Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells

Abstract

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.

Phage Interactions with the Nervous System in Health and Disease

“…Even though many different drugs that potentially target glioblastoma have been investigated, none of them have entered clinical practice, possibly because most glioblastomas are far from a single-pathway driven disease [ 195 ]. As reported, glioma stem-like cells are resistant to radiotherapy and chemotherapy for yet undefined reasons [ 199 , 200 , 201 , 202 , 203 , 204 , 205 ]. Especially in the case of brain tumors, the therapeutic effect is difficult to achieve because of the presence of the BBB, which impedes the penetration of drug particles.…”

“…Especially in the case of brain tumors, the therapeutic effect is difficult to achieve because of the presence of the BBB, which impedes the penetration of drug particles. Moreover, the glioma stem cells (GSCs) turned out to be capable of self-renewal and resistant to standard treatment [ 202 ]. This provides an unmet need to create innovative solutions that would allow difficulties in the delivery, pharmacokinetics, and therapeutic use of currently available drugs to be overcome, but also the development of new vectors and novel methods of administration, diagnosis, and monitoring of treatment responses.…”

“…Phage display technology has also been used to develop nanobodies targeting CA9 [ 233 ] and ABCC3 [ 234 ], expression of which was detected to correspond with the worse overall survival of patients with glioblastoma. Another approach could involve combining the use of phage display for identification of tumor-targeting peptides and CAR-T therapy for novel modifications of classic CAR-T, which seems to have potential in the development of novel cytotoxic therapies for glioblastoma, as demonstrated by Potez et al [ 202 ].…”