We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H-O(1-6)XXXXX-NH(2), O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture of these. The library consisted of 114 mixtures in total. Using a biotin-streptavidin assay, the binding of each sublibrary to NR6M, NR6W-A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify the most active amino acid residue at each position. On the basis of this knowledge, eight peptides were synthesized and tested for binding to EGFRvIII. We identified one peptide, H-FALGEA-NH(2), that showed more selective binding to the mutated receptor than the EGFRvIII specific peptide PEPHC1. This study demonstrates the value of using mixture-based combinatorial positional scanning libraries for the identification of novel peptide ligands targeted against the cancer-specific EGFRvIII. Our best candidate H-FALGEA-NH(2) will be radioactively labeled and evaluated as an imaging agent for positron emission tomography investigation for diagnosis, staging, and monitoring of therapy of various types of cancer.

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There is growing interest in the use of radiolabelled peptides as receptor targeting agents for diagnostic imaging of various cancer types using positron emission tomography. In this work, 1-18 F]FPyME) has been used for parallel fluorine-18 labelling of PEPHC1, a peptide selective towards the cancer-specific mutation of the epidermal growth factor receptor (EGFRvIII), and a number of truncated and mutated analogues. Conjugation of the peptides with [18 F]FPyME was achieved within 10 min in non-decay-corrected radiochemical yields of 30-50%. The high yield of the conjugation reaction combined with its short synthesis time allows the labelling of several peptides from a single batch of [18 F]FPyME.