Programmed cell death signaling via cell-surface expression of a single-chain antibody transgene.
BACKGROUND
The monoclonal antibody, 5H7, is specific for a monomorphic determinant on the a3 domain of human class I MHC (A, B, C). Immobilized 5H7 delivers programmed cell death (PCD) signals to human lymphoid tumor cells as well as peripheral blood mononuclear cells.


METHODS
The potential clinical utility of 5H7 was addressed by design of a single-chain variable antibody (scFv), termed 5H7scFv, which was coupled to glycophosphotidylinostitol (GPI), thereby providing membrane expression of the 5H7 idiotype (5H7scFv-GPI). Membrane expression of 5H7scFv-GPI conferred PCD-inducing properties to cells that do not normally have the capability to process and express whole antibody molecules. The initial construction was undertaken in a bacterial expression system, and appropriate protein folding was determined by binding to class I MHC-expressing cells.


RESULTS
5H7scFv-GPI-transfected Chinese hamster ovary cells demonstrated reconstitution of the 5H7 idiotype and binding to soluble HLA-A2. Cross-linking of class I MHC, via membrane expression of the scFv, provided effective PCD signaling in B and T lymphocyte tumor cells. Peripheral blood mononuclear cells were susceptible to 5H7scFv-GPI-induced PCD, and augmentation of PCD signals was noted with anti-CD3 and anti-CD28 preactivation. Responder cells demonstrated typical histologic features of PCD and Annexin V-fluorescein isothiocyanate binding.


CONCLUSIONS
Cell surface anchorage of scFv thus provides effective delivery of immune modulatory signals, which may be manipulated for various therapeutic strategies.
Kulkarni
S
S
Department of Surgery, University of Chicago, Illinois 60637, USA. skulkarn@surgery.bsd.uchicago.edu
Holman
P O
PO
Kopelan
A
A
van Seventer
G A
GA
van Seventer
J M
JM
Kranz
D M
DM
Woodle
E S
ES
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D017209:Apoptosis / Q000502:physiology*; D001666:Binding Sites, Antibody; D016466:CHO Cells; D018920:Coculture Techniques; D006224:Cricetinae; D005434:Flow Cytometry; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008221:Lymphoid Tissue / Q000166:cytology; D008565:Membrane Proteins / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D017304:Annexin A5; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D010716:Phosphatidylinositols