Oncolytic herpes simplex viruses designed for targeted treatment of EGFR-bearing tumors

Abstract

Oncolytic herpes simplex viruses (oHSVs) have emerged as leading cancer therapeutic agents. Effective oHSV virotherapy may ultimately require both intratumoral and systemic vector administration to target the primary tumor and distant metastases. An attractive approach to enhancing oHSV tumor specificity is engineering the virus envelope glycoproteins for selective recognition of and infection via tumor-specific cell surface proteins. We previously demonstrated that oHSVs could be retargeted to EGFR-expressing cells by the incorporation of a single-chain antibody (scFv) at the N terminus of glycoprotein D (gD). Here, we compared retargeted oHSVs generated by the insertion of scFv, affibody molecule, or VHH antibody ligands at different positions within the N terminus of gD. When compared to the scFv-directed oHSVs, VHH and affibody molecules mediated enhanced EGFR-specific tumor cell entry, spread and cell killing in vitro, and enabled long-term tumor-specific virus replication following intravenous delivery in vivo. Moreover, oHSVs retargeted via a VHH ligand reduced tumor growth upon intravenous injection and achieved complete tumor destruction after intratumoral injection. Systemic oHSV delivery is important for the treatment of metastatic disease, and our enhancements in targeted oHSV design are a critical step in creating an effective tumor-specific oHSVs for safe administration via the bloodstream.

Keywords: EGFR; GBM; HSV; MT: Regular Issue; glycoprotein; oncolytic; vector targeting.

Graphical abstract

Figure 1

Vector engineering (A) Schematic representation of the KG4:T124-ΔgD:GW vector, illustrating the unique long (U_L), unique short (U_S), terminal repeat long (TR_L), and terminal repeat short (TR_S) segments of the HSV genome. KG4:T124-ΔgD:GW contains BAC sequence between U_L37 and U_L38, two viral entry-enhancing mutations in the gB glycoprotein gene (gB:NT), a deletion encompassing the adjacent internal repeat sequences IR_L and IR_S (ΔJOINT), a GFP marker gene linked to the gC open reading frame via a T2A sequence (gC-T2A-GFP), and 4 copies of a miR-124 recognition sequence in the 3′ UTR of the ICP4 gene (ICP4:T124). A GW cassette replaces the coding sequence for the gD glycoprotein (ΔgD:GW). (B) Schematic of gD showing the locations of the SP, immunoglobulin-like fold (Ig fold), profusion domain (PFD), and transmembrane domain (TM). Residue 38 is deleted in all of the retargeted gD glycoproteins to ablate nectin-1 binding. The black box (Δx-24) indicates the residues removed to eliminate HVEM binding (the x position varies per construct); this is the insertion site for the targeting ligands shown below. (C) Glycoprotein incorporation into purified virus particles was assessed by western blot. 1 × 10⁸ gc of purified virus was loaded per lane and probed with antibodies recognizing the envelope glycoproteins gD and gB and the tegument protein VP16. Relative gD and gB band intensities normalized to VP16 and set to gD:scEΔ38 = 1× are shown below the lanes. Western blots were performed in duplicate and representative images and band intensities are shown.

Figure 2

The retargeted viruses enter cells via human EGFR Human EGFR-specific cell entry was assessed on HSV receptor-deficient J1.1-2 cells, J-C (nectin-1), and J-EGFR (hEGFR) cells. Cells were infected for 6 h at an MOI of 1,000 gc/cell and immunostained with antibody recognizing the IE protein ICP4 as a marker of virus entry (green) and counterstained with DAPI as a positive control (blue). Scale bars, 200 μM.

Figure 3

The retargeted viruses enter cells via the tumor-associated EGFRvIII mutant EGFRvIII-specific cell entry was assessed on HSV receptor-deficient B78H1 cells, B78-C (nectin-1), and B78-vIII (mutant EGFRvIII) cells. Cells were infected for 6 h (MOI of 1,000 gc/cell) and immunostained with antibody recognizing the IE protein ICP4 (green) and counterstained with DAPI as a positive control (blue). Scale bars, 200 μM.

