CN104263755B - A method of subjecting a monoclonal antibody cell strain to in-vivo tachytelic evolution by utilization of a DNA damage repair mechanism - Google Patents
A method of subjecting a monoclonal antibody cell strain to in-vivo tachytelic evolution by utilization of a DNA damage repair mechanism Download PDFInfo
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Abstract
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
The invention belongs to biological technical field, specially one kind are entered to monoclonal cell strain using DNA damage repair mechanism
The new method of the internal tachytelic evolution of row.
Background technology
Monoclonal antibody (monoclonal antibody, mAb), abbreviation monoclonal antibody, is only by can manufacture this antibody
Hybridoma after immunocyte and cancerous cell fusion produces.This hybridoma is made Single cell culture, list can be formed
Cell line, i.e. monoclonal.Using the method for culture or mouse peritoneal inoculation, just can obtain substantial amounts of, high concentration, very uniform
Antibody, its structure, amino acid sequence, specificity etc. are all consistent.Monoclonal antibody can be directly used for human diseasess diagnosis,
The research of prevention, treatment and immunologic mechanism, is that the immunologic diagnosises of human diseasess open bright prospects with immunization therapy.
Monoclonal antibody has several advantages as follows compared with polyclonal antibody.1. hybridoma is realized external immortalization and is persistently passed
, as long as there is not the gene mutation of cell strain it is possible to constantly produce high specific, the antibody of high homogeneity in generation.2. due to
The homogeneity antibody being likely to be obtained " endless " is so that antibody is possibly realized as medicine.3. standardized production can be realized, use
In the immunological analysis method with traget antibody as feature.4. because the high specific of monoclonal antibody and single creature learn work(
Can, can be used for the wide spectrums such as the detection of curative drug, radio-immuno-image, medical diagnosis reagent or even environmental pollution.Single
Although the preparation of clonal antibody has various optimizations in detail, technological frame relative maturity.
The techniqueflow of the design and development of humanization/full human monoclonal antibody includes target spot and selects and verify, be directed to
The preparation of target spot monoclonal antibody, antibody screening, antibody cloning, antibody humanization, antibody affinity maturation, human antibody preparation, animal mould
Type research and clinical trial etc..At present, also there is larger development space in a lot of aspects of antibody drug research and development.Wherein, antibody parent
Lifting with characteristics such as power and specificitys be antibody research and development the most key, be also be easiest to meet with technical bottleneck step.
The generation mechanism of antibody storage capacity mainly includes combining multiformity, junctional diversity and somatic hypermutation, and
High-affinity antibody is to produce under somatic hypermutation and selection of antigen.B cell occurs division to increase after identification antigen
Produce somatic hypermutation during growing, and mutation is concentrated mainly on weight, light chain variable district interior decision antibody and antigen parent
Complementary determining region (CDR) with power.Therefore, somatic hypermutation can produce the antibody of high-affinity, and has high-affinity
The B cell of antibody can preferential activation be bred under low concentration antigenic stimulus, gradually forms the B cell predominant cell of high-affinity
Group, secretion further produces high-affinity antibody.
Antibody library be using molecule clone technology amplification repertoire antibody weight, chain variable region gene, then with specific
Expression vector connect, be transformed in host cell and give expression to functional antibody, and by affine screen etc. method screen
The specific antibody of high-affinity.Antibody library experiences internal and external developmental stage, wherein in vivo with phage antibody library
Technology is most commonly seen, most commonly seen with ribosome and mRNA Antibody library in vitro.Antibody library method and technology are passed through simulation and are resisted
The important step that body generates in body, such as multiformity B cell colony, Immune Clone Selection, affinity maturation etc., accelerate antibody system
Standby speed, reduces production cost, screens and prepares high-affinity spy on a large scale for quick from the antibody library of big storage capacity
Heterogenetic antibody provides simplicity and efficient operating system.But antibody library method and technology yet suffer from urgently to be resolved hurrily at present
How problem, such as, while ensureing antibody library multiformity and assume efficiency, improve the problems such as storage capacity and antibody production.
And the light chain variable district of antibody is for the stability of antibody, affinity is extremely important, and this results in existing external antibody and develops
The impassable bottleneck of technology.
Content of the invention
For the problems referred to above, the invention discloses a kind of we have developed the internal antibody based on cell strain of monoclonal antibody
Evolution technology.The method using AID mutant and controls it the Mutation probability of variable region can be made to reach in the recruitment situation of CDR region
To 10-3-10-2In/generation, can be used for high-affinity antibody rapid screening technique.
For reaching above-mentioned purpose, the enforcement of the present invention adopts following scheme.
First on NCBI, inquiry determines the standard sequence of mice AID, DDB1 and SIRT2 gene, further according to nucleotide sequence
Design corresponding amplimer.These three genes are cloned into slow viruss skeleton carrier by the method for homologous recombination.AID gene
38 amino acids glycine (AID mutant) is converted to by directed mutagenesis method.
Further according to three plasmid slow viruss system approachs, packaging obtains viral supernatants, then carries out transfection acquisition respectively
Transfect into Plv-AID, Plv-DDB1 and Plv-SIRT2 simple substance grain, Plv-DDB1 and Plv-SIRT2 double-mass model and three plasmid corotation
Five stable cell lines.
