Synthesis, ¹⁸F-labeling and radiopharmacological characterization of a 30mer peptide as potential radiotracer for PET imaging of claudin-4

The cell surface receptor claudin-4 (Cld-4) represents a single-chain protein containing four transmembrane domains and constitutes cell–cell contacts of the tight-junction type by engaging in homophilic interactions. Cld-4 is upregulated in various tumors and represents a promising target for both diagnosis and treatment of solid tumors of epithelial origin. Therefore, the development of agents that allow imaging of Cld-4 in vivo such as ¹⁸F-labeled compounds for positron emission tomography (PET) appears to be attractive. A suitable ligand to target Cld-4 in vivo seems to be the C-terminal peptidic fragment of the C-terminal domain of the Clostridium perfringens enterotoxin cCPE(290-319). This fragment is of 30 amino acids in length and has the sequence SLDAGQYVLVMKANSSYSGNYPYSILFQKF corresponding to positions 290-319 of cCPE.
The synthesis of cCPE(290–319) and analogues derived thereof, such as N-terminally modified derivatives (fluorobenzoylated and FITC-conjugated) and variants in which critical amino acids (Tyr 306 and Leu 315) have been replaced, was envisaged to be accomplished by solid-phase peptide synthesis (SPPS). Among several approaches, sequential SPPS using three pseudoproline-dipeptide building blocks revealed to be the most efficient one to afford cCPE(290–319) and its derivatives. The affinity of the furnished peptides to a soluble protein construct that contains both extracellular loops of Cld-4 was studied by surface plasmon resonance (SPR), which allowed determining a K_d value of 1.4 lM for the N-terminally fluorobenzoylated cCPE(290-319). Labeling of cCPE(290–319) with fluorine-18 was achieved on solid phase using N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) and 4-[¹⁸F]fluorobenzoyl chloride as ¹⁸F-acylating agents. Most advantageous results were obtained when [¹⁸F]SFB was reacted with resin-bound cCPE(290–319) containing an N-terminal 6-aminohexanoic spacer. Stability assays in cell supernatants and plasma indicated no degradation of the resulting radiotracer in these physiological media. Cell binding of ¹⁸F-labeled cCPE(290–319) was investigated using the HT29, A375 and A431 tumor cell lines. Timedependent binding of the radiolabeled peptide to the Cld-4-positive A375 and A431 cells was observed, which was stronger than for the Cld-4-negative HT29 cell line. These findings are in accordance with results of confocal microscopy studies using FITC-conjugated cCPE(290–319) and A431 cells. The in vivo behavior of ¹⁸F-labeled cCPE(290–319) was studied in NMRI nu/nu mice and Wistar rats by dynamic PET imaging and radiometabolite analyses, respectively. These investigations have shown that 18F-labeled cCPE(290–319) is subject to substantial liver uptake and rapid metabolic degradation in vivo.
In conclusion, the synthesis and ¹⁸F-labeling of cCPE(290-319) were successfully established. Its binding to Cld-4 in vitro and in cellulo has been demonstrated. Initial radiopharmacological studies suggest the limited suitability of this peptide in its current non-stabilized form to target Cld-4 in vivo.