohol (GPI)-anchored, glycosylated cell surface protein, which can be hydrolyzed by furin protease into a 31kD N-terminal secreted protein and a 40kD C-terminal protein, the latter mainly in the membrane The combined GPI-anchored form exists and is limitedly distributed in normal mesothelial cells and surface epithelial cells, hence the name mesothelin. Mesothelin has limited expression in normal tissues and high expression in some tumors: such as pancreatic cancer (100% of cases), ovarian adenocarcinoma (70% of cases), lung adenocarcinoma (50% of cases) and malignant mesothelioma ( 100% of cases), etc. The mechanis...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K16/00
CPCC07K16/005C07K16/30C07K2317/524C07K2317/92
Inventor 龚睿曹广灿杨文娟石剑李璇杨春鹏张哲
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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