A TVA-single-chain antibody fusion protein mediates specific targeting of a subgroup A avian leukosis virus vector to cells expressing a tumor-specific form of epidermal growth factor receptor

Abstract

We have previously described an approach that employs retroviral receptor-ligand bridge proteins to target retroviral vectors to specific cell types. To determine whether targeted retroviral entry can also be achieved using a retroviral receptor-single-chain antibody bridge protein, the TVA-MR1 fusion protein was generated. TVA-MR1 is comprised of the extracellular domain of the TVA receptor for subgroup A avian leukosis viruses (ALV-A), fused to the MR1 single-chain antibody that binds specifically to EGFRvIII, a tumor-specific form of the epidermal growth factor receptor. We show that TVA-MR1 binds specifically to a murine version of EGFRvIII and promotes ALV-A entry selectively into cells that express this cell surface marker. These studies demonstrate that it is possible to target retroviral vectors to specific cell types through the use of a retroviral receptor-single-chain antibody fusion protein.

FIG. 1

Production of TVA-MR1. (A) TVA-MR1 was comprised of the extracellular domain of the TVA receptor for ALV-A fused, via a proline-rich linker region, to the N-terminal end of the MR1 single-chain antibody that binds specifically to EGFRvIII. (B) Extracellular supernatants collected from transfected 293 cells that expressed TVA-MR1 (lane 2) or did not express the bridge protein (lane 1) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The protein samples were then subjected to immunoblotting with SUA-rIgG (58) and a horseradish peroxidase-conjugated secondary antibody, and the TVA-MR1 protein was detected by enhanced chemiluminescence.

FIG. 2

TVA-MR1 binds specifically to the EGFRvIII protein. (A) 293T cells and 293T-EGFRvIII cells were incubated with extracellular supernatants that either lacked or contained TVA-MR1. The bound TVA-MR1 proteins were then detected by flow cytometry using SUA-rIgG and an FITC-conjugated secondary antibody. (B) Extracellular supernatants containing TVA-MR1 were preincubated with either no peptide (none), the MR1 epitope-containing peptide, or a scrambled version of this peptide. These samples were then incubated with 293T-EGFRvIII cells, and the bound TVA-MR1 proteins were then detected by flow cytometry as shown in panel A. The results obtained were compared with the background levels of fluorescence obtained with cells incubated in the absence of TVA-MR1. (C) 293T-TVA^syn cells were incubated without SUA-rIgG or with SUA-rIgG and with either no peptide (none), the MR1 epitope-containing peptide, or the scrambled peptide. The bound immunoadhesin was then detected by flow cytometry using an FITC-conjugated secondary antibody. Results of a representative experiment are shown (A through C).

FIG. 3

TVA-MR1 promotes ALV-A infection of 293T-EGFRvIII cells. 293T-EGFRvIII cells (A through C) and 293T cells (D through F) were incubated with extracellular supernatant that contained (+) or lacked (−) TVA-MR1 as indicated. These cells were incubated in the absence or presence of the ALV-A vector RCASBP(A)-EGFP, and then aliquots of each cell type (5 × 10⁴ 293T-EGFRvIII cells and 10⁵ 293T cells) were analyzed by flow cytometry.

FIG. 4

The TVA-MR1–EGFRvIII interaction is required for targeted viral entry. Extracellular supernatant containing TVA-MR1 was incubated with no peptide (none), the MR1 epitope-containing peptide, or the scrambled peptide before addition to 293T-EGFRvIII cells. The cells were then challenged with RCASBP(A)-EGFP and analyzed by flow cytometry 72 h later. For control purposes, viral infection of 293T-EGFRvIII cells was also assessed in the absence of any TVA-MR1.