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VEGF-resistant and PD-1-resistant difunctional antibody and application thereof

A bifunctional antibody, PD-1 technology, applied in the field of molecular immunology, can solve problems such as low production efficiency, difficulties in the development of bispecific antibodies, and poor pharmacokinetic performance

Active Publication Date: 2015-12-23
ANHUI RUBIOX VISION BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are more than 40 types of bifunctional antibody forms that have been developed, but due to problems such as low production efficiency and poor pharmacokinetic performance, the development of bispecific antibodies has always been difficult
At present, there is no anti-VEGF-anti-PD-1 bifunctional antibody at home and abroad

Method used

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  • VEGF-resistant and PD-1-resistant difunctional antibody and application thereof
  • VEGF-resistant and PD-1-resistant difunctional antibody and application thereof
  • VEGF-resistant and PD-1-resistant difunctional antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1V

[0030] Embodiment 1 Preparation of VEGF and PD1 protein

[0031] 1. Construction of expression vector for PD-1 antigen

[0032]The cDNA of human PD-1 was synthesized from Nanjing GenScript Company, the GeneID is 5133, and the cDNAID is NM_005018.2. An Fc purification tag was added after the synthetic extracellular region PD-1 gene, and two restriction enzyme sites of XbaI and BamHI were introduced at both ends and connected to the pTT5 expression plasmid, which was verified to be correct by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium until OD 600 When the temperature is 1, the bacteria are collected by centrifugation, and the plasmid is extracted with a large plasmid extraction kit (purchased from Qiagen).

[0033] 2. Construction of expression vector for VEGF antigen

[0034] The amino acid corresponding to the gene V...

Embodiment 2

[0037] Example 2 Preparation of bifunctional antibody BsAb7 and BsAb8 proteins

[0038] 1. Construction of bifunctional antibody expression vector

[0039] The cDNA of the light chain of the artificially synthesized antibody BsAb7 (its sequence is shown in SEQ ID NO.1), the cDNA of the artificially synthesized antibody BsAb7 heavy chain (its sequence is shown in SEQ ID NO.3), the cDNA of the artificially synthesized antibody BsAb8 heavy chain (its sequence As shown in SEQ ID NO.5), the synthesized cDNAs were respectively cloned into pTT5 plasmids, and the correct construction of the plasmids was confirmed by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium until OD 600 When the temperature is 1, the bacteria are collected by centrifugation, and the plasmid is extracted with a large plasmid extraction kit (purchased from Qiage...

Embodiment 3

[0042] Example 3 Determination of the affinity of bifunctional antibodies BsAb7 and BsAb8 to PD-1 and VEGF

[0043] The characterization affinity and binding kinetics of anti-PD-1 / VEGF antibodies BsAbB7 and BsAbB8 were analyzed by Biacore3000 instrument (purchased from GE). The recombinant fusion protein was covalently coupled to biotin using a biotin labeling kit (Pierce Company), and then flowed through an avidin-labeled SA chip (purchased from GE Company), so that the reaction value RU reached about 450. Binding was measured by flowing antibodies in PBS buffer at concentrations of 0.0133, 0.0266, 0.0532, 0.1064, 0.2128 μM and a flow rate of 50 μl / min. Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software. In order to minimize the role of avidity in estimating binding constants, only the initial data se...

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Abstract

The invention discloses a VEGF-resistant and PD-1-resistant difunctional antibody and application thereof, belonging to the technical field of molecular immunology. The VEGF-resistant and PD-1-resistant difunctional antibody contains a light chain an a heavy chain, wherein the light chain has an amino acid sequence as shown in SEQ ID NO.2, and the heavy chain has an amino acid sequence as shown in SEQ ID NO.4 or SEQ ID NO.6. Meanwhile, the invention provides a gene for encoding the difunctional antibody and the application of the difunctional antibody. The difunctional antibody provided by the invention can be combined with PD-1 and VEGF, has very high affinity, can be used for effectively simulating T cells to secrete IL2 and induce T cells to secrete IFN-gamma and can also be used for remarkably inhibiting the growth of tumor of a mouse so as to have a huge potential in application to preparation of anti-cancer drugs.

Description

technical field [0001] The invention relates to an anti-VEGF-anti-PD-1 bifunctional antibody and its application, belonging to the technical field of molecular immunology. Background technique [0002] According to the prediction of WHO experts, by 2020, the number of cancer cases in the global population will reach 20 million, and the death toll will reach 12 million. Therefore, tumor will become the biggest killer of human beings in this century and pose the most serious threat to human survival. . As China's population ages, more and more people will suffer from tumor diseases. Therefore, the development of anti-tumor drugs has become the focus of national health. [0003] Vascular endothelial growth factor, also known as VEGF. VEGF protein was successfully purified and identified by scientists from two biotechnology companies in the United States in 1989, and its gene sequence was cloned and determined, proving that VPF and VEGF are the same protein encoded by the same...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00G01N33/574
CPCC07K16/22C07K16/2818C07K2317/31C07K2317/70
Inventor 汪国兴胡思怡袁红武婷
Owner ANHUI RUBIOX VISION BIOTECH
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