Multi-target chimeric antigen receptor
A chimeric antigen receptor, multi-target technology, applied in cytokine/lymphokine/interferon receptors, hybrid peptides, receptors/cell surface antigens/cell surface determinants, etc.
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Embodiment 1
[0136] Example 1, Multi-target Chimeric Antigen Receptor Structure and Expression Vector Construction
[0137] 1. The multi-target chimeric antigen receptor (RaceCar) is a protein obtained by polymerizing the main peptide chain X and the auxiliary peptide chain Y;
[0138] The main peptide chain (X) includes the antigen-binding domain (A), the auxiliary peptide chain linking domain (B), the transmembrane domain (C) and the intracellular signaling domain (D), and the auxiliary peptide chain (Y) includes the antigen-binding Domain (E) and main peptide chain connecting domain (F); auxiliary peptide chain connecting domain (B) and main peptide chain connecting domain (F) complement each other to make main peptide chain X and auxiliary peptide chain Y polymerize to form a multi-target chimera Antigen receptor (RaceCar).
[0139] The multi-target chimeric antigen receptor (RaceCar-1) targeting CD19 and PD-L1 positive cells is polymerized from the main peptide chain X1 and the auxil...
Embodiment 2
[0164] Example 2, Flow Cytometry Verification of RaceCar's 293T Cell Expression
[0165] 1. Lenti-X-293T cells (Clontech, article number: 632180, hereinafter referred to as 293T) 1.2*10E 6 Spread in 6-well plate, 37°C, 5% CO 2 Incubate overnight.
[0166] 2. Transfect the vector pFUGW-RaceCar-1 constructed in Example 1 into the cells obtained in the above 1 using lipofectamine3000, at 37°C, 5% CO 2 Cultivate for 48h.
[0167] 3. The 293T cells transfected with pFUGW-RaceCar-1 were digested with trypsin and resuspended in PBS, and the cell density was controlled at 1*10E 6 / mL.
[0168] 4. Add 1.5uL APC Mouse anti-Human CD279 (BD, Cat. No. 558694) and FITC-labeled CD19-FC fusion protein (CD19-FC fusion protein nucleic acid sequence is sequence 35) to 200uL3 treated cells respectively, use FITC After 30 min in ice bath, desalt for labeling, 293F expression), and incubate on ice for 30 min. Centrifuge to remove the supernatant, and resuspend the cells with an equal volume o...
Embodiment 3
[0171] Example 3, Western blot verification of RaceCar expressed in 293T
[0172] 1. Take 5*10E 6 The 293T cells transfected with pFUGW-RaceCar-1 in Example 2 were centrifuged at 1500 R for 10 min and the supernatant was discarded.
[0173] 2. Add 200uL Ripa cell lysate (Biyuntian, Cat. No.: P0013B) and let stand on ice for 30 minutes. Centrifuge at 8000g for 5 minutes to take 20uL of the supernatant, add 5X protein electrophoresis buffer, and place in a metal bath at 95°C for 5 minutes to prepare protein electrophoresis samples.
[0174] 3. Carry out SDS-PAGE. After the electrophoresis is completed, cut off the part of the stacking gel, and transfer to the membrane at 100V, 200mA, and 1h.
[0175] 4. Place the PVDF membrane with the transferred protein in 5% skim milk / PBS solution and block overnight. PBST decolorization shaker washed 3 times, 15min each time.
[0176] 5. Put the PVDF membrane into 1uL antibody / mL1% skimmed milk / PBS solution, in which the antibody is PD1 / ...
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