22. A Novel Single-Chain Antibody for Selective Targeting of IL13Ra2-Expressing Brain Tumors

BACKGROUND: IL13Ra2 is overexpressed in majority of high grade astrocytomas and other malignancies, and has been validated as a target for therapeutic applications in various pre-clinical models. However, current IL13-based therapeutic agents lack specificity due to interaction with the IL13Ra1 receptor, which is widely expressed by normal cells. The generation of a targeting agent that strictly binds to IL13Ra2 would significantly expand the therapeutic potential for the treatment of IL13Ra2-expressing cancers. Recently, we developed and extensively characterized monoclonal antibody 47 (mAb47), which exclusively binds to a native form of human IL13Ra2. The goal of this study was to engineer a single-chain antibody (scFv) from the parental hybridoma cell line, test its targeting properties as a soluble agent, and create an adenovirus (Ad) with a modified fiber incorporating scFv47 as a targeting motif.METHODS: The phage-display approach was utilized for selection of functional combination of variable heavy and light chains from established hybridoma cells producing mAb47. Purified phages displaying scFv47 were tested for their interaction with IL13Ra2hFc recombinant protein. A competitive ELISA was utilized to verify if parental mAb47 and scFv47 share the same epitope. The soluble form of scFv47 expressed in E. coli and CHO cells was analyzed by SDS-PAGE, and tested for stability and targeting properties. To generate IL13Ra2-specific Ad, the fiber of a replication-deficient Ad5 encoding green fluorescent protein was replaced with a chimeric fiber gene composed of a T4 fibritin trimerization domain linked at its C-terminal to scFV47 (AdFFscFv47-CMV-GFP). To generate viral particles, a construct encoding the adenoviral genome was rescued in HEK293F28 cells, propagated, and purified. IL13Ra2+ and IL13Ra2- U251 cell lines were established via stable transfection with either control or IL13Ra2-specific shRNAs (U251-IL13Ra2. KO), respectively. The AdFFscFv47-CMV-GFP virus was tested for targeting properties in these U251 cell lines and IL13Ra2-expressing U87 glioma cells.RESULTS: The biopanning-selected pool of phages, as well several individual clones, demonstrated specific binding to IL13Ra2hFc protein, but not to hIgG in plate ELISA. Binding of scFv47-displayed phages to IL13Ra2 was completely abolished by the mAb47, but not control IgG or other tested IL13Ra2 mAbs, thus confirming the same IL13Ra2 epitope for scFv47 and parental mAb47. Similarly to phage-displayed scFv47, the soluble scFv47 showed specific binding to IL123Ra2, but not IL13Ra1. Interaction of Ad5FFscFv47-CMV-GFP was also specific to IL13Ra2-expressing U251 cells, as judged by flow cytometry for GFP expression in U251-IL13Ra2+ versus U251-IL13Ra2.KO cells. Furthermore, GFP expression in cells infected with Ad5FFscFv47-CMV-GFP strongly correlated with the level of surface expression of IL13Ra2. The specificity of viral infection was further validated in a U251 glioma model.CONCLUSION: Our data validate the scFv47 as a highly selective IL13Ra2 targeting agent and open venues for the exploration of scFv47-based viral, cell and protein therapeutics for the treatment of various IL13Ra2-expressing human malignancies.