Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.
Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcgamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to 20 times more IL-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with zeta-chain intracellular signaling domains only. IL-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3'-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.
Finney
H M
HM
Celltech Therapeutics Ltd., Slough, United Kingdom.
Lawson
A D
AD
Bebbington
C R
CR
Weir
A N
AN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D015703:Antigens, CD / Q000235:genetics; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics; D018106:CD28 Antigens / Q000235:genetics; D019672:Genes, T-Cell Receptor / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007376:Interleukin-2 / Q000096:biosynthesis; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D016254:Mutagenesis, Insertional; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D063268:Sialic Acid Binding Ig-like Lectin 3; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018106:CD28 Antigens; C571163:CD33 protein, human; C571165:Cd33 protein, mouse; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; C054979:antigen T cell receptor, zeta chain; D019869:Phosphatidylinositol 3-Kinases