Chimeric Antigen Receptor Recognizing Carcinoembryonic Antigen and Its Application
A chimeric antigen receptor, carcinoembryonic antigen technology, applied in the field of genetic engineering, can solve the stability of antigen chimeric receptors. There is no good solution, and there is no report showing the stability of chimeric antigen receptor cells in CEA solid tumors To achieve the effect of strengthening the ability to proliferate and kill tumors, the ability to remove tumor cells, and a wide range of options
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0066] Example 1 Construction of chimeric antigen receptor virus containing scFv of M5A, hMN-14, C2-45 and BW431 / 26
[0067] In order to prove that the anti-CEA CAR we screened has more advantages than the currently used anti-CEA CAR and other anti-CEA scFV CARs, it is necessary to simultaneously construct scFV-CARs of different anti-CEA humanized monoclonal antibodies and name them CEA-CAR- 1. CEA-CAR-2, CEA-CAR-3 and CEA-CAR-4 are as follows:
[0068] 1. Synthesis of gene sequences of chimeric antigen receptors targeting carcinoembryonic antigens containing different scFVs
[0069] Synthesize different single-chain antibody ScFv containing leader peptide (also known as signal peptide) (abbreviated as LP), anti-human CEA antigen, IgG4 hinge region (abbreviated as IgG4), CD28 transmembrane region or CD8 transmembrane region (abbreviated as TM) , whose structure is as figure 1 shown.
[0070] 2 Construction of lentiviral vectors expressing chimeric antigen receptors
[0071...
Embodiment 2
[0093] Example 2 Determination of affinity between scFv and target antigen (CEA)
[0094] The aforementioned four scFVs were tagged with His tags and cloned into plasmid vectors and transfected into HEK293 cells. The transfected cells were cultured for 6 days and all supernatants were collected. The supernatants were filtered through a 0.22um filter membrane and then purified by nickel column affinity chromatography. ScFV-His in the serum, the purity of scFV-His reached 95% after purification, and the concentration of scFV-His was determined by Bradford; the affinity of different scFV and CEA was measured by ForteBio RED96 interaction analyzer (Pall, CA, U.S.) was checked, and the experimental steps were carried out according to the instructions. The anti-human IgG-Fc (AHC) biosensor was purchased from Pall FortéBio, 1512121 according to the instruction manual; the concentration of recombinant CEA-Fc was 15ug / ml, purchased from Sino Biological Inc. Incubated in biosensors, s...
Embodiment 3
[0095] Example 3 Detection of Chimeric Antigen Receptor Maintenance in T Cells
[0096] 1 Isolation of Human Peripheral Blood Mononuclear Cells
[0097] Collect about 60ml of peripheral blood with anticoagulant-added blood collection tubes, divide into 30ml of 50ml centrifuge tubes, add 7.5ml of hydroxyethyl starch to dilute; naturally settle at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and The collected upper plasma was centrifuged at 1400rpm / min for 15min; then the pellet was resuspended with normal saline, added to the lymphocyte separation medium at a volume ratio of 1:1, gradient centrifuged at 400g / min, and the speed of the centrifuge was set to 1 , centrifuged for 20min; after centrifugation, the centrifuge tube was divided from top to bottom: the first layer: plasma layer; the second layer: annular milky white lymphocyte layer; the third layer: transparent separation liquid layer; the fourth layer: red blood cell layer; Take the se...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More