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 105-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. Efficient targeted retroviral gene delivery remains an important goal in gene therapy research, and we have been exploring the use of chimeric envelope glycoproteins as vector targeting elements. In this paper, we show that the topological relationship between the engineered domains in a chimeric envelope glycoprotein can have important functional consequences. Five different single-chain antibody fragments against human cell-surface antigens were displayed on retroviral vectors by fusing them close to the amino terminus of the Moloney murine leukemia virus (Mo-MLV) SU envelope glycoprotein. The spacing between the displayed antibodies and the SU glycoprotein was varied by fusing the antibody to different residues in SU and by insertion of peptide linker sequences between the domains. Expression and viral incorporation of the chimeric envelopes was remarkably efficient and the chimeric envelopes were shown to bind, via their displayed antibody domains, to human cells expressing the respective cognate antigens. However, specific antibody-mediated attachment to the human target cells did not in any case result in targeted gene delivery, irrespective of the spacing between the domains. However, as the interdomain spacing between the scFv and the SU glycoprotein increased, there was a dramatic enhancement of the infectivity of the engineered vectors for murine fibroblasts. The implications of these results are discussed in relation to future attempts to target retroviral gene delivery.