Bispecific antibody and preparation method and application thereof

A bispecific antibody and specific technology, applied in the fields of biotechnology and immunology, can solve the problems of limited curative effect of solid tumors

Active Publication Date: 2019-05-21
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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  • 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

[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 provides a bispecific antibody and a preparation method and application thereof. The bispecific antibody comprises a monoclonal antibody unit and a single-chain antibody unit, wherein the monoclonal antibody unit is composed of two intact light chain-heavy chain pairs, and is specifically bound with a tumor cell surface antigen; the single-chain antibody unit comprises two single-chain antibodies comprising heavy chain variable regions and light chain variable regions and is specifically bound with an immune cell surface antigen. The bispecific antibody is of a symmetrical structure formed through any one of the following connecting methods that (1) C ends of the two single-chain antibodies are connected with N ends of the two heavy chains of monoclonal antibodies through connecting peptides respectively; (2) N ends of the two single-chain antibodies are connected with C ends of the two heavy chains of the monoclonal antibodies through connecting peptides respectively. The bispecific antibody can be simultaneously bound with immune cells and tumor cells, mediate a guided immune reaction, and effectively kill and wound 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 Applications(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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