A novel method for rapid in vivo evolution of monoclonal antibody cell lines using DNA damage repair mechanisms
A technology of monoclonal antibody, DNA damage, applied in the field of biology
Active Publication Date: 2017-02-15
成都吉诺迈尔生物科技有限公司
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Problems solved by technology
2. Due to the possibility of obtaining "unlimited" homogeneous antibodies, it is possible to use antibodies as drugs
However, there are still problems to be solved in the method and technology of antibody libraries, such as how to improve the library capacity and antibody production while ensuring the diversity and presentation efficiency of antibody libraries.
Moreover, the light chain variable region of the antibody is very important for the stability and affinity of the antibody, which creates an insurmountable bottleneck in the existing in vitro antibody evolution technology
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[0014] The AID gene was cloned into a lentiviral vector (PLV-GFP) by homologous recombination. Identification of Plv-AID plasmids by enzyme digestion (such as figure 1 Shown in ) the size of the restriction fragment is about 600, which is in line with the expectation.
[0015] As above, the DDB1 and SIRT2 genes were cloned into the lentiviral vector PLV-GFP. Identification of Plv-DDB1 and Plv-SIRT2 plasmids (such as figure 1 Shown in ) the sizes of the enzyme-digested fragments were about 3500 and 1100, which were in line with expectations.
[0016] Plv-AID mutant, Plv-DDB1, and Plv-SIRT2 plasmids were co-transfected into 293T cells with the packaging plasmids PMD2G and PsPAX2 in the three-plasmid lentivirus system by the calcium phosphate method. After 72 hours, they were packaged into corresponding lentiviruses, and the packaging cells showed strong Fluorescence (such as figure 2 Indicated in ) (GFP fluorescence picture).
[0017] The above three lentiviruses were us...
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A method of subjecting a monoclonal antibody cell strain to in-vivo tachytelic evolution by utilization of a DNA damage repair mechanism is provided. The method includes following two parts: overexpression of an AID mutant, and increasing of recruitment of AID in an antibody genetic locus, thus increasing the mutation rate in an antibody CDR zone. The overexpression of the AID mutant relates to overexpression of the AID mutant in the monoclonal cell strain to increase the mutation rate in the antibody CDR zone and to allow the mutation rate to be 10<-3> per generation. In addition, increasing of recruitment of the AID in a CDR locus relates to chromatin conformation change triggering by depending on chromatin remodeling elements (such as SIRT2 and DDB1). Recruitment of the AID to the CDR zone depends on chromatin conformation changes and certain specific changes can increase the f recruitment of the AID in the CDR locus, and therefore the mutation rate in the antibody CDR zone is further increased and reaches 10<-2> per generation, and the method can be used for high-affinity rapid screening processes.
Description
technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a new method for rapidly evolving monoclonal cell strains in vivo by using a DNA damage repair mechanism. Background technique [0002] Monoclonal antibody (monoclonal antibody, mAb), referred to as monoclonal antibody, is produced only by hybridoma cells that can produce such antibodies after the fusion of immune cells and cancer cells. This hybridoma can be cultured as a single cell to form a single cell line, that is, a monoclonal. By culturing or intraperitoneally inoculating mice, a large amount, high concentration, and very uniform antibody can be obtained, and its structure, amino acid sequence, and specificity are all consistent. Monoclonal antibodies can be directly used in the diagnosis, prevention, treatment and immune mechanism research of human diseases, which has opened up broad prospects for the immunodiagnosis and immunotherapy of human diseases. [0003]...
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IPC IPC(8): C12N15/867
Inventor 刘聪章崇杰
Owner 成都吉诺迈尔生物科技有限公司