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

Anti-CD123 nano antibody and application thereof

A nanobody and chimeric antigen receptor technology, applied in the field of biomedicine, can solve problems such as failure to achieve ideals, and achieve high affinity effects

Active Publication Date: 2021-03-12
HUADAO SHANGHAI BIOPHARMA CO LTD
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although TKIs provide different treatment options for leukemia, they are still in the development stage, and more treatment strategies are needed clinically
[0004] The concept of Chimeric antigen receptor T cells (CAR-T) appeared as early as 1989, but it has not been able to achieve the desired effect in clinical trials.

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
  • Anti-CD123 nano antibody and application thereof
  • Anti-CD123 nano antibody and application thereof
  • Anti-CD123 nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction, panning and ELISA preliminary screening of phage nanobody library

[0073] (1) Construction of phage nanobody library

[0074] In this example, the Bactrian camel was first immunized with the extracellular region of the CD123 antigen, and after the titer was verified by ELISA, 200 mL of peripheral blood was extracted; lymphocytes were sorted from the peripheral blood, and peripheral blood mononuclear lymphocyte precipitates were obtained for RNA extraction;

[0075] Using the extracted RNA as a template, the Synthesize the first-strand cDNA with reverse transcriptase, and then use nested PCR to amplify the VHH gene; insert the amplified VHH gene into the pMECS phage display vector, electrotransform TG1 competent cells, take an appropriate amount of bacterial liquid for library identification, and the remaining Spread the culture evenly on the LB / AMPGLU plate;

[0076] After the bacteria grow, collect the lawn, add 1 / 3 volume of 50% glycerin, mix...

Embodiment 2

[0089] Example 2 FACS screening candidate clones

[0090] In this example, cell culture was carried out according to the standard cell culture protocol:

[0091] Use trypsin to digest the cells, prepare CD123-positive cells or CD123-negative cell suspensions, centrifuge at 300g for 5 minutes to remove the culture medium, and resuspend the cells with Flow Buffer to a cell concentration of 2×10 6 a / mL;

[0092] Add 2 × 10 to each well in a V-bottom 96-well plate 5 Centrifuge at 300g for 5 minutes to remove the supernatant, add VHH antibody crude extract to resuspend the cells, and incubate at 4°C for 1 hour;

[0093] Centrifuge at 300g for 5 minutes to remove the supernatant, resuspend the cells in Flow Buffer, add 100 μL of APC anti-his antibody (2 μg / mL) diluted in Flow Buffer, and incubate at 4°C for 1 hour;

[0094] After washing the cells 3 times with Flow Buffer, resuspend the cells in 200 μL Flow Buffer for flow cytometric detection.

Embodiment 3

[0095] Example 3 Expression, purification and affinity determination of VHH-mIgG2a Fc nanobody

[0096] In order to further identify the screened antibodies, this example constructs the vector C-4pCP.Stuffer-mCg2a-FC expressing VHH (with mouse Fc tag), the steps are as follows:

[0097] PCR amplification anti-CD123 heavy chain variable region coding gene, wherein, the upstream primer of CD123-9 (SEQ ID NO:6) is HD-F, the downstream primer is HD-B12-R2, CD123-12 (SEQ ID NO:6) 7) The upstream primer is HD-F, the downstream primer is HD-CD-12-R, the sequence is shown in Table 2, the PCR reaction system is shown in Table 3, and the reaction conditions are pre-denaturation at 95°C for 1min and denaturation at 95°C for 10s , 55°C annealing 10s, 72°C extension 10s, 30 cycles, 72°C extension 5min, 4°C storage.

[0098] Table 2

[0099]

[0100] table 3

[0101]

[0102] The empty vector was digested at 37°C for 6 hours, the system is shown in Table 4, and the digested vector ...

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 an anti-CD123 nano antibody and application thereof. The nano antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 and CDR3 as shown in SEQ ID NO: 3. The antibody screened from the camel VHH immune library has CDR regions shown as SEQ ID NO: 1-3, the nano antibody formed by combining the antibody with different framework regions FR has strong affinity with CD123, so that the constructed chimeric antigen receptor and chimeric antigen receptor immune cells have remarkable cytotoxicity on CD123 positive cells, and have wide application prospects in the field of tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to anti-CD123 nanometer antibody and application thereof. Background technique [0002] More than 30 years ago, the "3+7" regimen (daunorubicin chemotherapy for 3 days combined with cytarabine chemotherapy for 7 days) brought about 60% of AML patients into remission and became the standard induction regimen for children and adults with acute leukemia . In the 1990s, clinical experts began to focus on post-remission treatment options and their benefits, and a large number of studies were conducted, including high-dose cytarabine chemotherapy or hematopoietic stem cell transplantation (HSCT). Although the remission rate and overall survival rate of acute leukemia in children have reached greater than 90% and 60%, respectively, existing treatment options are still limited to anthracyclines, nucleoside analogs, and intensive post-remission therapy. In order to improve the prognosis ...

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 Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00C12N5/10A61K35/17A61P35/02
CPCC07K16/2866C07K14/7051C12N5/0634A61K35/17A61P35/02C07K2317/565C07K2317/569C07K2319/02C07K2319/03C12N2510/00
Inventor 狄升蒙侯莉石磊刘芳茅健余学军
Owner HUADAO SHANGHAI BIOPHARMA 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