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A kind of bispecific antibody and its preparation method and application

A bispecific antibody and single-chain antibody technology, applied in the fields of biotechnology and immunology, can solve problems such as limited efficacy in solid tumors

Active Publication Date: 2020-04-07
EXCYTE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunotherapy can only remove a small amount of scattered tumor cells, and has limited efficacy for advanced solid tumors

Method used

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  • A kind of bispecific antibody and its preparation method and application
  • A kind of bispecific antibody and its preparation method and application
  • A kind of bispecific antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Structure and sequence design of CD19×CD3 bispecific antibody

[0077] In this example, a bispecific antibody was designed using the tumor cell surface antigen CD19 and the immune cell surface antigen CD3 as targets.

[0078] Combining protein structure design software and a large number of manual experimental screening, the present invention screened and determined bispecific antibodies with symmetrical structures including single-chain antibody units and monoclonal antibody units in a variety of bispecific antibody structures that bind to CD19 and CD3 structure. Among them, the anti-CD19 monoclonal antibody unit is an IgG antibody, including two complete light chain-heavy chain pairs (that is, containing complete Fab and Fc domains, and the heavy chain and light chain are connected by disulfide bonds). - The CD3 single-chain antibody unit includes two single-chain antibodies (ScFv), each of which includes a heavy chain variable region and a light chain vari...

Embodiment 2CD19

[0087] Example 2 Preparation of CD19×CD3 bispecific antibody

[0088] 1. Design and synthesis of bispecific antibody coding genes

[0089] According to the amino acid sequences of the two bispecific antibodies YK001 and YK002 obtained in Example 1 and the codon preference of the host cell, the coding genes of the bispecific antibodies were designed. The specific sequences are as follows:

[0090] The heavy chain coding nucleotide sequence of the anti-CD19 monoclonal antibody of YK001 is shown in SEQ ID NO.2;

[0091] The heavy chain coding nucleotide sequence of the anti-CD19 monoclonal antibody of YK002 is shown in SEQ ID NO.21;

[0092] The light chain coding nucleotide sequence of the anti-CD19 monoclonal antibody is shown in SEQ ID NO.4 (YK001 and YK002 are the same);

[0093] The coding nucleotide sequence of the anti-CD3 single chain antibody in YK001 is shown in SEQ ID NO.14;

[0094] The coding nucleotide sequence of the anti-CD3 single chain antibody in YK002 is sh...

Embodiment 3

[0122] Example 3 Determination of Binding Activity of Bispecific Antibodies to Tumor Cells and Immune Cells

[0123] Using Raji (purchased from ATCC, CCL-86) as CD19-positive cells, and T cells as CD3-positive cells, the bispecific antibody of the present invention and the tumor cells expressing CD19 and the immune cells expressing CD3 were detected by flow cytometry. The binding activity of the cell to the target antigen.

[0124] 1. Detection of binding activity of bispecific antibody to Raji cells by flow cytometry

[0125] (1) Collect Raji cells: collect 1×10 6 cells / tubes.

[0126] (2) Rinse the cells: Rinse the cells once with 1ml staining buffer (PBS containing 0.5% w / v BSA+2mM EDTA), centrifuge at 350×g, 4°C for 5min, and resuspend the cells with 200μl staining buffer after centrifugation.

[0127] (3) Bs-antibody binding: add bispecific antibodies YK001 and YK002 to 5 μg / ml respectively, and incubate on ice for 45 minutes.

[0128] (4) Rinse the cells: add 1ml sta...

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Abstract

The invention relates to a bispecific antibody and its preparation method and application. The bispecific antibody of the present invention includes a monoclonal antibody unit and a single-chain antibody unit; wherein, the monoclonal antibody unit is two complete light chain-heavy chain pairs that specifically bind to tumor cell surface antigens; the single-chain antibody unit includes 2 A single-chain antibody, the single-chain antibody includes a heavy chain variable region and a light chain variable region, and specifically binds to an immune cell surface antigen; the bispecific antibody provided by the present invention is a symmetrical structure connected by any of the following methods : (1) The C-terminals of the two single-chain antibodies are connected to the N-terminals of the two heavy chains of the monoclonal antibody through connecting peptides; (2) The N-terminals of the two single-chain antibodies are connected to the N-terminals of the monoclonal antibodies through connecting peptides C-terminal junction of the 2 heavy chains. The bispecific antibody of the present invention can simultaneously bind immune cells and tumor cells, mediate a directed immune response, and effectively kill tumor cells.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and immunology, in particular to a bispecific antibody binding to CD19 and CD3 and its preparation method and application. Background technique [0002] Antibody drugs are biomacromolecular drugs prepared by antibody engineering technology based on cell engineering technology and genetic engineering technology. They have the advantages of high specificity, uniform properties, and directional preparation for specific targets. Monoclonal antibodies are mainly used clinically in the following three aspects: tumor treatment, immune disease treatment and anti-infection treatment. Among them, tumor treatment is currently the most widely used field of monoclonal antibodies. Among the monoclonal antibody products that have entered clinical trials and are on the market, the number of products used for tumor treatment accounts for about 50%. Monoclonal antibody treatment of tumors is an immunotherap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00A61P37/02G01N33/68
CPCA61P37/02A61P35/00C07K2317/31C07K16/2803C07K16/2809C07K2317/622C07K2317/60C07K2319/00C07K2317/92C07K2317/73G01N2333/70596G01N2333/7051C07K16/468
Inventor 袁清安孟庆武白丽莉赵立坤李延虎
Owner EXCYTE LLC
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