![]() Humanization of the murine scFv 763.74(A) was performed by grafting its CDRs into the stable human framework rFW1.4 ( 13). The variable regions of the immunoglobulin genes expressed by the 763.74 hybridoma were amplified with a set of proprietary primers from cDNA generated from the hybridoma cells using an RT-PCR protocol and sequenced using a standard dye-terminator capillary sequencing method (Synbuild). Glioblastoma-derived neurospheres (GBM-NS) were generated as previously described ( 12). All tumor cell lines were routinely tested to exclude contamination with Mycoplasma and assessed for the expression of transgenes and tumor markers by flow cytometry to confirm identity. Cells were kept in culture for less than 6 consecutive months, after which, aliquots from the original expanded vial (cultured for four to six passages after being received) were used. WM115 cells were modified to express the fusion protein firefly luciferase and enhanced GFP (eGFP-FFluc ref. All cells were maintained in culture with the appropriate media, either RPMI-1640 (Gibco), DMEM (Gibco), or MEM (Gibco) supplemented with 10% FBS (Sigma), 1% l-glutamine (Gibco), and 1% penicillin/streptomycin (Gibco) in a humidified atmosphere containing 5% CO 2 at 37☌. The 293T cells used for the production of retroviral vectors were obtained from the ATCC in early 2000. The tumor cell lines MDA-MB-468 and MDA-MB-231 were obtained from German Collection of Microorganism and Cell Cultures GmbH (ACC 738 and ACC 732). Ferrone (Massachusetts General Hospital). The tumor cell lines WM115 and SK-MEL-2 were obtained from the ATCC between 20, whereas M14 cells were provided by Dr. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. ![]() This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. ![]()
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