title: Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.
abstract: The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized 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 signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (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 tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, 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 nonexpressing scFvs 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 10(5)-fold enrichment of positive clones after 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 scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.
authors.affiliation: 1 Clinical Immune and Molecular Monitoring Laboratory, Clinical & Experimental Pharmacology Group, University of Manchester, Manchester Academic Health Science Centre , Christie NHS Foundation Trust, M20 4BX, Manchester, United Kingdom .
authors.forename: Grazyna
authors.initials: G
authors.lastname: Lipowska-Bhalla
authors.forename: David E
authors.initials: DE
authors.lastname: Gilham
authors.forename: Robert E
authors.initials: RE
authors.lastname: Hawkins
authors.forename: Dominic G
authors.initials: DG
authors.lastname: Rothwell
mesh_terms: D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods; D019169:Jurkat Cells; D019151:Peptide Library; D011946:Receptors, Antigen / Q000235:genetics / Q000276:immunology*; D012190:Retroviridae / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D019151:Peptide Library; D011946:Receptors, Antigen; D057127:Single-Chain Antibodies