Humanized respiratory syncytial virus-resisting neutralizing gentically engineered Fab antibody

A genetically engineered antibody and genetic engineering technology, applied in the fields of bioengineering and biopharmaceuticals, can solve the problems of reduced antibody treatment potency, low success rate, and high cost

Inactive Publication Date: 2007-06-27
首都儿科研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty in screening and preparing human monoclonal antibodies by hybridoma technology, the success rate is low, the selection range is small, the expression cell line is unstable, the antibody titer is low, and the cost is expensive. The highest is only 90% humanization, and the remaining mouse-derived hypervariable regions will also produce severe human anti-mouse antibody rejection during long-term application of antibodies, resulting in reduced or invalid antibody therapeutic potency, and may even give patients with severe side effects

Method used

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  • Humanized respiratory syncytial virus-resisting neutralizing gentically engineered Fab antibody
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  • Humanized respiratory syncytial virus-resisting neutralizing gentically engineered Fab antibody

Examples

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example 1

[0017] Example 1: PCR amplification of the human IgG Fab segment gene: separate lymphocytes from the anticoagulated blood collected in the convalescent phase of RSV infected sick children (determined through virus isolation and double serum antibody determination) with lymphocyte separation fluid, use TriZol (Gibco, BRL, USA) to extract total cellular RNA, reverse-transcribe the extracted RNA into cDNA by reverse transcriptase (Gibco, BRL, USA) with Olig-dT primers, and use a set of specific IgG Fab Gamma chain, Kampa chain and Lamda chain Primers (for the sequence of primers, see the document "Acquisition and Expression of the Fab Segment Gene of Human Neutralizing Anti-Hantaan Virus Monoclonal Antibody", Acta Virology, Volume 13, Issue 4, Page 297-308, December 1997, Table 1), for human The source light and heavy chain Fab genes were PCR amplified. The PCR conditions are: 94°C for 1 minute, 54°C for 1 minute, 72°C for 2 minutes, 35 cycles (PE480), the above PCR products were...

example 2

[0018] Example 2: The establishment of phage antibody gene library: the Kamba and Lamda chain PCR products synthesized by different primers are mixed, the Fd chain synthesized by different primers is mixed, respectively cloned into phage vector pComb3 with SacI / XbaI and XhoI / SpeI, and cloned into The pComb3 vector DNA ligation products of the light and heavy chain genes were precipitated with ethanol, suspended in 10ul distilled water, and electroporated into 200ul electroporated bacteria XLI-Blu prepared in advance (electroporation conditions: Bio-Red electroporation instrument, 0.2cm electroporation cup, 2.5K volts), add 10ml of SOC culture solution after electroporation, add 10ml of SB culture solution with ampicillin and tetracycline at 37°C for 1 hour, add 80-100ml of the aforementioned SB, and add it after 2 hours at 37°C Helper phage VCSM131×10 12 PFU / ml, kanamycin (70ug / ml) was added after 1 hour, and cultured on a shaker at 37°C overnight. Precipitate the phage super...

example 3

[0019] Example 3: Preparation of RSV antigens used for antibody library enrichment screening: Respiratory syncytial virus was cultivated on Hep-2 cells until the cells appeared pathological changes and reached "++++", according to the literature (Crowe J.E.J., P.T.Bui, A.R.Davis, et al.A further attenuated derivative of a cold-passaged temperature-sensitive mutant of human respiratory syncytial virus retains immunogenicity and protective efficacy against wild-type challenge in seronegative chimpanzees. Vaccine, 1994, 12:783-79) method via sucrose pad Layer ultracentrifugation to purify the RSV antigen, and the purified RSV antigen can be directly used to coat the ELISA plate.

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Abstract

The present invention is humanized respiratory syncytial virus-resisting genetic engineering antibody including two Fab section antibody gene strains, the gene product and the application. The recombinant antibodies are functional antibodies combined specifically to respiratory syncytial virus and named as RSVFab58 and RSVFab88. The potential application is that by means of the Fab antibody or IgG whole antibody gene, the antibody may be produced in prokaryotic cell, yeast cell, insect cell and any other expression system for preventing and treating lower respiratory infection, especially serious lower respiratory infection, caused by respiratory syncytial virus.

Description

technical field [0001] The invention belongs to the technical fields of bioengineering and biopharmaceuticals. Background technique [0002] The invention relates to the preparation and application of human genetic engineering monoclonal antibody for prevention and treatment, especially the genetic engineering monoclonal antibody specific for respiratory syncytial virus. [0003] Respiratory syncytial virus (RSV) is the most important pathogen of lower respiratory tract infection in infants and young children in the world and in my country, especially severe lower respiratory tract infection (pneumonia, bronchiolitis), and is also a major risk factor for respiratory tract infection in immunosuppressed patients and the elderly. It can cause 6.5 million infants and young children to become ill every year in the world, and 1 million people die. However, there is no specific and effective drug treatment for severe infection caused by RSV. For a long time, people have been worki...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395A61P31/12
Inventor 钱渊梁米芳肖玮
Owner 首都儿科研究所
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