Targeting adenoviruses with factor x-single-chain antibody fusion proteins

Abstract

Abstract It has been shown that blood clotting factors, including factor X (FX), bind to the adenovirus serotype 5 (Ad5) hexon protein and target the virus to liver hepatocytes after intravenous injection. These factors bind to hexon via their conserved vitamin K-dependent gamma-carboxyglutamic acid (GLA) domains with subnanomolar affinity. In this work, we have used this strong interaction to retarget Ad to new receptors, using the GLA domain of FX fused to single-chain antibody variable fragment (ScFv). We demonstrate that fusion of the GLA domain of human FX to receptor-specific ScFvs will target Ad5 vectors to cells expressing these receptors. Fusion of an alphaHer2 ScFv to GLA increased in vitro transduction of Her2-positive versus Her2-negative cells when compared with untargeted virus. Similar results were obtained with ScFvs against the epidermal growth factor receptor (EGFR) and against the stem cell marker ATP-binding cassette protein G2 (ABCG2). Direct expression of GLA fusion protein from replication-defective or replication-competent Ad increased infection and killing of cancer cells in vitro and in vivo. These data demonstrate the potential of using GLA domains to bridge secreted ligands with intracellularly produced Ad5 vectors for vector targeting.

FIG. 1.

(A) Schematic of natural binding of FX to the adenovirus hexon. (B) Schematic of the engineered GLA–ScFv fusion protein strategy for generating targeting ligands. (C) GLA fusion protein constructs and controls designed for Her2, EGFR, and ABCG2 targeting. ABCG2, ATP-binding cassette protein G2; Ad5, adenovirus serotype 5; EGF, epidermal growth factor; EGFR, EGF receptor; GFP, green fluorescent protein; His6, hexahistidine; SA, streptavidin; ScFv, single-chain antibody variable fragment.

FIG. 2.

(A) Secreted GLA-αHer2-GFP fusion proteins bind to Her2-positive SK-BR-3 cells in vitro. Supernatant from 293 cells stably transfected to express various GLA fusion proteins or controls was incubated with 1 × 10⁶ MDA-MB-435 or SK-BR-3 cells. For Her2 expression analysis, cells were incubated with biotinylated Herceptin followed by streptavidin Alexa Fluor 488 incubation. After 1 hr of incubation on ice, cells were washed and analyzed by fluorescence-activated cell sorting (FACS) for green fluorescence. Incubation of Ad-Red virus with GLA-αHer2-GFP protein before infection increased the transduction of Her2-positive cells. Ad-Red viral particles (1 × 10⁷) were incubated with supernatant from cells expressing GLA-αHer2-GFP or control protein for 1 hr and then applied to cells for 30 min at 37°C. Cells were then washed and plated overnight. After 24 hr, cells were trypsinized and analyzed by FACS. (B) Percent dsRed positive. (C) Mean fluorescence index (MFI). ***p < 0.001.

FIG. 3.

In vivo effects of GLA-αHer2 on SKOV-3 infection by Ad5. (A and B) Nude mice injected intraperitoneally with 4 × 10⁶ SKOV-3 cells 28 days before treatment were injected with 1 × 10⁹ Ad-GL-RC virus that had been mixed with control 293 cell supernatant or with the supernatant from GLA-αHer2-expressing cells. (A) Luciferase images were taken on day 24 after virus injection. (B) Two mice from each group in (A) were imaged after the abdominal cavity was opened to facilitate viewing 10 days after viral injection. (C) Nude mice injected intraperitoneally with 4 × 10⁶ SKOV-3 cells 21 days before virus treatment were injected with 1 × 10⁹ Ad-GL-RC or Ad-GL-FB-RC virus with or without prior incubation with GLA-αHer2 supernatant. Survival was monitored over time after this single treatment and was analyzed by Kaplan–Meier survival analysis. Statistical comparisons described in text were performed by log-rank analysis. Color images available online at www.liebertonline.com/hum.

FIG. 4.

GLA can be fused to other targeting ScFvs and used to target Ad5. (A) αEGFR ScFv fused to GLA in cell supernatant was used to target Ad-Red to EGFR-overexpressing cells lines SKOV-3 and MDA-MD-468. Supernatant from 293 cells not secreting the targeting protein was incubated with Ad-Red and used as a negative control. dsRed expression was analyzed after 24 hr by FACS. (B) Purified GLA-αEGFR protein was incubated with Ad-GL-RC or Ad-GL-HB-RC at the indicated GLA-to-hexon molar ratios for 1 hr at 4°C before infection of EGFR-expressing SKOV-3 cells. After 24 hr, in vitro luciferase expression was measured.

FIG. 5.

Luciferase imaging of mice 24 hr after injection with Ad-GL virus. Warfarin was administered to the indicated animals on days 1 and 3 before viral injection. Ad-GL was mixed with either buffer, bovine serum albumin, or purified GLA-αEGFR protein 1 hr before injection. Color images available online at www.liebertonline.com/hum.

FIG. 6.

(A) In vitro luciferase expression from SKOV-3 cells infected with Ad-GL. SKOV-3 cells have low CAR expression and are not readily infected by Ad at low MOI. A549 cells infected with either Ad-GLA-αEGFR-RD or Ad-LacZ-RD control virus provided secreted protein via a permeable Transwell. Protein secreted by Ad-GLA-αEGFR-RD-infected A549 cells enhanced the infection of SKOV-3 cells by Ad-GL-RC. n = 4. (B) In vitro cell-killing assay for Ad-GLA-αEGFR-RC virus. SKOV-3 cells plated on a 96-well plate were infected with serially diluted Ad-GLA-αEGFR-RC or control Ad-GL-RC virus. Two weeks after infection, cell viability was assessed by MTT assay (n = 4).

FIG. 7.

Treatment of SKOV-3 ovarian cancer xenografts with GLA-expressing viruses. Subcutaneous tumors were initiated and treated six times with the indicated virus combinations described in text. Black lines represent mice treated with buffer. Blue lines represent mice treated with oncolytic Ad-GL-RC in combination with replication-defective gene-expressing viruses. Ad-LacZ-RD was the negative control replication-defective virus for Ad-GLA-αEGFR-RD in these groups. Red lines represent mice treated with oncolytic Ad-GL-RC in combination with oncolytic replication-competent viruses. Ad-RC was the negative control replication-competent virus for replication-competent Ad-GLA-αEGFR-RC. Dashed lines represent negative control viruses. Solid lines represent GLA-expressing viruses. (A) Mean tumor volumes. Note: Lines terminate when the first mouse was killed, because tumor averages after this point are skewed from the beginning averages. (B) Kaplan–Meier survival analysis through 60 days posttreatment. Color images available online at www.liebertonline.com/hum.