Anti-vegf/pigf bispecific antibody, its preparation method and use

A bispecific antibody and carrier technology, applied in the biological field, can solve the problems of limited number of antibodies and high cost of antibodies

Active Publication Date: 2019-06-14
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in clinical practice, on the one hand, due to the high cost of the combined application of the two antibodies, and the limited number of antibodies available for combination therapy, on the other hand, clinically, the combination therapy of two different targeting antibody drugs has problems in terms of safety. many risks

Method used

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  • Anti-vegf/pigf bispecific antibody, its preparation method and use
  • Anti-vegf/pigf bispecific antibody, its preparation method and use
  • Anti-vegf/pigf bispecific antibody, its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Cloning of Human Antibody Light and Heavy Chain Constant Region Genes

[0041]Lymphocytes from healthy people were separated with lymphocyte separation medium, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). ) reports, respectively designed primers to amplify the antibody heavy chain and light chain constant region genes. All PCR reactions were hot-started, and the reaction conditions were: 94°C for minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minute and 10 seconds, 30 cycles; 72°C for 10 minutes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (purchased from Life Technology, the same below), and the correct clone was confirmed after sequencing verification. SEQ ID NO: 1 and SEQ ID NO: 2 show the nucleotide and amino acid sequences of the light chain constant region (CL), respectively. SEQ ID NO:3 and SEQ ID NO:4 show the nucleotide and amino acid ...

Embodiment 2

[0042] Example 2. Construction of anti-VEGF / PIGF bispecific antibody expression vector

[0043] Light chain VL1 (from anti-VEGF antibody, nucleotide sequence SEQ ID No.7), VL2 (from anti-PIGF antibody, nucleotide sequence SEQ ID No.5), heavy chain VH1 (from anti-VEGF antibody, nucleotide sequence The sequence of SEQ ID No.11), the nucleotide sequence of VH2 (from the anti-PIGF antibody, the nucleotide sequence of SEQ ID No.9) was synthesized from the whole gene by Shanghai Bioengineering Technology Service Co., Ltd. The light chain VL1 and VL2 nucleotide sequences were connected in series with Linker1 by the method of overlapPCR (see SEQ ID No.13 for the Linker1 nucleotide sequence), and were cloned into the pGEM-T vector. Then the nucleotide sequence of the double variable region gene (VL1-VL2) of the light chain and the nucleotide sequence of the constant region of the light chain of the human antibody were cloned into the pGEM-T vector by overlapPCR, and the positive clones...

experiment example 1

[0047] Experimental example 1. Biacore method to detect the affinity experiment of anti-VEGF / PIGF bispecific antibody and VEGF

[0048] The affinity of the anti-VEGF / PIGF bispecific antibody to VEGF (purchased from: R&D Company) was based on the BIAcore-2000 TM The association rate constants and dissociation rate constants measured by the surface plasmon resonance system were calculated. The biosensor chip CM5 was activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and hydroxysuccinimide for covalent coupling of VEGF. The VEGF buffer was exchanged for 20 mM sodium acetate (pH 4.8) and then diluted to about 50 µg / ml. An aliquot of the sample (35 µl) was injected at a flow rate of 2 µl / min to achieve approximately 700-1400 resonance units (RU) of coupled protein, followed by injection of 1M ethanolamine as a blocking agent. For the detection of the affinity between the anti-VEGF / PIGF bispecific antibody and VEGF, the anti-VEGF / PIGF bispecific anti...

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Abstract

The invention belongs to the field of biological technologies, and more specifically discloses an anti-VEGF / PIGF (vascular endothelial growth factor / placenta growth factor) bispecific antibody as well as a preparation method and an application thereof. The anti-VEGF / PIGF bispecific antibody can be combined with VEGF and PIGF, and can effectively inhibit the formation of tumor internal blood vessels, thereby inhibiting the growth of tumors more effectively.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the invention discloses a bispecific antibody capable of simultaneously binding to two different targets, its preparation method and its application in tumor treatment. Background technique [0002] Angiogenesis plays a very important role in the occurrence and development of tumors. When the tumor volume exceeds 1mm 3 At this time, the nutrients obtained by relying on tissue penetration cannot support the nutritional needs of the tumor for continued development. Therefore, tumor tissue needs to obtain various nutrients needed for its development through the formation of new blood vessels. Tumor cells in a state of hypoxia and nutrient deficiency will secrete a variety of factors that promote angiogenesis, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), human placental growth factor (PIGF), tumor necrosis factor -α (TNF-α), etc. Under the acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C07K16/22C12N15/13C12N15/85C12N5/10C12P21/02A61K39/395A61P35/00
Inventor 周远锋张成海张玉晶
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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