A strategy for systemic delivery of the oncolytic herpes virus HSV1716: redirected tropism by antibody-binding sites incorporated on the virion surface as a glycoprotein D fusion protein

Abstract

We report on the ability of single-chain variable fragment (scFv) incorporated into the viral envelope to alter the tropism of herpes simplex virus (HSV) 1716. Using recombinant viruses expressing fusion proteins comprising cellsurface antigen-specific scFvs N terminus linked to amino acids 274-393 of gD, we demonstrated that the tropism of these HSV1716 variants was modified such that infection was mediated by the cognate antigen. Thus, an HSV1716 variant that expressed an anti-CD55 scFv targeting moiety linked to these gD residues was able to infect non-permissive Chinese hamster ovary cells expressing CD55 and this infection was specifically blocked by an anti-CD55 monoclonal antibody. Similarly, the infection efficiency of an HSV1716 variant for semi-permissive human leukaemic, CD38-positive cell lines was greatly improved by an anti-CD38 scFv targeting moiety linked to gD residues 274-393, and this enhanced infectivity was abrogated specifically by an anti-CD38 monoclonal antibody. Finally, intravenous/ intraperitoneal injection of an HSV1716 variant displaying an anti-epidermal growth factor receptor (EGFR) scFv linked to residues 274-393 of gD enhanced destruction of subcutaneous EGFR-positive tumours in nude mice compared to unmodified HSV1716. Therefore, targeting of HSV1716 oncolysis to specific cell types through the display of entry mediating scFv/gD fusion proteins represents an efficient route for systemic delivery.

Inhibition of human tumor growth in mice by an oncolytic herpes simplex virus designed to target solely HER-2-positive cells

“…However, R-LM249 inability to infect cells that express HER-2 at low-intermediate level ensures that any nontumor cell constitutively expressing this molecule is spared by the virus. Such high safety profile cannot be obtained with recombinants that carry 2 copies of the glycoprotein, 1 WT and 1 retargeted (34).…”

Expression of inhibitor of growth 4 by HSV1716 improves oncolytic potency and enhances efficacy

“…The Ing4 expression cassette that included the CMV-IE promoter from plasmid was then transferred into the RL1 shuttle vector, pRL1-del/gfp, used for the production of HSV1716 variants by homologous recombination. 26 The presence of the Ing4 expression cassettes in the RL1 loci of HSV1716 was confirmed by both Southern blotting and by PCR using primers that amplify across the ICP34.5-deleted region of HSV1716 (data not shown). To confirm Ing4 expression, BHK cells were infected with 1 PFU (plaqueforming unit) per cell HSV1716Ing4 and, after 24 h, whole-cell extracts were analyzed by SDS-PAGE/western blotting using an Ing4 antibody (Abcam, Cambridge, UK).…”

“…Ing4 DNA was ligated into each vector, and the TKp-Ing4, gCp-Ing4, UL38p-Ing4 and CMV-Ing4 expression cassettes were cloned into the customized Gateway entry vector pENT1Agfp for production of recombinant HSV1716 variants by site-specific recombination. 26 Temporal expression of Ing4 by CMV-Ing4, TK-Ing4, gC-Ing4 or UL38-Ing4 was confirmed by reverse transcriptase PCR using RNA extracted from infected BHK cells and the original Ing4 primers. Strong Ing4 expression was observed with CMV-Ing4 at 3 h post infection and this was maintained at 6 and 9 h post infection.…”

“…26 The gC promoter was amplified from HSV-1 DNA by PCR as a SacI/KpnI fragment using primers (RE site in bold, promoter sequence underlined), 5 0 -GTGAGCTCCCGAAGACCGCCGGTGTG-3 0 and 5 0 -AGGGTACCGGCAAAGCGA GACCGGG-3 0 . The TK promoter was amplified from HSV-1 DNA by PCR as a SalI/PstI fragment using primers 5 0 -AGAAGTCGACTATGATGACACAA ACCCCGCC-3 0 and GACTGCAGTGCGGCACGCTGTTGACGCT-3 0 .…”

“…The CMV-Ing2 or CMVIng3 expression cassettes from their respective pcDNA4 constructs were excised and cloned into the customized Gateway entry vector pENT1Agfp used for production of recombinant HSV1716 variants by site-specific recombination. 26 …”