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Preparation method and application of high-throughput fully-humanized antibody

A fully human antibody, high-throughput technology, applied in botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as poor affinity, inability to ensure identical antibodies, and good cell accuracy

Active Publication Date: 2018-03-06
ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The development of antibody phage display library technology makes it possible to obtain antibodies in vitro without immunization, but the antibodies obtained are not natively paired, have low efficiency, have not been selected by human immune surveillance / tolerance, and are not truly fully human antibodies , has potential autoimmune reaction and immunogenicity, and often has poor affinity; fully human antibodies produced by EBV transformed B cell cloning technology are difficult to produce stable cell lines, and the antibody production efficiency is low; human produced by transgenic mice The source antibody is human antibody heavy chain and light chain gene knockout mouse, and the cloned antibody is obtained by traditional mouse monoclonal antibody technology after immunization of mice
However, the process of endogenous production and maturation of B cells / antibodies containing human antibody heavy chain and light chain genes occurs in mice, and is subject to immune surveillance / tolerance selection in mice, so it is not a real Fully human antibody with potential for autoimmune response and immunogenicity
[0003] CN102464717A discloses a preparation method of human-derived antinuclear antibody, which is essentially a phage display library technology. All the obtained cells are mixed together to isolate the heavy chain and light chain genes, and then randomly paired and combined. Antibodies identical to human natural antibodies
[0004] CN101451134A discloses a method for amplifying human antibody heavy chain and light chain gene fragments from human trace B cells, which uses magnetic bead sorting to obtain B cells, only uses one cell marker, and does not have flow sorting The accuracy of the cells obtained by using several cell marker combinations is good; in addition, this method uses 10 B cells for common gene amplification, and randomly pairs the amplified heavy chain and light chain genes, but still cannot Ensure that you get antibodies that are exactly the same as the body's natural antibodies

Method used

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  • Preparation method and application of high-throughput fully-humanized antibody
  • Preparation method and application of high-throughput fully-humanized antibody
  • Preparation method and application of high-throughput fully-humanized antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1 Sample Collection and PBMC Separation

[0118] According to different experimental purposes, through literature research, understand the pathogen information, pathogenesis, disease progression process, body immune response, main antigen structure and neutralizing epitope that cause these infectious diseases, autoimmune diseases or tumors and the development of corresponding treatments and vaccines. Then 5-10ml of blood at the time point with the highest neutralizing antibody titer was drawn for PBMC separation.

[0119] PBMC separation and cryopreservation: Freshly obtained blood samples were centrifuged at 2400 rpm for 7 minutes at room temperature, and another centrifuge tube was added to an equal volume of Ficoll (GE) for use; after centrifugation, the upper layer plasma was transferred to a new centrifuge tube for storage at -80°C, the middle layer Take the leukocytes into another centrifuge tube, add an equal volume of 1640 medium (Gibco) containing 5% F...

Embodiment 2

[0121] 1. Sorting plasma cells

[0122] In view of the significant increase in antigen-specific plasma cells after immunization, flow cytometry will be used to sort individual plasma cells for samples with clear immunization and appropriate sampling time. PBMC staining: mark the flow tube according to the staining scheme, and set the single-staining positive control, negative control and Isotype control; the plasma cell staining scheme is:

[0123] Combination 1: AqVD-Amycan- / CD19-BV605+ / CD3 / 14 / 16 / 235a-PE-Cy5- / CD20-PerCP- / IgD-PE- / CD138-FITC- / CD38-AF647+ / CD27-BV421+;

[0124] Combination 2: AqVD-Amycan- / CD19-PE+ / CD3 / 14 / 16 / 235a-PE-Cy5- / CD20-PerCP- / IgD-BB515- / CD138-PE-CF594- / CD38-AF647+ / CD27-BV421+;

[0125] Combination 3: AqVD-Amycan- / CD19-PerCP+ / CD3 / 14 / 16 / 235a-PE-Cy5- / CD20-PE-Cy7- / IgD-PE- / CD138-FITC- / CD38-BV605+ / CD27-BV421+.

[0126] Plasma cell sorting as figure 1 shown.

[0127] 2. Sorting memory B lymphocytes

[0128] Since the plasma cells will return to the basic leve...

Embodiment 3

[0134] Example 3 Single B Lymphocyte Gene Amplification

[0135] The isolated single peripheral nuclear cells were subjected to cloning of variable region genes of immunoglobulin heavy and light chains.

[0136] 1. Reverse transcription to synthesize cDNA:

[0137] Synthesize cDNA in a 20 μl reaction system in a 96-well PCR plate, 50ng / μl Random hexamer Primers, 1.5μl 25mMdNTPs, and 50U Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), and react on a PCR machine at 65°C 1 hour;

[0138] 2. Nested PCR amplification of antibody variable region genes:

[0139] Use the nested PCR primers provided by the present invention to carry out antibody variable region genes. Synthesize IgH, Igλ, and Igκ variable region genes in a 96-well plate with 50 μl reaction system for PCR reaction. The first round of PCR: 5 μl RT reaction product in 50 μl system, 5 units of HotStarTaq Plus enzyme (QIAGEN), 0.2 mM dNTPs, and 0.5μM IgH (VH1.1-VH7.1), Igκ (VK0.1-VK0.5) or Igλ (VL1.1-V...

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Abstract

The invention relates to gene engineering and antibody preparation and specifically discloses a preparation method and application of a high-throughput fully-humanized antibody. The preparation methodcomprises the following steps: separating peripheral blood monouclear cells; separating single cells of plasma cells or antigen-specificity memory B blymphocytes; amplifying the heavy-chain and light-chain gene variable regions of single B cell antibody by use of a primer provided by the inventor; establishing an expression system containing antibody heavy-chain and light-chain genes by use of alinear expression system containing a heavy-chain fragment or light-chain fragment constant area; finally, separating and purifying a fully humanized monoclonal antibody. By adopting the primer combination provided by the invention to amplify the genes in the antibody heavy-chain and light-chain variable areas, natural pairing of the light-chain and heavy-chain variable areas can be maintained, and the preparation method has the advantages of high gene diversity, high titer, full humanization, high antibody affinity, strong specificity, no heterologous serum reaction, no risk of propagating other infectious diseases, etc.

Description

technical field [0001] The present invention relates to genetic engineering and antibody preparation, in particular to the preparation method and application of high-throughput fully human monoclonal antibody. Background technique [0002] Compared with traditional small molecule chemical drugs, monoclonal antibody (Monoclonal antibodies, MAbs) drugs have higher targeting and specificity, and are a class of biotechnology drugs with the fastest compound growth rate in recent years. An important tool for human diseases such as infectious diseases, autoimmune diseases and tumors. At present, the methods for producing human antibodies mainly include various antibody phage display library technologies, EBV transformed B cell cloning technology and transgenic mouse technology. The development of antibody phage display library technology makes it possible to obtain antibodies in vitro without immunization, but the antibodies obtained are not natively paired, have low efficiency, h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N15/85C07K16/00C12N15/11C12Q1/6806
CPCC07K16/00C07K2317/21C07K2317/52C07K2317/56C12Q1/6806
Inventor 廖化新
Owner ZHUHAI TRINOMAB BIOTECHNOLOGY CO LTD
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