Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications

Abstract

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

Hitting the Target but Missing the Point: Recent Progress towards Adenovirus-Based Precision Virotherapies

“…The rate-limiting step is the lack of efficacious tumour targeting peptides that can be incorporated into the viral capsid efficiently to retarget the Ad5 NULL platform towards tumour cells. Previously, we and others have utilised methods such as bacteriophage (phage) biopanning to identify peptides that can bind TAAs [ 153 , 154 , 155 ].…”

“…This has been tried with several different peptides targeting cancer-specific markers, such as folate receptor α (FRα) commonly upregulated in ovarian cancers [ 159 ]. However, after binding, the FRα mediated cell entry mechanism does not allow for correct intracellular trafficking, showing retention of targeted virotherapies in late endosomes in FRα positive ovarian cancer cells, with limited successful transduction [ 153 ]. This highlights another potential consideration when developing targeting approaches for adenoviral-based oncolytics—not all TAA receptor pathways will be compatible with clathrin-mediated endocytosis pathways and thus represent viable entry routes for adenoviral-based virotherapies.…”

Hitting the Target but Missing the Point: Recent Progress Towards Adenovirus-Based Precision Virotherapies

“…The rate-limiting step is the lack of efficacious tumour targeting peptides that can be incorporated into the viral capsid efficiently to re-target the Ad5NULL platform towards tumour cells. Previously, we and others have utilised methods such as bacteriophage (phage) biopanning approaches have been used to identify peptides that can bind TAAs (Hulin-Curtis et al, 2020;Maruta et al, 2002Maruta et al, , 2007Uusi-Kerttula et al, 2015).…”

Hitting the Target but Missing the Point: Recent Progress Towards Adenovirus-Based Precision Virotherapies

“…The rate-limiting step is the lack of efficacious tumour targeting peptides that can be incorporated into the viral capsid efficiently to re-target the Ad5NULL platform towards tumour cells. Previously, we and others have utilised methods such as bacteriophage (phage) biopanning approaches have been used to identify peptides that can bind TAAs [155][156][157].…”

“…This has been tired with several different peptides targeting cancer-specific markers, such as folate receptor α (FRα) commonly upregulated in ovarian cancers [162]. However, after binding, the FRα mediated cell entry mechanism does not allow for correct intracellular trafficking, showing retention of targeted virotherapies in late endosomes in FRα positive ovarian cancer cells, with limited successful transduction [155]. This highlights another potential consideration when developing targeting approaches for adenoviral based oncolytics -not all TAA receptor pathways will be compatible with clathrin-mediated endocytosis pathways and thus represent viable entry routes for adenoviral based virotherapies.…”