Figure 4

Viruses retargeted by the SD antibodies or affibody molecules demonstrate enhanced cell-to-cell spread Vero donor cells were infected at an MOI of 10,000 gc/cell, extracellular virus was inactivated, and equal numbers of infected donor cells were added onto monolayers of uninfected (A) Vero, (B) U251, (C) SNB19, or (D) A549 acceptor cells. Plaques were imaged at 2 dpi and plaque size was quantified using ImageJ software. Averages were calculated ±SEM, and 1-way ANOVA analyses were used to determine differences observed between groups (n = 5–15; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 5

Viruses retargeted via the SD antibodies or affibody molecule demonstrate enhanced cell entry (A) Vero, (B) U251, (C) SNB19, and (D) A549 cells were infected at 1,000 gc/cell, fixed at 6 hpi, and stained for ICP4 and DAPI. Fluorescent images were captured and quantified with ImageJ software. Data are presented as the percentage of ICP4⁺ cells relative to the total DAPI⁺ cells. Averages were calculated ±SEMs (n = 5). One-way ANOVA analyses were used to determine differences observed between groups. Statistical significance (∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001) is shown for gD:scEGFRΔ2-24 relative to the other viruses. Statically significant differences (p < 0.0001) were also observed between gD:wt and gD:SD2Δ6-24(SNB19 and A549); gD:wt and gD:SD3Δ6-24 (SNB19 and A549); gD:wt and gD:SD1Δ6-24 (A549) but are not illustrated.

Figure 6

Virus-mediated cell death in vitro (A) U251, (B) SNB19, or (C) A549 cells were infected at 1,000 gc/cell, and cell viability was assessed at 24, 48, and 72 hpi by alamarBlue assay. Data are presented as the percentage of viable cells relative to uninfected cells at each time point; average ±SEM (n = 5–8). Statistics were determined by 2-way ANOVA. The viability of U251, SNB19, and A549 cells infected with gD:wt, gD:SD1Δ6-24, gD:SD2Δ6-24, gD:SD3Δ6-24, and gD:ZEGFRΔ7-24 was significantly reduced compared to gD:scEGFRΔ2-24. For U251 cells, p < 0.0001 for all time points. For SNB19 cells, p < 0.0001 at 24 and 48 hpi for each comparison; at 72 hpi, p < 0.001 for gD:SD1Δ6-24, gD:SD2Δ6-24, and gD:wt, p < 0.01 for gD:SD3Δ6-24, and p < 0.05 for gD:ZEΔ7-24. For A549 cells, at 24 hpi, p < 0.0001 for gD:SD2Δ6-24, p < 0.01 for gD:wt and gD:SD1Δ6-24, and p < 0.05 for gD:SD3Δ6-24; at 48 hpi, p < 0.0001 for gD:SD2Δ6-24, p < 0.001 for gD:wt, and p < 0.01 for gD:SD1Δ6-24 and gD:ZEΔ7-24; at 72 hpi, p < 0.01 for gD:SD1Δ6-24, gD:wt, and gD:SD2Δ6-24. (D) Infected Vero donor cells were mixed with U251 acceptor cells (1 Vero cell per 10 U251 cells), and cell viability was evaluated by alamarBlue assay. The gD:ZEGFRΔ7-24 virus significantly reduced cell viability when compared to gD:scEGFRΔ2-24 at 24, 48 (p < 0.05), and 72 hpi (p < 0.001). gD:SD2Δ6-24 significantly reduced cell viability at 48 and 72 hpi when compared to gD:scEGFRΔ2-24 (p < 0.001).

Figure 7

Treatment of U251 subcutaneous tumors by systemic or intratumoral vector delivery U251 cells were implanted in the right hind flank of BALB/c athymic nude mice, and when tumors reached a volume of ∼100 mm³, the mice were treated with 1 × 10¹⁰ gc of the indicated virus or vehicle control (PBS). Black arrows indicate the days of treatment. (A–C) The i.v. delivery of a single dose of Fluc_gD:scE (scE), Fluc_gD:SD2 (SD2), or Fluc_gD:ZE (ZE) (n = 5 mice per group). (D–F) Four doses of SD2 delivered i.t. or i.v. (n = 3 mice per group). (A and F) Fluc expression from the viral backbone was quantified by BLI in a time course beginning 1 day after vector delivery and expressed as p/s (mean ± SEM). (B and D) Tumor growth was assessed by creating a growth curve of tumor volume (mm³; mean ± SEM) and by (C and E) determining the AUC for the growth curves generated in (B) and (D). Statistical differences were determined by 2-way ANOVA (A, B, D, and F) and by 2-tailed nonparametric Mann-Whitney test (C and E) (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).