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Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application

A technology for bispecific antibodies and uses, which can be applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., and can solve problems such as adjustment disorders, safety risks, and high cost

Active Publication Date: 2012-03-14
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, monoclonal antibody combinations have many limitations
First of all, the safety and effect of the clinical application of two antibodies at the same time have not been reported in detail so far, and there may be safety risks; in addition, the combined application of the two antibodies also has the defects of regulatory barriers and high cost, which limits its clinical application

Method used

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  • Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application
  • Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application
  • Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Cloning of Human Antibody Light and Heavy Chain Constant Regions and Fc Region Genes

[0050] Isolate healthy human lymphocytes with lymphocyte separation fluid, extract total RNA with Trizol reagent (Invitrogen company product), according to literature (Cell, 1980, 22:197-207) and literature (Nucleic Acids Research, 1982, 10: 4071-4079 ) primers were designed to amplify the heavy chain and light chain constant region genes of the antibody respectively. The PCR reactions were all hot-started. The reaction conditions were: 94°C for minutes; 94°C for 45 seconds; 60°C for 45 seconds; 30 cycles; 10 minutes at 72°C. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (promega company product). After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 1 and SEQ ID NO: 2 show the nucleotide and amino acid sequences of the heavy chain constant region (CH), respectively. SE...

Embodiment 2

[0051] Example 2: Construction of novel bispecific antibodies

[0052]Specific construction method: According to the sequence reported in (Cancer Res.57 (1997) 4593-4599), the heavy and light chain variable region genes of Bevacizumab were synthesized from the whole gene, and the light and heavy chain variable region genes of Hu1A12, please refer to Chinese patent 200710039873.3 , WO2008128455. The heavy chain variable region of Bevacizumab was connected in series with the N-terminus of the heavy chain variable region of hu1A12 cloned by PCR through the Linker (ASTKGP, AST or GGGGS) or directly to construct VEGF / OPN-BsAb, The heavy chain variable regions of VEGF / OPN-BsAb1, VEGF / OPN-BsAb2, and VEGF / OPN-BsAb3 are then linked to the heavy chain constant regions of human IgG1 antibodies to construct VEGF / OPN-BsAb, VEGF / OPN- The heavy chain gene of BsAb1, VEGF / OPN-BsAb2, VEGF / OPN-BsAb3; the light chain variable region of Bevacizumab was passed through the Overlap PCR method via Li...

experiment example 1

[0057] Experimental example 1: Detection of binding activity of bispecific antibody to VEGF and OPN

[0058] Dilute VEGF (product of R&D Company) with 0.05mmol / L sodium carbonate-sodium bicarbonate buffer solution (pH 9.6) to 2ug / ml, 50ul / well, and coat overnight at 4°C. After blocking with 10% skimmed milk at room temperature for 2 hours, different concentrations of bispecific antibodies and bevacizumab (product of Roche Company) were added, 50ul / well, and 3 parallel wells were taken for each concentration, and incubated at room temperature for 2 hours. Discard the supernatant, wash with PBS three times, add HRP-labeled goat anti-human IgG monoclonal antibody (product of KPL Company) diluted according to the titer, 50ul / well, and incubate at room temperature for 45min. After fully washing with PBS and TMD color development, the absorbance (A450) value at 450 nm was measured in a microplate reader (Elx type automatic enzyme colorimeter, American BIO-TEK company). Experimental...

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Abstract

The invention, belonging to the technical field of biology, particularly discloses an anti-VEGF / anti-OPN double variable region IgG molecule like bispecific antibody VEGF / OPN-BsAb, its preparation method and application in preparing antitumor drugs. The antibody disclosed herein has amino acid sequences represented by SEQ ID No:10 and SED ID No:12, can be combined with VEGF and OPN, and has anti-neoplastic treatment effect similar with combing with Bevacizumab and hu1A12.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a class of anti-human VEGF and OPN bispecific antibody, its preparation method and its application in the preparation of antitumor drugs. Background technique [0002] Malignant tumor is a disease that seriously endangers human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve. [0003] Angiogenesis plays an important role in the growth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C07K16/18C12N15/11C12N15/63C12N5/10C12P21/00A61K39/395A61P35/00
Inventor 戴建新李博华赵健张大鹏王皓郭亚军
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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