The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747Biochem. 269, -2754. Selected substrates were then transplanted into the reactive site loop of a1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific a1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.Keywords: phage-display; protease; human kallikrein; inhibitor; a1-antichymotrypsin.Prostate cancer is currently the most commonly diagnosed cancer in American men. This pathology is the second leading cause of cancer death after lung cancer and the majority of the patients with locally advanced prostate cancer have an increased risk for disease progression. In this progression, proteases are believed to play a pivotal role in the malignant behaviour of cancer cells, including rapid tumor growth, invasion and metastasis. Human glandular kallikrein (hK2) protein is a trypsin-like serine protease expressed predominantly in the prostate epithelium. First isolated from human seminal plasma [1], hK2 has emerged recently as a diagnostic marker for prostate cancer. When tested in combination with assays for various forms of prostate specific antigen (PSA), hK2 seemed to be better suited to distinguish malignant from benign prostate disease than the well established marker PSA (prostate specific antigen or hK3) [2][3][4]. In addition to its role as a marker, the proteolytic activities suggest that hK2 could contribute to cancer progression. Several potential functions for this enzyme have been proposed, including the activation of urokinase-type plasminogen activator Taking into account its prostate tissue-specific expression and the involvement of all its potential substrates in cancer development, hK2 can be considered as a potential therapeutic target.The serpins (serine protease inhibitors) are a large family of proteins implicated in the regulation of complex physiological processes. These proteins of about 45 kDa can be subdivided into two groups, one being inhibitory and the other noninhibitory. Serpins contain an exposed flexible reactive-site loop (RSL), which is implicated in the interaction with the putative target protease. Following the binding to the enzyme and cleavage of the P1-P'1 scissile bond of the RSL, a covalent com...

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy. ' 2006 Wiley-Liss, Inc.

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