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Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu¹

Joan T. Garrett^*,, Sharad Rawale^*, Stephanie D. Allen^*,, Gary Phillips, Guido Forni^#, John C. Morris^** and Pravin T. P. Kaumaya^2,*,,,¶,||

^* Departments of Obstetrics and Gynecology, Chemistry Biology Interface Program, Ohio State Biochemistry Program, Center for Biostatistics, ^¶ Molecular Virology, Immunology, Medical Genetics, and ^|| Arthur G. James Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; ^# Molecular Biology Center, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy; and ^** Metabolism Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563–626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563–598 and 597–626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597–626 and 613–626 recognizing HER-2. The 597–626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597–626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Cancer Institute Grant CA 84356 (to P.T.P.K.).

² Address correspondence and reprint requests to Dr. Pravin T. P. Kaumaya, The Ohio State University, Suite 316 Medical Research Facility, 420 West 12th Avenue, Columbus, OH 43210. E-mail address: kaumaya.1{at}osu.edu

³ Abbreviations used in this paper: ECD, extracellular domain; ADCC, Ab-dependent cell-mediated cytotoxicity; MVF, measles virus fusion protein; CDC, complement-dependent cytotoxicity; CD, circular dichroism; ESI-MS, electrospray ionization mass spectroscopy; HER-2, human epidermal growth factor receptor; PEO, polyethyleneoxide.

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