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Building method and application of antibody library

A technology of antibody library and construction method, which is applied in the fields of application, chemical instruments and methods, antibody mimics/scaffolds, etc., can solve the problems of limited and variable available targets, complex antigens, etc., and achieve improved recognition accuracy, high affinity, good specific effect

Active Publication Date: 2019-04-05
SHENZHEN ISTIRBIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In view of the deficiencies in the prior art and actual needs, the present invention provides a method for constructing an antibody library and its application. The method is designed based on the principle that the synNotch system controls intracellular gene expression, and the extracellular recognition domain of the synNotch system is changed to The coding domain of the extracellular antibody library, the regulated target gene is changed into a screening marker gene, an antibody screening system activated by antigen is obtained, and the polyclonal antibody technology for screening complex antigens is obtained, so as to solve the problem of complex, diverse, variable and complex tumor antigens. A problem with limited available targets, with broad application prospects and huge market value

Method used

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  • Building method and application of antibody library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Screening of known CD19 and GPC3 antibodies

[0093] The schematic diagram of the synNotch system controlling gene expression in cells is shown in figure 1 , when the extracellular single-chain antibody of the synNotch system recognizes and binds to the antigen, the synNotch system undergoes inducible cleavage of the transmembrane region, thereby releasing the intracellular transcriptional domain into the nucleus, and combining with the upstream cis-activator to activate the expression of the regulated target gene ;

[0094] The schematic diagram of the antibody screening system based on synNotch is shown in figure 2 , based on the synNotch system, the extracellular recognition domain is changed to an extracellular antibody library coding domain, and the regulated target gene is changed to a screening marker gene, thereby obtaining an antibody screening system activated by antigen;

[0095] Screen antibodies specific to CD19 and GPC3 from the known CD19 and...

Embodiment 2

[0109] Example 2. Preparation of antibody library and screening of CD19 antibody expressing cells by flow cytometry

[0110] (1) Construction of a monoclonal cell line stably transfected with a cis-activator and a screening marker gene

[0111] In this embodiment, the cis-activator pTet is used. After receiving the tTA signal, pTet starts the expression of the screening marker green fluorescent protein gene, and artificially synthesizes the entire sequence ( Figure 15 ), constructed into a lentiviral vector. Pack the lentivirus and transfect it into 293T cells. After 3 days, the transfected cells are monoclonalized. Take out a part of the monoclonal cells to be screened and transfect the lentivirus expressing tTA. After 2-3 days, observe with a fluorescence microscope. Select a cell line with green fluorescent expression but no fluorescent expression before transfection, which is a monoclonal cell line that is stably transfected with a cis-activator and a screening marker ge...

Embodiment 3

[0122] Example 3. Preparation of antibody library and drug screening of CD19 antibody expressing cells

[0123] (1) Construct a monoclonal cell line stably transfected with a cis-activator and a selection marker gene;

[0124] In this example, the cis-activator pTet, the screening marker uses iCasp9 negative selection system (SEQ ID NO.21), green fluorescent protein, and puromycin resistance gene, and the three are connected with a 2A sequence that can be automatically broken;

[0125] By artificially synthesizing the entire coding sequence ( Figure 21 ), constructed into a lentiviral vector, packaged lentivirus, transfected into 293T cells, and 3 days later, the transfected cells were monoclonalized; a part of the monoclonal cells to be screened were respectively taken out and transfected to express tTA in Example 1 After 2-3 days, observe with a fluorescence microscope, and select a cell line with green fluorescent expression but no fluorescent expression before transfecti...

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Abstract

The invention provides a building method and application of an antibody library. The method is designed on the theory that a synNotch system controls the gene expression in cells; through groping by alarge number of experiments, the whole scheme flow process is optimized; through repeated design and verification, the extracellular recognition structural domain of the synNotch system is changed into the antibody library; a regulated and controlled target gene is changed into a screened marking gene, so that a simple, compact and efficient antibody screening method with various advantages is obtained; a polyclone antibody technology aiming at complex antigen can be screened; the problems of tumor antigen complexity, diversity, mutability and limited available targets are solved; wide application prospects and huge market values are realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing an antibody library and its application. Background technique [0002] Antibodies are immunoglobulins expressed by B cells that bind to specific antigens. In most animals, antibodies consist of pairs of heavy and light chains. Each chain is composed of two different regions, a variable region (Fv) and a constant region (Fc). Among them, the Fv regions of the heavy and light chains are responsible for binding to the target antigen, known as antigen-binding determinants. The antibody formed by connecting the heavy chain variable region and the light chain variable region through a short peptide (linker) of 15-20 amino acids is called a single-chain antibody (scFv). Antibody drugs dominate in the two fields of tumors and autoimmune diseases. Especially in the field of tumor treatment, immunotherapy such as monoclonal antibodies, monoclonal antibody-based anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/65C12N15/867C12N5/10
CPCC12N15/86C07K16/00C07K2319/22C07K2319/21C07K2319/43C07K2319/42C07K2319/41C12N2740/15043C07K16/005C07K16/2803C07K16/303C07K2317/622C12N2740/16043C07K2319/71C07K2319/035C12Q1/6897C12N15/1086C12N15/1037
Inventor 姚永超李友佳殷莎刘广杰
Owner SHENZHEN ISTIRBIO CO LTD
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