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Anti-CD3/anti-CD19 dual-specific antibody and application thereof

A bispecific antibody and single-chain antibody technology, applied in the field of anti-CD3/anti-CD19 bispecific antibody, can solve the problem of weakening the immune effect of tumors, and achieve the effect of avoiding side effects and high affinity

Inactive Publication Date: 2015-11-04
SHANDONG BAYONN PHARMA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The monoclonal antibody binds to the effector cell surface active receptor FcγRI / FcγRIII through the Fc segment, thereby mediating the killing effect, but the T cells with immune killing effect cannot be effectively mediated because of the lack of the above-mentioned receptors on the surface, thus weakening the body's ability to fight against tumors. immune effect

Method used

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  • Anti-CD3/anti-CD19 dual-specific antibody and application thereof
  • Anti-CD3/anti-CD19 dual-specific antibody and application thereof
  • Anti-CD3/anti-CD19 dual-specific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Obtaining of anti-human CD3 and CD19 single-chain antibodies

[0032] 1. Preparation of cDNA

[0033] Collect 10 ml of peripheral blood from 50 people with liver cancer, kidney cancer, melanoma, and healthy people, anticoagulate with sodium heparin, separate mononuclear cells (PBMC) by density gradient centrifugation, and sensitize with CD3 or CD19 antigen in vitro. After PBMC were sensitized in vitro, press 4×10 6 Add 1ml of EBV culture supernatant to PBMC, incubate at 37°C for 3h, discard the supernatant, add RPMI1640 complete medium containing 100ml / L FCS, and after 2 weeks of culture, replace with RPMI1640 complete medium containing 200ml / L NBS. The culture supernatant of transformed cells was screened by indirect ELISA method, CD3 or CD19 was used as the tumor cell antigen solid phase plate, the collected B cell culture supernatant was used as the primary antibody, and HRP-labeled goat anti-human IgG / M (HRP-GAH-IgG / M) as the secondary antibody. Cells...

Embodiment 2

[0045] Example 2: Expression and purification of single-chain bispecific antibodies against CD3 and CD19

[0046] The bispecific single chain antibody construct comprises the specific binding domains of human CD3 and human CD19, wherein the corresponding branchable heavy chain region (V H ) and the corresponding variable light chain region (V L ) from N-terminal to C-terminal in the following order: V H (CD19)-V L (CD19)-V H (CD3)-V L (CD3) or V H (CD3)-V L (CD3)-V H (CD19)- L (CD19).

[0047] 1. Expression of anti-CD3 / anti-CD19 mini bifunctional antibody. A single colony of Escherichia coli containing the recombinant plasmid was selected and inoculated into 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl) containing ampicillin (0.1 mg / mL), and cultured with shaking at 37°C for 8 h, Transfer to Ap5 medium containing ampicillin (0.1mg / mL) at a ratio of 1:2 (0.6g / L yeast extract, 11g / L acid hydrolyzed casein, 1.5g / L glucose, 1.2g / L NaCl, 3.73g / L KCl, 1.07g / L NH...

Embodiment 3

[0055] Example 3: Determination of the binding activity of anti-CD3 / anti-CD19 bispecific antibody by indirect immunofluorescence

[0056] To test the function of the constructs with respect to CD19 and CD3 binding capacity, flow cytometry analysis (FACS) was performed. For this, CD19-positive Nalm6 cells (human B-cell precursor leukemia) and CD3-positive Jurkat cells (human T-cell leukemia) were used. Incubate 200,000 Nalm6 cells and 200,000 Jurkat cells with 50ul purified anti-CD3 / anti-CD19 bifunctional antibody on ice for 30min, centrifuge at 300×g, 4°C for 5min, discard the supernatant, wash the cells with cold PBS, repeat 3 times. Cells were incubated with mouse anti-His-tag monoclonal antibody (diluted 1:1000) for 1 h at 4°C, the antibody specifically binds to the cell-bound construct through the C-terminal histidine tag of the construct, 300×g, Centrifuge at 4°C for 5 minutes, discard the supernatant, wash the cells with PBS, and repeat 3 times to remove unbound mouse ...

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Abstract

The invention discloses an anti-CD3 / anti-CD19 dual-specific antibody and an application thereof. The anti-CD3 / anti-CD19 dual-specific antibody comprises the combined structural domain of a completely-humanized anti-CD3 single-chain antibody and a completely-humanized anti-CD19 single-chain antibody. The variable region amino acid sequence of heavy chains and light chains of the completely-humanized anti-CD3 single-chain antibody is shown as the SEQ ID NO:7-8. The variable region amino acid sequence of heavy chains and light chains of the completely-humanized anti-CD19 single-chain antibody is shown as the SEQ ID NO:15-16. The anti-CD3 / anti-CD19 dual-specific antibody has high affinity to CD3 molecules and CD19 molecules, and has the effect of conducting mediated activation on T lymphocytes to kill tumor cells. Moreover, the heavy chains and the light chains of the antibody molecules are completely humanized, and thus plenty of side effects are avoided.

Description

technical field [0001] The invention relates to the technical field of antibody drug development and production. Specifically, the present invention relates to anti-CD3 / anti-CD19 bispecific antibodies and applications thereof. Background technique [0002] B lymphocytic leukemia and malignant lymphoma are malignant tumors that originate in the bone marrow hematopoietic system and lymph nodes and spread throughout the body. Although traditional radiotherapy and chemotherapy have certain curative effects, they are not selective and cause great damage to normal tissues. In recent years, biotherapeutic methods have been widely used in tumor treatment, especially monoclonal antibodies such as rituximab , and received good results because of its specific targeting and high affinity. The monoclonal antibody binds to the effector cell surface active receptor FcγRI / FcγRIII through the Fc segment, thereby mediating the killing effect, but the T cells with immune killing effect can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/63A61K39/395A61P35/00A61P35/02
Inventor 张守涛闫庆连董相陈董成涛张凤展
Owner SHANDONG BAYONN PHARMA TECH
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