Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.
Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.
Ager
S
S
Cambridge Centre for Protein Engineering, England.
Nilson
B H
BH
Morling
F J
FJ
Peng
K W
KW
Cosset
F L
FL
Russell
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D017252:CD3 Complex / Q000235:genetics* / Q000276:immunology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005347:Fibroblasts; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000276:immunology* / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D016254:Mutagenesis, Insertional; D010957:Plasmids
D000954:Antigens, Surface; D017252:CD3 Complex; D006023:Glycoproteins