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Method for preparing bispecific antibodies

A bispecific antibody and preparation process technology, applied in chemical instruments and methods, specific peptides, peptides, etc., can solve problems such as binding effects

Pending Publication Date: 2022-05-06
HANGZHOU JINGYINKANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, changing the variable domain of the Fab may have a greater impact on its binding to the antigen

Method used

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  • Method for preparing bispecific antibodies
  • Method for preparing bispecific antibodies
  • Method for preparing bispecific antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] Example 1. Design of an IgG1 (Kappa) CH1 and CL domain "cysteine ​​mutation" library

[0158] The CH1 domain sequences of IgG1, IgG2, IgG3 and IgG4 are highly conserved. The light chain is linked to the heavy chain by an interchain disulfide bond in the CH1-CL domain. In the present invention, we hope to replace the natural interchain disulfide bonds with engineered interchain disulfide bonds by mutating the CH1-CL domain of IgG. Using the Fab crystal structure of human IgG1 (kappa) from the Protein Data Bank (PDB code: 1VGE) as a representative structure, cysteine ​​mutations in the CH1 and CL domains that might interact to form interchain disulfide bonds were analyzed and designed location. The cysteine ​​pairs that form natural interchain disulfide bonds are mutated to valine, and new cysteine ​​pairs are introduced at different positions in the CH1-CL domain to form engineered interchain disulfide bonds, by Therefore, an IgG1 (kappa) mutant library was designed. ...

Embodiment 2

[0162] Example 2 Construction and expression of IgG1 (Kappa) "cysteine ​​mutation" library

[0163] Trastuzumab (Trastuzumab, ERBB2 Ab) heavy chain (HoleRF-His) containing T366S / L368A / Y407V "hole" mutations, H435R, Y436F (RF) mutations [EU numbering] and a 6xHis tag at the C-terminus, used as Representative of IgGl (Kappa) heavy chain (SEQ ID NO: 1). Trastuzumab heavy chain (HoleRF-His) codon-optimized DNA was synthesized and used as template for PCR amplification (SEQ ID NO: 2).

[0164]PCR 1, TraH F (SEQ ID NO:3, the primer has an Xho I restriction enzyme site) and TraH R (SEQ ID NO:4, the primer has a Not I restriction enzyme site) primer pair for Trastuzumab PCR amplification of the heavy chain (HoleRF-His) using synthetic DNA as template. The PCR product was digested with Xho I and Not I, and inserted into the same digestion site in pPIC9 (Invitrogen) to construct the expression vector of Trastuzumab heavy chain (HoleRF-His), which was named pPIC9-TraH ( Hole RF-His). ...

Embodiment 3

[0195] Example 3. Screening and identification of IgG1 (Kappa) "cysteine ​​mutation" library

[0196] The concentration of the bispecific antibody in the culture supernatant was measured by ELISA. Briefly, 100 μL / well of 5 μg / mL AGL protein (Leeanntech), 50 mM sodium carbonate buffer (pH 9.6) was added to a 96-well plate (Maxisorp Nunc-Immuno, Thermo Scientific), and coated overnight at 4°C. After washing the plate 3 times with PBS-T (PBS containing 0.05% Tween-20), the plate was blocked with 2% non-fat dry milk in PBS-T for 1 hour at 37°C. After washing the plate 3 times with PBS-T, add 100 μL / well of human IgG standard (initial concentration 2.5ug / ml) and culture supernatant diluted 1:2 in PBS-T, at 37°C Incubate for 1 hour. The plate was washed 3 times with PBS-T, and 100 μL / well of AGL-HRP (Leeanntech) diluted 1:5000 in PBS-T and 0.5% skimmed milk powder was added. The plate was incubated at 37° C. for 1 hour, washed 3 times with PBS-T, and 100 μL / well of TMB (Leeanntec...

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Abstract

The invention provides a method for improving the correct pairing rate of a heavy chain and a light chain in a preparation process of a bispecific antibody, which comprises the following steps of: eliminating a natural interchain disulfide bond on a CH1-CL structural domain of a Fab arm through amino acid substitution, and simultaneously forming an engineered interchain disulfide bond through amino acid substitution. The method of the invention further comprises the step of reversing the charge of a pair of amino acids of the CH1-CL domain of the Fab arm by amino acid substitution. The method provided by the invention can significantly improve the correct pairing rate of the heavy chain and the light chain in the production of the bispecific antibody, and is suitable for various antibody types.

Description

technical field [0001] The invention relates to the field of biotechnology; specifically, the invention relates to a new method for preparing bispecific antibodies and its application. Background technique [0002] Recombinant monoclonal antibodies (mAbs) that bind antigens with high efficiency and specificity offer exciting opportunities in the fields of biomedicine and biotechnology. A number of therapeutic monoclonal antibodies have been successfully used to treat a variety of diseases, including cancer, immune disorders, and infections. Furthermore, bispecific antibodies (bsAbs) targeting more than one antigen have shown great potential to maximize the benefits of antibody therapy. [0003] Antibodies are proteins that recognize and specifically bind to antigens (commonly known as immunoglobulins, Immunologlobulin, abbreviated as Ig). In most mammals, including humans and mice, immunoglobulins consist of two identical heavy chains and two identical light chains. Each ...

Claims

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Application Information

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IPC IPC(8): C07K16/46
CPCC07K16/468C07K2317/55C07K16/46C07K16/00
Inventor 姜有为徐飞李发慧郑奔刘金贵
Owner HANGZHOU JINGYINKANG BIOTECH CO LTD
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