Isolation of tumour antigen specific scFv's using a chimeric antigen receptor bicistronic retroviral vector in a mammalian screening protocol.
AffiliationUniversity of Manchester, CIMML, Manchester, United Kingdom
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AbstractThe clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realised with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest generation receptors eliciting greater signalling and proliferation potential. However, the antigen binding scFv domain of the receptors is critical in determining the activity of the chimeric receptor expressing T cells; as this determines specificity and affinity to the tumour antigen. In this study, we describe a mammalian T-cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFv's. This approach involves expression of the scFv library in a CAR, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A-cassette ensures the exclusion of non-expressing scFv's and the screening using a chimeric receptor in a mammalian T-cell line ensures selection in the optimum context for therapeutic use. Proof of principle experiments show that the protocol was capable of a 105-fold enrichment of positive clones following three rounds of selection. Furthermore, an antigen specific clone was successfully isolated from a partially enriched scFv library confirming the strength of the protocol. This approach has the potential to identify novel scFv's of use in adoptive T cell therapy and, potentially, wider antibody-based applications.
CitationIsolation of tumour antigen specific scFv's using a chimeric antigen receptor bicistronic retroviral vector in a mammalian screening protocol. 2013: Hum Gene Ther Methods
JournalHuman Gene Therapy Methods