Validation of a humanized anti-EGFR variant III chimeric antigen receptor for a Phase I trial of CART-EGFRvIII in glioblastoma
Le résumé fourni par la source
Chimeric antigen receptors (CARs) are synthetic molecules designed to re-direct T cells to specific antigens; CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of novel surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFR variant III) results from an in-frame deletion of a portion of the extracellular domain. In glioblastoma, the EGFRvIII mutation is oncogenic, portends a poor prognosis, and is thought to be enriched in glioblastoma stem cells. However, because the neoepitope of EGFR variant III is based on a small peptide sequence, an antibody or single-chain variable fragment (scFv) directed to this epitope must be rigorously tested to confirm lack of cross-reactivity to the ubiquitously expressed wild-type EGFR. We chose a vector backbone encoding a second generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFv’s and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low affinity scFv was chosen based on its specificity for EGFR variant III over wild type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead candidate CAR in vitro against EGFR expressing keratinocytes and in vivo in immunodeficient mice grafted with normal human skin; a cetuximab-based CAR served as a positive control. EGFRvIII-directed CAR-T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFR variant III+ glioblastoma. We have designed a phase I clinical study of CAR T cells transduced with humanized scFv directed to EGFR variant III in patients with either residual or recurrent glioblastoma (NCT02209376).
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