Targeting urothelium: ex vivo assay standardization and selection of internalizing ligands.
OBJECTIVE
With the goal of targeting the human bladder using phage display technology we designed and tested a tissue binding assay on intact urothelium ex vivo. This approach may form the molecular basis for clinical development of peptide or peptidomimetic guided intravesical compounds.


METHODS
We screened 2 phage display random peptide libraries on human urothelium. Select peptides were tested for their binding ability to human urothelium, 2 human transitional cell carcinoma cell lines and a nontransitional cell carcinoma cell line. Next we standardized an ex vivo binding assay, validated binding of selected phage to whole urothelium, and evaluated whether receptor mediated internalization into urothelium derived cells occurred. Finally we tested if the presence of the glycosaminoglycan layer had any effect on the binding of the urothelium targeted phage.


RESULTS
Phage selected and recovered in the screening were isolated and sequenced. Displayed peptide sequences were searched against online protein databases. Five classes of peptide motifs were characterized based on their ability to bind to normal urothelium but not to control cell lines. Remarkable consistency and reproducibility were observed in the ex vivo binding assays. Two classes of peptide motifs sharing the sequence Ile/Leu-Ser-Gly-Leu bound to normal urothelium and to 2 transitional cell carcinoma cells but not to nontransitional cell carcinoma cells in a glycosaminoglycan independent manner and mediated internalization into cells of urothelial origin.


CONCLUSIONS
We introduce a strategy for screening combinatorial peptide libraries on bladder mucosa, a standard model for ex vivo intact urothelium binding assays and a panel of urothelium binding peptides that may be suitable for translation into targeted intravesical therapy applications.
Ardelt
Peter U
PU
Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, USA.
Wood
Christopher G
CG
Chen
Limor
L
Mintz
Paul J
PJ
Moya
Catherine
C
Arap
Marco A
MA
Wright
Kenneth C
KC
Pasqualini
Renata
R
Arap
Wadih
W
D002295:Carcinoma, Transitional Cell / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D019151:Peptide Library; D018000:Receptors, Peptide / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000378:metabolism* / Q000473:pathology; D019459:Urothelium / Q000378:metabolism* / Q000473:pathology
D019151:Peptide Library; D018000:Receptors, Peptide