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BackgroundGlioblastoma (GBM) is both the most common and aggressive type of primary brain tumor, associated with high mortality rates and resistance to conventional therapy. Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and in vivo phage display biopanning.Methods and resultsWe have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining.ConclusionThese results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM.

Glioblastoma (GBM) is the most aggressive primary brain tumor with high mortality rates and resistance to conventional therapy. Their resistance to conventional therapy has been attributed to the presence of cancer stem cells (CSCs), a sub-population of tumor cells capable of self-renewal and tumor initiation. Developing novel strategies to specifically target GSCs may allow more effective therapeutic strategies. Using in vivo phage display biopanning, we have identified several peptides with the potential to selectively target and bind GSCs. We wished to leverage the GSC targeting properties of the peptides to augment therapeutic delivery vehicles for the development of novel targeting strategies. We used a combination of GSC targeting peptides to modify the antigen-binding domain of chimeric antigen receptors, by arranging the peptides in tandem at the N-terminus of the CAR molecule. These tandem peptides were tested for binding to GSCs in vitro and in vivo. The functionality of the CAR-T cells was evaluated by measuring cytokine release in the supernatant after overnight co-culture through ELISA. Apoptosis was evaluated by flow cytometry with Annexin V staining. Two different GSC-targeting peptide CAR-T cells demonstrated specific targeting GSCs. Following co-culture with GSCs, GSC targeting CAR-T cells were activated with release of Interferon gamma and subsequently induced GSCs specific apoptosis. These results demonstrate the use of phage display biopanning to isolate GSC targeting peptides which may be used to develop novel GBM specific cytotoxic therapies.