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Fully humanized anti-tetanus bispecific antibody as well as construction method and application thereof

A bispecific antibody and fully humanized technology, applied in the biological field, can solve the problems of unreported monoclonal antibody sequences, sequences that have not been cloned and expressed, and have no production and application value, so as to reduce human allergies and overcome the source The effect of limited and significant economic and social benefits

Active Publication Date: 2020-05-08
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the use of hybridoma monoclonal technology and phage display technology, people have gradually used these technologies for research in this field, but there are still some problems in these researches, such as: ① Some articles did not report the sequence of the monoclonal antibody screened Or no sequencing was performed; ②The screened sequences were not cloned and expressed, but only sequence analysis was done; ③The cloned and expressed sequences were only subjected to in vitro ELLISA and / or in vitro binding experiments, and the protective effect in vivo was unknown; ④Some sequences were expressed Afterwards, in vivo neutralization and protection experiments in mice were carried out. A common idea is to mix multiple monoclonal antibodies similar to the "cocktail" method. There are often two options for this idea. One is to express the monoclonal antibodies separately and then mix them; the other is to mix them. express monoclonal antibody
The former is more than twice the production cost of recombinant monoclonal antibody, and has no production application value
And the latter has the shortcoming that the product purity is low and the yield is low and the cost is very high

Method used

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  • Fully humanized anti-tetanus bispecific antibody as well as construction method and application thereof
  • Fully humanized anti-tetanus bispecific antibody as well as construction method and application thereof
  • Fully humanized anti-tetanus bispecific antibody as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Recombinant expression and purification of tetanus toxoid and mutants.

[0032] Expression sequence plasmid construction

[0033] The tetanus toxoid (Genbank: P04958) protein sequence 864-1315 (amino acid sequence as shown in SEQ ID No.1) is named as TTC, and the corresponding amino acids are mutated to obtain (Y1290A)TTC, (R1226A) respectively ) TTC and (Y1290A / R1226A) TTC mutants. Based on the codon preference characteristics of Escherichia coli, these mutants are optimized to be suitable for expressing codons in Escherichia coli: TTC (nucleotide sequence as shown in SEQ ID No.2, optimized according to the codon preference of Escherichia coli The nucleotide sequence of the tetanus toxin heavy chain C fragment, TGGCCA at the 5-terminal is the MSCI restriction site, and CTCGAG at the 3-terminal is the XhoI restriction site), (Y1290A)TTC, (R1226A) and (Y1290A / R1226A)TTC, Please Shanghai Jierui Biosynthetics synthesized these sequences separately, and then add...

Embodiment 2

[0045] Example 2 Construction, screening and sequencing of fully humanized phage immune antibody library

[0046] 2.1 Construction of fully humanized phage immune antibody library

[0047] 1) PCR amplification of human tetanus VH and VL genes

[0048] Total RNA was extracted from the peripheral blood lymphocytes of 3 healthy plasma donors with high tetanus antibody titers according to the Invitrogen RNA extraction kit instructions. Using purified RNA as a template, cDNA was synthesized by reverse transcription with OligodT primers. According to the human antibody sequence, the antibody light and heavy chain variable region framework primers (nature biotechnology 14:309-314 (1996)) were used. The primers covered 7 kinds of VH family genes, 7 kinds of Vκ family genes, 8 kinds of Vλ family genes, antibody genes Covering more than 95% of the diversity of human antibody genes. PCR conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 57°C fo...

Embodiment 3

[0067] Example 3 Bispecific antibody expression, purification and identification

[0068] 3.1 Construction of vector pCHO 1.0-hygromycin

[0069] Take a section of nucleotide sequence 2118-3141 of pcDNA3.1 / Hygro (the nucleotide sequence encoding the hygromycin gene, as shown in SEQ ID No.3), and replace the section 8860-9459 of the vector pCHO 1.0 to make it PGK The promoter (phosphoglycerate kinase promoter) expresses hygromycin.

[0070] bispecific expression vector

[0071] 3.2.1 Fc + hinge region knob mutant (or hole mutant) sequence

[0072] The amino acid sequence 221-446 (EU coding system) of human IgG1 (IMGT named IGHG1) contains three regions: 221-234 is the Hinge region, and 234-341 is the C H 2 area and 342-446 for C H 3 area. In the present invention, refer to literature (nature biotechnology (1998), 16 (7): 677-681) to C H The amino acids in the 3 regions are mutated, and one chain expresses the knob mutant of S354C and T366W (see SEQ ID No.6 for its polypept...

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Abstract

The invention provides a fully humanized anti-tetanus bispecific antibody. The bispecific antibody includes a single-stranded unit A and a single-stranded unit B, both the single-stranded units A andB contain a single-stranded variable fragment (scFv), a human IgG1 hinge region and an Fc fragment, wherein the scFv of the single-stranded unit A has specific binding ability to a region centered onTrp 1289 of tetanus toxoid, and the scFv of the single-stranded unit B has specific binding ability to a region centered on Arg1216 of the tetanus toxoid. The invention also provides a construction method and application of the bispecific antibody. After neutralization experiments in mice, results show that the antibody has a neutralizing protection effect, and the bispecific antibody can be usedas a diagnostic reagent to detect the quality of a tetanus toxoid antigen, and can replace the tetanus antitoxin, equine tetanus immunoglobulin or human tetanus immunoglobulin to be used for passive immunization.

Description

technical field [0001] The invention belongs to biotechnology and relates to the technical field of genetic engineering. More specifically, the invention discloses a bispecific anti-tetanus toxoid antibody, especially a fully humanized anti-tetanus bispecific antibody; in addition , the present invention also relates to the construction method of the fully humanized anti-tetanus bispecific antibody, and the application of the fully humanized anti-tetanus bispecific antibody in passive treatment after tetanus infection. Background technique [0002] Tetanus is a specific infection caused by Bacillus tetani invading wounds, and its main pathogen is the tetanus toxoid produced by Bacillus tetani. Tetanus toxoid is a protein with a molecular weight of approximately 150 kDa consisting of 1315 amino acids. After the proteolytic enzyme of Bacillus tetani, the polypeptide is cleaved between 456 and 467 amino acid residues into about 50KD light chain (LC) and 100KD heavy chain (LN),...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K1/22C07K1/18G01N33/569A61K39/40A61P31/04
CPCA61P31/04C07K16/1282C07K2317/35C07K2317/622C07K2317/76C07K2319/30G01N33/56911Y02A50/30
Inventor 李鑫饶深铖范铁炯孙九如柏伟
Owner SHANGHAI SERUM BIOTECH
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