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Antibody for resisting anti-cancer embryo antigen as well as preparation method and application thereof

An antibody and amino acid technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of patients without particularly effective treatment methods and low survival rate

Active Publication Date: 2020-03-06
HUADAO SHANGHAI BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Digestive system tumors mainly include esophageal cancer, gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, bile duct cancer, etc. At present, the most effective method is still surgery, and there is almost no particularly effective treatment method for those patients who lack surgical indications. Said that the 5-year survival rate is still very low, so there is an urgent need to develop new treatments for these tumors

Method used

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  • Antibody for resisting anti-cancer embryo antigen as well as preparation method and application thereof
  • Antibody for resisting anti-cancer embryo antigen as well as preparation method and application thereof
  • Antibody for resisting anti-cancer embryo antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Panning and ELISA preliminary screening of natural human phage antibody library

[0084] Panning of phage antibody library

[0085] Prepare 3 EP tubes for magnetic bead blocking and wall blocking (A: 1ml PBS, 2% Blotting-GradeBlocker, 50μl streptavidin magnetic beads, 100μl phage library; B: 1ml PBS, 2%Blotting-GradeBlocker, 100 μl streptavidin magnetic beads; C: 1 ml PBS, 2% Blotting-Grade Blocker) at room temperature for 1-2 h. Discard the solution in tube C and the magnetic beads in tube A, collect the phage in tube A and add them to tube C, and at the same time add biotin-labeled antigen CEA (final concentration 20 μg / ml), incubate at room temperature for 2 h. Collect the magnetic beads in tube B and discard the solution, add the mixture in tube C and incubate on a rotary shaker for 15 minutes. Put the EP tube on the magnetic stand and let it stand for 30s, so that the magnetic beads are adsorbed to the tube wall. Discard the supernatant, and add MPBST (PBS+2%Blo...

Embodiment 2

[0093] FACS screening of candidate clones

[0094] Cell culture was performed according to standard cell culture protocols, and single-cell suspensions were prepared by trypsinizing the cells. Centrifuge (300g, 5min) to remove the culture medium and resuspend the cells to 2*10 6 cell / ml. Add 2*10 to each well of the round bottom 96-well plate 5 cell suspension of cells. After centrifugation at 300 g for 5 min, the supernatant was removed, and the cells were resuspended with 100 μl of periplasmic protein extract, and incubated at 4°C for 1 h. After centrifugation at 300g for 5min, the supernatant was removed, the cells were resuspended in Flow solution, and repeated 3 times to remove periplasmic protein extracts. Dilute anti-myc antibody to 2 μg / ml with blocking solution, resuspend cells in 100 μl per well, and incubate at 4°C for 1 hour. After washing the cells with Flow Buffer for 3 times, add 100 μl of Alexa-488 anti-mouse antibody to resuspend the cells, and incubate a...

Embodiment 3

[0102] Off-rate ranking candidate clones

[0103] Antibody affinity depends on the degree of fit between the variable region of the antibody and the antigen, and is generally assessed by affinity. The dissociation constant (Koff) is generally the main kinetic mechanism affecting antibody affinity, so by measuring the dissociation rate, the level of antibody affinity can be initially compared. At the same time, Koff is concentration-independent, so Koff can be measured without purification of antibodies. Determining the Koff of candidate clones by Octet Red and sorting will help to further select clones for subsequent analysis. Bacterial periplasmic extracts were prepared according to standard operating procedures. The biotin-labeled antigen (5 μg / ml) was immobilized on the streptavidin SA biosensor, the periplasmic extract was diluted 2-fold with 1×kinetic buffer, the binding time of antigen and scFv antibody was 300s, and the dissociation time was 300s. SA biosensors were ...

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PUM

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Abstract

The invention provides an anti-CEA antibody. The anti-CEA antibody comprises a heavy chain variable region and a light chain variable region, wherein a complementary determining region of the heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No. 1, CDR-H2 with an amino acid sequence shown as SEQ ID No. 2 and CDR-H3 with an amino acid sequence shown as SEQ IDNo. 3; a complementation determining region of the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID No. 4, CDR-L2 with an amino acid sequence shown as SEQ ID No. 5 and CDR-L3 with an amino acid sequence shown as SEQ ID No. 6. The CEA scFv is screened by an inventor of the invention by utilizing a phage display technology, so that the CEA-resistant single-chain antibody with high affinity is obtained. An anti-CEA chimeric antigen receptor gene is introduced into T cells through a genetic engineering method to prepare CEA CAR-T, so that the CEA CAR-T cellsspecifically recognize digestive tract tumor cells expressing CEA and kill the digestive tract tumor cells, and the anti-tumor effect of the CEA CAR-T cells is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-carcinoembryonic antigen antibody and its preparation method and application. Background technique [0002] The concept of Chimeric antigen receptor T cells (CAR-T) has appeared as early as 1989, but it has not achieved the desired effect in clinical trials. In the following two decades, scientists continued to try and optimize this, until 2011, CD19 (B lymphocyte antigen CD19, CD19) targeted CAR-T cells were used in relapsed / refractory chronic B lymphocytic leukemia Clinical research, and achieved amazing curative effect. So far, the application of CAR-T cells in tumor treatment has opened a new chapter. [0003] Chimeric antigen receptors are artificially synthesized fusion proteins that function similarly to T cell receptors, mainly including signal peptides, antigen recognition regions, hinge regions, transmembrane regions, and intracellular signal regions. After bindi...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N5/10A61K45/00A61P35/00G01N33/574
CPCA61K45/00A61P35/00C07K14/7051C07K16/3007C07K2317/622C07K2317/92C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N5/0646C12N2510/00G01N33/57473G01N33/57484
Inventor 狄升蒙刘芳茅健余学军
Owner HUADAO SHANGHAI BIOPHARMA CO LTD
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