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Heterodimer formed by CH3 structural domain transformation induction as well as preparation method and application thereof

A technology of heterodimers and structural domains, applied in the field of heterodimers and their preparation, to achieve the effects of increased yield, simple construction method, and less point mutations

Active Publication Date: 2021-07-16
周易
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the method disclosed in this patent can increase the formation of heterodim

Method used

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  • Heterodimer formed by CH3 structural domain transformation induction as well as preparation method and application thereof
  • Heterodimer formed by CH3 structural domain transformation induction as well as preparation method and application thereof
  • Heterodimer formed by CH3 structural domain transformation induction as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Design, Construction, Expression and Purification of the First Round of Mutation Candidate Combinations

[0084] 1. Design of the first round of CH3 domain amino acid modification

[0085] The vast majority of antibodies on the market belong to the IgG1 or IgG4 subtype, and the amino acid sequences of the Fc segments of IgG1 and IgG4 are highly conserved. In the present invention, IgG1 is preferably used as a template to design amino acid modifications of the CH3 domain, and these amino acid modifications are also applicable to IgG4 subtypes unless otherwise specified.

[0086] The CH3 domain will form a homodimer, and the crystal structure of the CH3 domain of the antibody Fc segment of IgG1 (pdbcode: 4BSW) is as follows figure 1 As shown, K409 of CH3_A is located in the cavity surrounded by F405, D399 and K370 of CH3_B, and forms an ionic interaction with the side chain of D399. The above sites are highly conserved on IgG4, and the only difference from IgG...

Embodiment 2

[0094] Example 2. Design, construction, expression and purification of the second round of mutation candidate combinations

[0095] In Example 1, the formation of homodimers (scFv-Fc / scFv-Fc) was inhibited by electrostatic repulsion by introducing positively charged amino acids into the CH3_B chain. This example aims to further explore ways to reduce the mutual attraction between CH3_B chains and inhibit the formation of homodimeric proteins by introducing a combination of other charged amino acid mutations. Based on the existing K409F and F405E point mutation combinations in the CH3_A domain, the following charged amino acid mutation combinations are further introduced into the CH3_A domain and CH3_B domain:

[0096] CH3_A domain: K392D, CH3_B domain: D399K;

[0097] CH3_A domain: K439D, CH3_B domain: E356K+E357K;

[0098] CH3_A domain: L368D, CH3_B domain: S364R;

[0099] CH3_A domain: L368D, CH3_B domain: S364K;

[0100] CH3_A domain: K360E, CH3_B domain: Q347R;

[010...

Embodiment 3

[0107] Example 3 Effect of transfection ratio on scFv-Fc / Fc heterodimer formation

[0108] In order to further investigate the effect of the co-transformation ratio of the recombinant vector of the A chain and the recombinant vector of the B chain on the ratio of homodimers and heterodimers, the co-transformation expression vectors used in the better mutation combination 4 were recombined with CH3_A The ratio of carrier: CH3_B recombinant vector = 1:1 and CH3_A recombinant vector: CH3_B recombinant vector = 1.5:1 was transfected with PEI to 293E cells in suspension culture, and after culturing for 5-6 days, the cell supernatant was collected. The respective transient products were obtained by Protein A affinity chromatography. The compositions of homodimeric proteins (scFv-Fc / scFv-Fc, Fc / Fc) and heterodimeric proteins (scFv-Fc / Fc) were detected by SDS-PAGE electrophoresis under non-reducing conditions. For specific results, see Figure 4 . It can be seen from the results th...

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Abstract

The invention belongs to the field of antibody engineering, and provides a heterodimer as well as a preparation method and application thereof. According to the heterodimer, various interactions between interface amino acids, such as ionic interaction, hydrophobic interaction, spatial interaction and the like, are comprehensively considered, a knob-into-hole model and electrostatic interaction are combined, and an optimal CH3 mutation sequence is screened, which is more likely to form a heterodimer rather than a homodimer, therefore, the yield of heterodimer molecules is greatly improved. Compared with reference literatures CN106883297A and US20150307628A1, the construction method of the knob-into-hole model provided by the invention is simpler, point mutation is less, and the purity of the heterodimer can be improved to 95% or more under the condition that disulfide bonds are not introduced.

Description

technical field [0001] The invention belongs to the field of antibody engineering, and in particular relates to a heterodimer induced by transformation of CH3 domain and its preparation method and application. Background technique [0002] Bispecific antibodies can be constructed in a variety of ways, among which IgG bispecific antibodies have similar structures, physicochemical properties and Fc segment functions as ordinary antibodies. Generally, IgG type bispecific antibodies consist of two heavy chains with different amino acid sequences (i.e. heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (i.e. light chain against antigen A). chain LC_A and light chain LC_B against antigen B). When 4 polypeptide chains are combined, homodimers and heterodimers will be formed between the two heavy chains, and mismatches will also be formed between the light and heavy chains, so there will be 8 different...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/85
CPCC07K16/468C07K16/00C12N15/85C07K2317/526C07K16/46A61K39/395
Inventor 周易
Owner 周易
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