Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof

A nanobody and antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problems of restricting the research and development of VEGFR2 antibody drugs

Inactive Publication Date: 2015-11-25
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the technical bottlenecks of traditional antibody affinity maturation, protein expressio

Method used

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  • Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof
  • Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof
  • Anti-VEGFR2 human source nano antibody NTV1 and preparation method therefor and use thereof

Examples

Experimental program
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preparation example Construction

[0080] This embodiment provides a preparation method for preparing anti-VEGFR2 nanobody, comprising the following steps:

[0081] a. Use phage display technology to screen antibodies

[0082] The biotin-VEGFR2 fusion antigen was coated on the surface of the magnetic beads and incubated at 4°C for 2 hours. After washing with PBS three times, block with PBS containing 2% skimmed milk powder at 37°C for 1 hour. Set capacity to 1 x 10 12 The cloned human nanobody phage antibody library was added to the blocked immunotube and combined for 1 hour at 37°C. Wash 10 times with PBST and 5 times with PBS. Add 1 mL of glycine elution buffer (pH2.2), shake at room temperature for 10 min, suck out the eluate, and neutralize to pH7.4 with Tris. Aspirate the eluate and infect with 10 times the volume of TG1 at room temperature for 10 minutes, measure the titer, transfer to 37°C and add helper phage M13KO7 for shaking culture overnight. The above process is carried out for four rounds. T...

experiment example

[0089] Experimental example: Determination of biological activity of nanobodies

[0090] In this experimental example, the MTT assay was used to determine the ability of nanobodies to inhibit the proliferation of human umbilical vein endothelial cells (HUVECs), and the effect of nanobodies on angiogenesis in HUVECs was determined using a microtubule formation assay.

[0091] MTT experiment: Incubate HUVECs with a special medium for HUVECs cells (5% FBS-ECM) at 37°C for 24 hours, then add the corresponding concentration of Nanobodies, add MTT dye to the cell culture plate after 72 hours of incubation, and use enzymes after 4 hours Read absorbance at 570nm.

[0092] NTV1 at concentrations of 1 nM, 10 nM, 100 nM and 1000 nM was added to HUVECs at a concentration of 5 × 10 3 In a 96-well plate of cells / well, read at a wavelength of 570 nm after 72 hours, and set a blank control. Each concentration was repeated 3 times, the standard deviation was calculated, and the statistical a...

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Abstract

The invention discloses an anti-VEGFR2 human source nano antibody NTV1 which comprises a frame region and a complementarity-determining region, wherein the nano antibody has an amino acid sequence shown in SEQ ID NO: 1. The invention further provides a preparation method for the anti-VEGFR2 human source nano antibody NTV1. The preparation method comprises the following steps: a, screening a nano antibody combined with VEGFR2 by adopting a phage display technology; b, carrying out expression and purification of the nano antibody; and c, determining the interaction between an antigen and the antibody by adopting an AlphaScreen technology to further screen the antibody with the highest affinity . The human source nano antibody NTV1 with higher affinity and good anti-tumor activity is obtained. The VEGFR2 nano antibody egg white which is high in affinity and stable and uniform is obtained by expression and optimization, a human source anti-VEGFR3 nano antibody drug is successfully developed, and a novel field of drug research and development of a targeted VEGFR2 antibody is opened up.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals. More specifically, the present invention relates to a VEGFR2 nanobody, a preparation method of the antibody and a use of the antibody in the preparation of anticancer drugs. Background technique [0002] Malignant tumors are currently one of the major diseases that seriously affect human health and threaten human life. As of 2012, more than 8.2 million people in the world died of cancer. The World Health Organization and government health departments of various countries have listed cancer as a top priority. Task. However, the treatment of tumors is still a difficult problem in today's medical field, and researchers have been looking for new directions for tumor treatment. Through the study of tumors, it has been found that angiogenesis is a necessary condition for the occurrence and development of tumors, and vascular endothelial growth factor receptor (VEGFR), as the receptor of the key fact...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/63C12P21/08A61K39/395A61P35/00
Inventor 马琳徐华强谷凯张成海陈学涛侯丽蒋轶
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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