Chimeric antigen receptor effector cell switches with humanized targeting moieties and/or optimized chimeric antigen receptor interacting domains and uses thereof
A technology of chimeric antigen receptors and effector cells, which is applied in the field of chimeric receptor effector cell design and R-T cell design, which publicly provides chimeric receptor effector cells, separation, and receptors, and can solve necrosis, death, Problems such as B cell hypoplasia
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example 1
[0629] Design of humanized CAR-EC switch
[0630] background
[0631] As a proof-of-concept, the anti-CD19 murine clone FMC63 was used as a switch for switchable CAR-EC programming in pilot studies. The FMC63 clone was originally described by H. Zola and colleagues (1) in 1991 and used in the most well-studied conventional CAR-T cells from Carl June and colleagues (2-4). To reduce the possibility of immunogenicity in human applications, the murine framework regions of the FMC63 antibody were partially replaced with human sequences.
[0632] General process:
[0633] Briefly, the humanization process was performed as follows: The murine FMC63 sequences of the variable light (VL) and variable heavy (VH) domains were submitted to the IgBLAST program available on the NCBI website, The website is available on the World Wide Web at: ncbi.nlm.nih.gov / igblast / (incorporated herein by reference in its entirety); see also Ye et al., Nucleic Acids Research, July 2013; 41 (Web Ser...
example 2
[0662] Expression and purification of humanized CAR-EC switch
[0663] Briefly, in order to express and purify the humanized CAR-EC switch, a heavy chain variant as described in Table 4 was combined with a light chain variant as described in Table 5 according to the scheme shown in Table 6 Pairing was performed in which the light chain included the GCN4 peptide linked by a GGGGS linker in LCNT format as shown in Table 7, and the resulting switch was expressed and purified according to the following protein expression method.
[0664] Protein Expression in Expi293F Cells (30ml Culture Volume)
[0665] Expi293F cells (ThermoFisher, Waltham, MA, Cat. No. A14524) set up with the ExpiFectamine 293 Transfection Kit (ThermoFisher, Waltham, MA) were used to transfect Expi293F cells (ThermoFisher, Waltham, MA) using a modified version. Fisher, catalog number A14527).
[0666] Briefly, 24 hours before transfection, Expi293F cells (Thermo Fisher, Waltham, MA, cat# A14527) were incuba...
example 3
[0675] Determining CAR-EC switch binding efficiency
[0676] To determine the relative binding efficiencies of the humanized variants, flow cytometry-based binding was performed as follows:
[0677] Preparation of cells for flow cytometry analysis of CAR-EC switch binding efficiency.
[0678] Collect CD19+RS4; 11 cells ( CRL-1873 TM ) and centrifuged at 300xg for 5 minutes at 4°C, and then the cells were centrifuged at 5x10 6 Cells / ml were resuspended in ice-cold FACS buffer (PBS, 5% fetal bovine serum, 1 mM EDTA). Dispense 100 μL of the cell suspension into each well of the 96-well plate, and then wash the cells using the following washing method: a) add 200 μL of FACS buffer to each well of the well plate; b) at 4°C at 300×g Centrifuge the plate for 5 minutes; c) remove the supernatant from the plate into the sink with a "flick" motion; d) loosen the cell pellet by gentle vortexing of the plate; and e) resuspend the cell pellet in an appropriate volume of buffer (des...
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