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. 2015 May;26(5):320-9.
doi: 10.1089/hum.2015.015.

Incorporation of Peptides Targeting EGFR and FGFR1 Into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies

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Free PMC article

Incorporation of Peptides Targeting EGFR and FGFR1 Into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies

Hanni Uusi-Kerttula et al. Hum Gene Ther. .
Free PMC article

Abstract

Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCAR(low)/EGFR(high) cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of circumventing preexisting Ad5 immunity that warrants further investigation.

Figures

<b>FIG. 1.</b>
FIG. 1.
Generation of recombinant cancer-targetable Ad5 vectors. (A) Overview of viral modification and production using recombineering. (1) Design of the selection cassette, (2) temperature-induced (42°C) recombineering of the selection cassette into Escherichia coli strain SW102 by electroporation, (3) recombineering of the target sequence by electroporation, (4) verification of the correct clone by sequencing, purification of DNA by maxiprep, and generation of P1 virus stocks in permissive T-REx-293 cells, (5) propagation of high titer P2 stocks, (6) purification of viral particles by CsCl gradient ultracentrifugation and dialysis, and (7) titration by microBCA assay. (B) Amino acid sequence alterations within the Ad5 fiber knob domain. Peptide sequences GE11, YHWYGYTPQNVI; M*, MQLPLAT; LS, LSPPRYP; and scramble, LMTLAQP, were genetically inserted into fiber knob HI loop after Thr541. For the purpose of native hCAR-binding ablation, KO1 mutation (S408E, P409A) was introduced into the AB loop. (C) The inserted peptides (shown in green) GE11, M*, LS, scramble, and the HI loop (magenta) are highlighted. Inset: side view of the fiber knob, showing native (gray) or mutated hCAR-binding site (yellow). EGFR, epidermal growth factor receptor; FGFR1, fibroblast growth factor receptor 1; hCAR, coxsackie and adenovirus receptor; KO1, hCAR-binding mutation. (D) Verification of fiber integrity by Western blotting. Color images available online at www.liebertpub.com/hum
<b>FIG. 2.</b>
FIG. 2.
Transduction efficiency of targeted Ad5 vectors and effect of human coagulation factor X (hFX). (A) hCAR expression (green) as compared with isotype control normal mouse IgG (gray) on different cell lines, measured by flow cytometry. (B) Transduction efficiency on CHO-K1 and CHO-CAR cell lines in serum-free medium (n=4). (C) Transduction efficiency on OVCAR3 cells (n=3). (D) Transduction efficiency in serum-free medium (hFX) on T24 cell line (n=4). (E) Transduction efficiency on OVCAR3 and T24 cell lines in serum-free medium or supplemented with 10 μg/ml of human coagulation factor X (hFX) (n=4). p-Values indicate comparison to the same virus in serum-free conditions (hFX). (F) Transduction efficiency in presence of hFX (+) on T24 cell line (n=4). p-Values indicate comparison to the same virus in serum-free conditions (Fig. 2D). *p<0.05, **p<0.01, ***p<0.001, ns=not statistically significant, p>0.05. Error bars represent SD. Color images available online at www.liebertpub.com/hum
<b>FIG. 3.</b>
FIG. 3.
Transduction efficiency on primary epithelial ovarian cancer (EOC) cells and vector neutralization by ascitic fluid. (A) Inhibition of Ad5.Luc vector transduction in the presence of increasing concentrations of ascitic fluid, relative to serum conditions. The approximate dilution that neutralizes 50% and 90% of transduction is indicated with dotted lines. (B) Vector transduction efficiency in four different virus–medium preincubation conditions: serum medium; with 10 μg/ml of human coagulation factor X (hFX+); with 2.5% OAS000 supernatant; or with 2.5% OAS001 supernatant. (C) Neutralizing effect of ovarian ascites fluid supernatant on Ad5 vector transduction. Transduction levels (%) are shown relative to Ad5.Luc transduction in serum-free conditions. OAS000, ovarian ascites supernatant patient #000; OAS001, ovarian ascites supernatant patient #001. Error bars represent the SD (n=3).

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