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Bispecific antibody and preparation method and application thereof

A bispecific antibody and heterodimer technology, applied in the direction of antibodies, chemical instruments and methods, antibody mimics/scaffolds, etc., can solve the problem of short half-life, small molecular weight, reduced production complexity and easy penetration through tissues and Tumor cells reach the target and other issues, to achieve the effect of high safety and efficient tumor killing effect

Active Publication Date: 2019-12-10
EXCELMAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bispecific antibodies that do not contain Fc, such as BiTE, DART, TandAbs, Bi-Nanobody, etc., have small molecular weight, can be expressed in prokaryotic cells, reduce production complexity, and easily pass through tissues and tumor cells to reach targets, but cannot Mediates Fc-related biological functions, will be quickly cleared in the body, and the half-life is very short. If the antibody introduces a non-natural peptide chain connection fragment or additional structure, the relative molecular weight and physical and chemical properties are quite different from natural IgG antibodies, and Easier to form multimers, resulting in immunogenicity [15] (Kontermann RE, et al. Drug discovery today 2015; 20(7):838-847)

Method used

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  • Bispecific antibody and preparation method and application thereof
  • Bispecific antibody and preparation method and application thereof
  • Bispecific antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Embodiment 1: expression vector construction

[0110] (1) Sequence design of CD20×CD3 BsAb

[0111] The bispecific antibody in the practice of the present invention comprises 3 or 4 polypeptide chains ( figure 1 ), respectively named as the first heavy chain (its variable region has the amino acid sequence shown in SEQ ID No.: 2), the first light chain (its variable region has the amino acid sequence shown in SEQ ID No.: 4), the first Second heavy chain (its variable region has the amino acid sequence shown in SEQ ID No.: 6), the second light chain (its variable region has the amino acid sequence shown in SEQ ID No.: 10) and scFv-Fc chain (having the amino acid sequence shown in SEQ ID No.: 10) The amino acid sequence shown in SEQ ID No.:11, SEQ ID No.:12, SEQ ID No.:13 or SEQ ID No.:14; or its variable region has SEQ ID No.:2, SEQ ID No.:4 , SEQ ID No.:6, SEQ ID No.:8 or the amino acid sequence shown in SEQ ID No.:10), forming an immunoglobulin domain specifically bi...

Embodiment 2

[0119] Example 2: Transient transfection and expression of bispecific antibodies

[0120]The endotoxin-free plasmid maxi kit (Endo-Free-Plasmid Maxi Kit (100), purchased from OMEGA, catalog number D6926-04) was used for massive extraction of plasmids, and the operation steps were performed according to the instructions provided by the kit. HEK293 cells were cultured to a cell density of 2.0-3.0×10 6 cells / mL, the cell suspension was centrifuged for 5min at a speed of 1000rpm, the old culture supernatant was discarded, and the cells were resuspended with fresh medium (OPM-291CD05Medium, purchased from Shanghai OPM Biotechnology Co., Ltd.) to a density of 1.0×106 / mL. Co-transfection was carried out according to the plasmid combinations provided in Table 1, and the transfected cell suspension was placed in a culture shaker at 37°C, 5% CO2, and 120rpm for 5-7 days in the dark, and supplemented with supplements on the 4th day. material.

[0121] Table 1 Transient transfection of...

Embodiment 3

[0124] Example 3 Purification of double antibodies

[0125] The expression supernatant was collected by centrifugation, the cell supernatant was filtered with a 0.22 μm filter membrane, and the protein A affinity chromatography filler (MabSelect SuRe TM , purchased from GE Healthcare, Cat. No. 17-5438-02) to capture the BsAb protein in the supernatant, using equilibration buffer (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 1.8mM KH 2 PO 4 ) after washing away non-specifically bound proteins (about 10 column volumes), elute with elution buffer (100mM glycine, pH 3.0-pH 3.5) for 5-10 column volumes, collect the eluate, and wash with neutralization buffer solution (1M Tris-HCl, pH 9.0) to adjust the pH to neutral. Eluted proteins were analyzed by SDS-PAGE. figure 2 -A, 2-B show that after one-step purification of the four-chain bispecific antibody by protein A, both V1 and V2 can reach high purity, and reducing electrophoresis shows that the ratio of the two heavy chains to ...

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Abstract

The invention relates to a bispecific antibody and a preparation method and application thereof. The bispecific antibody contains a structural domain capable of being bound to CD20, a structural domain capable of being bound to CD3 and a heterogeneous dipolymer Fc region, and the structural domain capable of being bound to the CD20 and the structural domain capable of being bound to the CD3 are independently selected from an Fab region, an ScFv region or an sDab region. The bispecific antibody can stably and efficiently target the CD20 and the CD3, and can mediate T-cell specific killing tumorcells.

Description

technical field [0001] The present invention relates to an antibody and its preparation method and application, in particular to a bispecific antibody and its preparation method and application. Background technique [0002] Specific antigens on the surface of B cells, such as CD19, CD20, CD22, CD52, etc., are potential therapeutic targets for B cell-related diseases. Among them, CD20 protein is also called B lymphocyte-restricted differentiation antigen, Bp35 or B1, which is human MS4A1 gene The four transmembrane, highly hydrophobic, non-glycosylated phosphoprotein encoded by the expression has a molecular weight of about 35kD [1-3] (StashenkoP, et al.J Immunol 1980,125(4):1678-1685; Einfeld DA, et al.EMBO J 1988,7(3):711-717; Oettgen HC, et al.Hybridoma 1983,2(1 ):17-28). CD20 is specifically expressed on the surface of pre-B cells and mature B cells, but not in hematopoietic stem cells, progenitor cells, plasma cells and other normal somatic cells. CD20 is also expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395A61P35/00A61P35/02A61P19/02A61P29/00A61P37/02A61P25/00
CPCC07K16/2887C07K16/2809C07K2317/33C07K2317/31C07K2317/732C07K2317/92C07K2317/94C07K2319/30A61K2039/505A61P35/02C07K2317/526C07K2317/622C07K2317/52C07K2317/522C07K2317/565
Inventor 张文军李峰华亚南刘嘉熙方春华
Owner EXCELMAB INC
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