title: The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.
abstract: Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.
authors.affiliation: Cancer Research UK, Department of Medical Oncology, University of Manchester and Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Guest
authors.forename: Robert E
authors.initials: RE
authors.lastname: Hawkins
authors.forename: Natalia
authors.initials: N
authors.lastname: Kirillova
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Cheadle
authors.forename: Jennifer
authors.initials: J
authors.lastname: Arnold
authors.forename: Allison
authors.initials: A
authors.lastname: O'Neill
authors.forename: Joely
authors.initials: J
authors.lastname: Irlam
authors.forename: Kerry A
authors.initials: KA
authors.lastname: Chester
authors.forename: John T
authors.initials: JT
authors.lastname: Kemshead
authors.forename: David M
authors.initials: DM
authors.lastname: Shaw
authors.forename: M J
authors.initials: MJ
authors.lastname: Embleton
authors.forename: Peter L
authors.initials: PL
authors.lastname: Stern
authors.forename: David E
authors.initials: DE
authors.lastname: Gilham
mesh_terms: D018941:Antigens, CD19 / Q000276:immunology / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000276:immunology*; D017252:CD3 Complex / Q000235:genetics* / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000276:immunology / Q000494:pharmacology; D005110:Extracellular Space; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology / Q000494:pharmacology; D019006:Neural Cell Adhesion Molecules / Q000276:immunology / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D014162:Transfection
chemical_list: D018941:Antigens, CD19; D000951:Antigens, Neoplasm; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D002272:Carcinoembryonic Antigen; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D019006:Neural Cell Adhesion Molecules; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C063709:trophoblastic glycoprotein 5T4, human