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Anti-VEGF/ANG2 bispecific antibody, and application thereof

A bispecific antibody and antibody technology, applied in the direction of antibodies, anti-tumor drugs, hybrid immunoglobulins, etc., can solve the problems of decreased maturity, late TNM staging, increased blood vessel density, etc., to achieve inhibited growth and good tumor Activity, the effect of avoiding the increase of dosage

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

Etoh et al.’s research on gastric cancer cases also found that the high expression of Ang2 in the tissue is accompanied by increased blood vessel density and decreased maturity, and the clinical TNM stage of the patient is late, and the postoperative survival rate is low (Etoh et al.2001)

Method used

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  • Anti-VEGF/ANG2 bispecific antibody, and application thereof
  • Anti-VEGF/ANG2 bispecific antibody, and application thereof
  • Anti-VEGF/ANG2 bispecific antibody, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0069] 1. Nucleotide sequence design and synthesis of anti-VEGF-ANG2 bispecific antibody (bsAb):

[0070] According to the bsAb heavy chain HC VEGF (GGGGS) 3 -HC ANG2 (Reverse) amino acid sequence and connection form Design heavy chain nucleotide sequence, and add Sac I restriction site, KOZAK sequence and leader sequence at the 5' end of the sequence, and add Xho I restriction site at its 3' end point. Design three oligonucleotide primers containing EcoR I restriction site at the 3' end to synthesize heavy chain HC respectively VEGF -(GGGGS) 3 and HC ANG2 (Reverse) fragment, the full-length 2616bp bsAb heavy chain gene was synthesized by PCR direct ligation method (SDLPCR).

[0071] According to the amino acid sequence of human anti-VEGF-A IgG1 light chain and human anti-ANG2 IgG1 light chain amino acid sequence, design the 5' end with Not I restriction site, KOZAK sequence and leader sequence, and the 3' end with Psi I restriction site PCR The primers are used to ampl...

Embodiment 2

[0083] Antigen-antibody binding test (ELISA method):

[0084] 1. Coating: Dilute the antigen (hVEGF-A or ANG2) to a protein content of 5 μg / ml with 0.05M pH9.1 carbonate coating buffer. Add 0.1ml to the reaction well of each polystyrene plate, overnight at 4°C. The next day, the solution in the well was discarded, and washed 3 times with washing buffer, 3 minutes each time.

[0085] 2. Adding samples: Add 0.1ml of a certain dilution of the antibody to be tested (anti-VEGF Fab, anti-ANG2 monoclonal antibody or anti-VEGF-ANG2bsAb) to the above-mentioned coated reaction wells, and incubate at 37°C for 1 hour. Then wash. (Make blank wells, negative control wells and positive control wells at the same time).

[0086] 3. Add enzyme-labeled antibody (goat anti-human IgG-gamma): Add 0.1 ml of freshly diluted enzyme-labeled antibody (diluted after titration) to each reaction well. Incubate at 37°C for 0.5-1 hour and wash.

[0087] 4. Add substrate solution for color development: A...

Embodiment 3

[0093] Anti-VEGF / ANG2 bispecific anti-human nasopharyngeal carcinoma CNE xenograft tumor growth inhibition test in nude mice

[0094] Take the tumor tissue in the vigorous growth period and cut it into 1.5mm 3 Left and right, under sterile conditions, inoculated subcutaneously on the right side of nude mice. Use a vernier caliper to measure the diameter of the transplanted tumor in mice, and wait until the tumor grows to 100-200mm 3 Animals were then randomly grouped. Using the method of measuring the tumor diameter, dynamically observe the anti-tumor effect of the tested animals. Tumor diameter was measured once every 2 days, and the weight of the mouse was also weighed at the same time as each measurement. The experimental group was injected with anti-VEGF / ANG2 bispecific antibody through the tail vein, once a day, and the negative group was given the same amount of normal saline at the same time. Tumor volume calculation formula:

[0095] TV=0.52×a×b 2

[0096] Where...

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Abstract

The invention relates to bispecific monoclonal antibody medicaments, and especially relates to a human vascular endothelial growth factor (VEGF / VEGF-A) resistant and human angiopoietin 2 (ANG2) resistant bispecific monoclonal antibody medicine, which is used for resisting neovascularization. The anti-VEGF / ANG2 bispecific antibody is characterized in that: a VEGF monoclonal antibody is adopted as a basis, and a second antigen binding site is replaced by an antibody segment specifically bound to ANG2.

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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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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