The further RNA extracts kit by using OMEGA company extract Plv-AID mutant, Plv-DDB1 and
In the cell line of Plv-SIRT2 simple substance grain and three plasmid corotation, RNA carries out reversion acquisition CDNA.In the method using PCR further
Check antibody mutation rate in Plv-AID mutant, Plv-DDB1 and Plv-SIRT2 simple substance grain and the cell line of three plasmid corotation
Situation of change.Determine antibody mutation evolution rate highest in the cell line of three plasmid corotation.
Further by chromosome co-precipitation (ChIP) experiment, verify DDB1, SIRT2 albumen can directly affect AID
(Fig. 4, display in 5 can reach 10 for the lifting further of raising on antibody gene VDJ, therefore induction intracellular antibody mutation rate-2/ generation).
The impact to chromatin conformation of mutant and DDB1, SIRT2 albumen of AID gene is utilized, in gene in the present invention
The acceleration of intracellular antibody evolution is achieved on group DNA sequence level, is a kind of invention of novelty.Research and development to therapeutic antibodies
And large-scale production is significant.
Specific embodiment
By AID gene cloning with homologous recombination method to slow virus carrier (PLV-GFP).Enzyme action identification Plv-AID plasmid is (such as
Shown in Fig. 1) endonuclease bamhi size be 600 about, with expection be consistent.
Ibid, by DDB1 and SIRT2 gene cloning to slow virus carrier PLV-GFP.Enzyme action identifies Plv-DDB1 and Plv-
SIRT2 plasmid (as shown in fig. 1) endonuclease bamhi size is respectively 3500 and 1100 about, is consistent with expection.
By the packaging matter in Plv-AID mutant, Plv-DDB1 and Plv-SIRT2 plasmid and three plasmid slow viruss systems
Grain PMD2G and PsPAX2 calcium phosphate method cotransfection 293T cell, is packaged into corresponding slow viruss, incasing cellss present after 72 hours
Intense fluorescence (as shown in Figure 2) (GFP fluorescence picture).
By three of the above slow virus infection monoclonal cell strain VEGFR-E3, after one week, carry out following experiment：Using OMEGA
The E.Z.N.A that company producesTMTotal RNA Kit II (R6934-02) extracts Plv-AID mutant, Plv-DDB1 and Plv-
RNA in the cell line cell of SIRT2 simple substance grain and three plasmid corotation, obtains cDNA after reversion, with universal primer
GCTAGTGAGCTCTTTGCCCAG and AAAAAAAAAAAAAAAAAAAAA carries out polymerase chain reaction,PCR amplification to V1, V2, V3 area
And be sequenced (PCR)；Result shows, in the case of these three genes of overexpression, antibody CDR region series jump rate improves 3-12 times；
Under the conditions of the common overexpression of three kinds of genes, mutation rate more rises to 102Again (as shown in Figure 3).
Carry out chromatin immune co-precipitation with AID antibody, measure what AID albumen infected in Plv-DDB1 and Plv-SIRT2
The ability recruited to CDR (CDR3) Chromatin domains in VEGFR-E3 monoclonal cell strain.Result display overexpression DDB1 and
In the case of SIRT2, AID improves about 3 times (as shown in Figure 4) to the ability that antibody gene CDR (CDR3) region is recruited.
Brief description：
Fig. 1 is slow virus carrier Plv-AID mutant, Plv-DDB1 and Plv-SIRT2 enzyme action qualification figure.
Fig. 2 is expression green fluorescence after Plv-AID mutant, Plv-DDB1 and Plv-SIRT2 plasmid transfection incasing cellss
Albumen figure.
Fig. 3 is born of the same parents after hybridoma monoclonal cell strain (VEGFR-E3) infection DDB1, SIRT2 and AID mutant slow viruss
Interior antibody CDR site mutation rate increases figure.
Fig. 4 is ChIP experimental verification DDB1 and SIRT2 albumen can improve the enrichment figure in antibody gene CDR region for the AID.
Claims (1)
1. a kind of method making cell strain of monoclonal antibody carry out internal antibody tachytelic evolution using DNA damage repair mechanism, its
It is characterised by：Overexpression AID mutant and raising AID raising in VDJ site, overexpression DDB1 and SIRT2 gene, described
AID mutant is the mutant that the 38th amino acids of AID replace with glycine.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102482639A (en) * | 2009-04-03 | 2012-05-30 | 医学研究会 | Mutants of activation-induced cytidine deaminase (aid) and methods of use |
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| CN102482639A (en) * | 2009-04-03 | 2012-05-30 | 医学研究会 | Mutants of activation-induced cytidine deaminase (aid) and methods of use |
Non-Patent Citations (2)
| Title |
|---|
| "基因工程抗体亲和力成熟的策略";胡晓林 等;《免疫学杂志》;20061130;第22卷(第6期);第702-704页 * |
| "基因工程抗体亲和力成熟研究进展";于礼 等;《中国医药生物技术》;20120831;第7卷(第4期);第286页 * |
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