ORIGINAL ARTICLE AHNP-streptavidin: a tetrameric bacterially produced antibody surrogate fusion protein against p185 her2/neu K Masuda, M Richter, X Song, A Berezov, K Masuda, R Murali, MI Greene and H Zhang Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The anti-p185 her2/neu peptidomimetic (AHNP) is a small exo-cyclic peptide derived from the anti-p185 her2/neu rhumAb 4D5 (h4D5). AHNP mimics many but not all of the antitumor characteristics exhibited by h4D5. However, the pharmacokinetic profiles of AHNP are less than optimal for therapeutic or diagnostic purposes. To improve the binding affinity to p185 her2/neu and the antitumor efficacy, we have engineered a fusion protein containing AHNP and a nonimmunoglobulin protein scaffold, streptavidin (SA). The recombinant protein, AHNP-SA (ASA) bound to p185 her2/neu with high affinity, inhibited the proliferation of p185 her2/neu -overexpressing cells, and reduced tumor growth induced by p185 her2/neu - transformed cells. These data suggest that the bacterially produced tetrameric ASA can be used as an antibody- surrogate molecule. This class of molecule will play a role in the diagnosis and treatment of p185 her2/neu -related tumors. Our studies establish a general principle by which a small biologically active synthetic exo-cyclic peptide can be engineered to enhance functional aspects by structured oligomerization and can be produced recombinantly using bacterial expression. Oncogene (2006) 25, 7740–7746. doi:10.1038/sj.onc.1209745; published online 19 June 2006 Keywords: AHNP; P185 her2/neu ; streptavidin; tetramer Introduction Monoclonal antibodies represent mainstream agents for molecular diagnosis and have recently become de facto cancer therapeutics. There is a great interest in devel- oping smaller versions of therapeutic antibodies for several reasons: namely to improve tumor penetration, eliminate the humanization process, and reduce the cost of production (Maynard and Georgiou, 2000; Hudson and Souriau, 2003). Previously, we established a general principle that a single CDR3 loop from the heavy chain of an antibody is sufficient to mimic its parental monoclonal antibody function (Murali and Greene, 1998). The CDR peptide is much smaller than the antibody but retains binding affinity to the antigen as well as the biological activity of the antibody. Our studies with CDR mimetics demonstrated that the constrained CDRs could mediate binding in a context- independent manner and formed the theoretical basis for the technique of CDR grafting (Bruck et al., 1986; Williams etal., 1989; Park etal., 2000). p185 her2/neu (Her2, Neu, c-ErbB2) is overexpressed in many breast and ovarian cancers (Slamon etal., 1989), lung tumors (Kern etal., 1990) as well as tumors of the pancreas (Williams etal., 1991). Extensive studies have shown that p185 her2/neu plays a dominant role in mediat- ing the malignant phenotype (Kokai etal., 1988; Cohen et al., 1989; Lodato et al., 1990). The evolution of targeted therapy began with the development of mono- clonal antibodies to p185 her2/neu (Drebin etal., 1984, 1985, 1986, 1988) that directly lead to the reversion of the malignant phenotype. The anti-p185 her2/neu antibody ‘trastuzumab’ (h4D5 or Herceptin, Genentech, San Francisco, CA, USA), which represented a humanized antibody of similar specificity, has been approved by FDA to treat breast cancers (Sliwkowski etal., 1999). Earlier, we reported the design of a p185 her2/neu binding peptidomimetic (BiP) anti-p185 her2/neu peptidomimetic (AHNP) (FCDGFYACYMDV) (Park et al., 2000; Berezov et al., 2001). AHNP was rationally derived from the structure of the CDR-H3 loop of the anti- p185 her2/neu antibody h4D5, and possessed the ability to disable p185 her2/neu tyrosine kinase activity in vitro and in vivo similar to, but with far less affinity, than the monoclonal antibody. Unlike the intact phenotype- reverting monoclonals, AHNP was unable to down- regulate p185 her2/neu from the cell surface, indicating that the small molecule functioned to directly disable the p185 her2/neu complex. These characteristics suggested a potential for antibody-mimicking peptidomimetics to serve as reagents for tumor therapy. Streptavidin (SA), a tetrameric protein produced by Streptomyces avidinii that binds to biotin with an extremely high affinity (K D B10 15 M), represents an ideal scaffold to enhance the functional potential of AHNP. With a high affinity for biotin, the fusion protein will be able to carry biotinylated therapeutics (such as cytotoxins) or labeling reagents to tumor cells with p185 her2/neu overexpression. This strategy has already Received 14 March 2006; revised 26 April 2006; accepted 26 April 2006; published online 19 June 2006 Correspondence: Dr MI Greene, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 252 John Morgan Building, 36th Hamilton Walk, Philadelphia, PA 19104-6082, USA. E-mail: greene@reo.med.upenn.edu Oncogene (2006) 25, 7740–7746 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc