Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

CD317 single-chain antibody 317scfv, its coding sequence, preparation method and application

A 317scfv, single-chain antibody technology, applied in the field of tumor treatment, can solve the problems of limited treatment effect of solid tumors, discounted ADCC effect, and inability to combine radiotherapy and chemotherapy, and achieves reduction of antigenic epitopes, wide clinical application, and good penetration effect. Effect

Active Publication Date: 2020-11-17
SHENZHEN BINDEBIOTECH CO LTD
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The anti-tumor mechanism of existing antibodies targeting CD317 is through antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADCC), but cancer patients, especially those who have undergone radiotherapy and chemotherapy After that, a large number of immune cells in tumor patients died, so the effect of ADCC was greatly reduced, and it could not be used in combination with radiotherapy and chemotherapy
In addition, traditional antibodies have a large molecular weight and poor penetration. It is difficult to enrich the antibody with sufficient concentration inside the tumor during the treatment of solid tumors, so the therapeutic effect on solid tumors is relatively limited.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • CD317 single-chain antibody 317scfv, its coding sequence, preparation method and application
  • CD317 single-chain antibody 317scfv, its coding sequence, preparation method and application
  • CD317 single-chain antibody 317scfv, its coding sequence, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 PCR Amplification, Cloning Identification and Construction of Expression Vector of Single-chain Antibody 317 scFv Gene

[0032] (1) PCR amplification of the single-chain antibody 317 scFv gene: collect the hybridoma cell line (1C12C6), extract cellular RNA, and the specific steps are as follows:

[0033] ① Take the treated cells, remove the medium, add 1mL Trizol to dissolve the cells.

[0034] ②Put the cells dissolved with Trizol into the EP tube, add 200 μL of chloroform, mix well, and centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0035] ③ Take 500 μL of supernatant, add 500 μL of pre-cooled isopropanol, mix well, and centrifuge at 12,000 rpm at 4°C for 15 minutes.

[0036] ④Remove the supernatant, wash the precipitate with 75% ethanol, and centrifuge at 12000rpm at 4°C for 8min.

[0037] ⑤ On the ultra-clean bench, after drying the precipitate, add 20 μL DEPC water.

[0038] ⑥Detect RNA concentration and purity.

[0039] Use a reverse transcription ...

Embodiment 2

[0046] Example 2 Expression of single-chain antibody 317 scFv in eukaryotic cells HEK293F

[0047] (1) Expression of single-chain antibody 317 scFv in eukaryotic cells HEK293F: pcDNA3.1(+) control plasmid and pcDNA3.1(+)-317 scFv recombinant plasmid were transfected into eukaryotic cells using PEI25000 transfection reagent HEK293F, at 8% CO 2 , 37°C constant temperature incubator shaking culture (120rpm) for 72h; collect the cell expression supernatant, centrifuge at 3000g for 5min, carefully absorb the supernatant, and filter through a 0.45μm filter membrane.

[0048] (2) Detect the expression of 317 scFv by Dot Blot: Take 50 μL of the cell expression supernatants of the control group and the pcDNA3.1(+)-317 scFv group collected in the above step (1), and drop evenly on the On the PVDF membrane activated by water and methanol, 5% skimmed milk powder was blocked at room temperature for 1 h, and the anti-His tag antibody with HRP from Sigma was added (diluted concentration was...

Embodiment 3

[0049] Example 3 Effect of single-chain antibody 317 scFv on inhibiting tumor cell growth

[0050] (1) The effect of single-chain antibody 317 scFv on inhibiting the growth of tumor cells: strict counting of liver cancer cell line Hep G2 and breast cancer cell line MCF-7 (TC20 TM Automated Cell Counter), with 1×10 per well 5 Cells were seeded in a 12-well plate; cervical cancer cell line Hela was seeded at 2×10 5 Each cell was inoculated in a 12-well plate; 500 μL of DMEM medium was removed from each well overnight, and 500 μL of the sterile HEK293F expression supernatant collected in Example 2 (pcDNA3.1 (+) control group, hereinafter referred to as Ctrl group) or pcDNA3.1(+)-317 scFv supernatant (hereinafter referred to as 317 scFv group), mix gently and store in 5% CO 2 , 37 ℃ constant temperature incubator after continuing to culture for 48h (each experimental group set up 3 duplicate wells), photographed under the microscope, and collected cell counts (TC20 TM Automated...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a CD317 single-chain antibody 317 scFv and a coding sequence, preparing method and application thereof. The single-chain antibody 317 scFv is independent of ADCC, and directly acts on tumor cells for inhibiting growth of the tumor cells, the single-chain antibody 317 scFv, radiotherapy and chemotherapy and the like can be jointly used, a Fc part does not exist, the molecularweight is small, and the penetration effect is also better.

Description

technical field [0001] The invention relates to the technical field of tumor treatment, in particular to CD317 single-chain antibody 317 scFv, its coding sequence, preparation method and application. Background technique [0002] The treatment of tumors, especially solid tumors, is a worldwide problem. Conventional treatment methods such as surgery, radiotherapy, and chemotherapy cannot completely remove or completely kill tumor cells. Tumor metastasis or recurrence occurs from time to time, and the clinical effect is poor. In addition, The specificity of conventional radiotherapy / chemotherapy is extremely poor. While killing tumor cells, it also causes great damage to normal cells and has serious adverse reactions. Cancer patients are often forced to stop receiving treatment because they cannot tolerate it; another On the one hand, chemotherapy and radiotherapy are particularly likely to damage the immune system, which plays an important role in the anti-tumor mechanism, an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85A61K39/395A61P35/00
Inventor 万晓春李欣章桂忠李俊鑫刘绿艳
Owner SHENZHEN BINDEBIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com