Bispecific antibody to VEGF/PDGFR beta and application thereof

A bispecific antibody, antibody technology, applied in the direction of antibodies, anti-tumor drugs, hybrid immunoglobulins, etc., can solve the problems of toxicity and high toxicity, achieve good tumor activity, inhibit growth, and avoid the increase of drug dosage Effect

Inactive Publication Date: 2011-11-23
CHANGZHOU ADAM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these small molecule inhibitors have certain toxicity to the human body, especially when they are combined with chemotherapy (Sosman et al.2007, Roodhart et al.2008)

Method used

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  • Bispecific antibody to VEGF/PDGFR beta and application thereof
  • Bispecific antibody to VEGF/PDGFR beta and application thereof
  • Bispecific antibody to VEGF/PDGFR beta and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Preparation method of anti-VEGF-PDGFRbeta bispecific antibody

[0071] 1. Nucleotide sequence design and synthesis of anti-VEGF-PDGFRbeta bispecific antibody (bsAb):

[0072] According to the bsAb heavy chain VH VEGF -CH1 VEGF -CH2 VEGF -CH3 VEGF -(GGGGS) 3 -VH PDGFRbeta -(GGGGS) 3 -VL PDGFRbetaThe heavy chain nucleotide sequence was designed according to the amino acid sequence and connection form, and a Sac I restriction site, KOZAK sequence and leader sequence were added at the 5' end of the sequence, and an Xho I restriction site was added at its 3' end. Design three oligonucleotide primers containing EcoRI restriction site at the 3' end to synthesize the heavy chain VH respectively VEGF -CH1 VEGF , CH2 VEGF -CH3 VEGF -(GGGGS) 3 and VH PDGFRbeta -(GGGGS) 3 -VL PDGFRbeta Fragment, the full-length 2118bp bsAb heavy chain gene was synthesized by PCR direct ligation method (SDL PCR).

[0073] According to the amino acid sequence of human anti-VEGF-A IgG1...

Embodiment 2

[0080] Antigen-Antibody Binding Experiment

[0081] Antigen-antibody binding experiments were performed using Biacore T 100 system (GE Healthcare) with high-density VEGF-A (1000RU) immobilized on a CM4 chip through covalent binding, and bsAb was diluted to 100nM and injected into the system so that it flowed at a flow rate of 10uL / min Over-immobilize the VEGF-A surface on the chip for a total of 5 minutes. PDGFRβ (500nM) was then infused at a flow rate of 30uL / min for 10 minutes. The binding curves in the experimental results were automatically generated by Biacore Evaluation Software vl.1.1. The curve obtained from the co-binding experiment is basically consistent with the curve generated by the binding of the bispecific antibody to VEGF-A or PDGFRβ respectively, indicating that the antibody can specifically bind to VEGF-A and PDGFRβ at the same time. See Figure 4 . Anti-VEGF-PDGFRβ bispecific antibody (bsAb) simultaneously binding hVEGF-A and PDGFRβ assay (Biacore). In ...

Embodiment 3

[0083] Comparison of Antibody Inhibition Blood Vessel Experiments

[0084] HUVECs cells were inserted into 96-well plates with a density of about 1000 cells per well, cultured in EGM-2MV medium at 37°C for 48 hours, and then cells were placed in serum-free medium containing 1 times insulin-transferrin-sodium selenium Incubate at 37°C. After 24 hours, the original medium was removed, replaced with anti-VEGF-A full antibody (bevazimumab), anti-VEGF-A Fab or bsAb containing 2.6nM hVEGF-A and different concentrations (0.0005nM-500nM), and cultured at 37°C for 24 hours. After that, 1uCi / well of 3H-thymidine was added to the cells and cultured at 37°C for 24 hours.

[0085] HBVPs cells were inserted into 96-well plates with a density of about 500 cells per well. The cells were cultured at 37°C for 48 hours in a medium containing serum and PGS, and then cultured at 37°C for 24 hours in a serum-free medium. Add 0.4nM PDGFR β to stimulate cell growth, and at the same time add differ...

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Abstract

The invention relates to a medicine of a bispecific monoclonal antibody, and especially to a medicine of a bispecific monoclonal antibody to human vascular endothelial growth factor (VEGF/VEGF-A) and platelet-derived growth factor receptor (PDGFR) for resistance to angiogenesis of tumor. The bispecific antibody to VEGF/PDGFR beta provided in the invention is characterized in that: a monoclonal antibody to VEGF is used as the base for the antibody and a single chain antibody to PDGFR beta is connected with the terminal of FC segment of the monoclonal antibody to VEGF to form the bispecific antibody to VEGF/PDGFR beta. The bispecific antibody related to in the invention is obtained by employing technical means like gene engineering and constructing antibody segments which identify VEGF and PDGFR beta in a same antibody molecule that can be specifically bound with the two antibody segments; the effect of the bispecific antibody on inhibiting angiogenesis of tumor issue is obviously superior to that of a single antibody to VEGF; and the bispecific antibody has good activity in resisting tumors.

Description

technical field [0001] This patent relates to bispecific monoclonal antibody drugs, especially anti-tumor angiogenesis anti-human vascular endothelial growth factor (VEGF / VEGF-A) and human platelet-derived growth factor receptor (PDGFR) bispecific monoclonal antibody drugs ; Background technique [0002] Tumor growth accompanied by neovascularization has been reported in the literature more than a century ago (Ferrara 2002). But it was not until 1939 that Ide and his colleagues proposed for the first time that there may be some tumor-derived angiogenesis stimulator that provides blood vessel supply for tumor growth (Ide et al.1939). Years later, Algire et al. argued that "the rapid spread of tumors depends on a rich vascular supply" (Algire et al. 1945), after observing that increased vascular density preceded rapid tumor growth. In the 1960s, experiments by Greenblatt and Shubik (Greenblatt et al.1968) and Ehrmann and Knoth (Ehrmann et al.1968) successively provided preli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395A61P35/00
Inventor 霍世元叶亚东滕凌朱文华潘鹂路易斯易格那罗
Owner CHANGZHOU ADAM BIOTECH
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