[CANCER RESEARCH 61, 5349 –5354, July 15, 2001] Advances in Brief Growth Suppression of Intracranial Xenografted Glioblastomas Overexpressing Mutant Epidermal Growth Factor Receptors by Systemic Administration of Monoclonal Antibody (mAb) 806, a Novel Monoclonal Antibody Directed to the Receptor 1 Kazuhiko Mishima, Terrance G. Johns, Rodney B. Luwor, Andrew M. Scott, Elisabeth Stockert, Achim A. Jungbluth, Xiang-Dong Ji, Padma Suvarna, Joseph R. Voland, Lloyd J. Old, H-J. Su Huang, and Webster K. Cavenee 2 Ludwig Institute for Cancer Research, San Diego Branch [K. M., H-J. S. H., W. K. C.], Center for Molecular Genetics [W. K. C.], Department of Medicine [H-J. S. H., W. K. C.], and Cancer Center [W. K. C.], University of California at San Diego, La Jolla, California 92093-0660; Ludwig Institute for Cancer Research, Melbourne Branch, Austin Hospital, Heidelberg, Victoria 3084 Australia [T. G. J., R. B. L., A. M. S.]; Ludwig Institute for Cancer Research, New York Branch, New York, New York 10021-6007 [E. S., A. A. J., L. J. O.]; and Becton-Dickinson PharMingen, San Diego, California 92121 [X-D. J., P. S., J. R. V.] Abstract A mutant epidermal growth factor receptor (variously called EGFR, de2–7 EGFR, or EGFRvIII) containing a deletion of 267 amino acids of the extracellular domain is frequently highly expressed in human malig- nant gliomas and has been reported for cancers of the lung, breast, and prostate. We tested the efficacy of a novel monoclonal anti-EGFR anti- body, mAb 806, on the growth of intracranial xenografted gliomas in nude mice. Systemic treatment with mAb 806 significantly reduced the volume of tumors and increased the survival of mice bearing xenografts of U87 MG.EGFR, LN-Z308.EGFR, or A1207.EGFR gliomas, each of which expresses high levels of EGFR. In contrast, mAb 806 treatment was ineffective with mice bearing the parental U87 MG tumors, which ex- pressed low levels of endogenous wild-type EGFR, or U87 MG.DK tu- mors, which expressed high levels of kinase-deficient EGFR. A slight increase of survival of mice xenografted with a wild-type EGFR-overex- pressing U87 MG glioma (U87 MG.wtEGFR) was effected by mAb 806 concordant with its weak cross-reactivity with such cells. Treatment of U87 MG.EGFR tumors in mice with mAb 806 caused decreases in both tumor growth and angiogenesis, as well as increased apoptosis. Mecha- nistically, in vivo mAb 806 treatment resulted in reduced phosphorylation of the constitutively active EGFR and caused down-regulated expression of the apoptotic protector, Bcl-X L . These data provide preclinical evidence that mAb 806 treatment may be a useful biotherapeutic agent for those aggressive gliomas that express EGFR. Introduction mAbs 3 with specificity for tumor-associated antigens have long been considered promising agents for the treatment of cancer (1–3). Such mAbs may have intrinsic antitumor activity, they may be used in combination with other therapies, or they can be used to deliver conjugated cytotoxic agents (2). Receptors for certain growth factors are potential targets for this approach because their location on the cell surface may make them accessible to antibodies the subsequent bind- ing of which may block the growth-regulatory biological functions of the receptor (3–5). One notable example is the EGFR family member, HER-2/Neu (ErbB2), a transmembrane tyrosine kinase receptor fre- quently overexpressed in breast cancers (6, 7). The anti-HER-2 anti- body, Herceptin, causes growth inhibition of HER-2-expressing cells in vitro and in vivo (8) and shows antitumor effects in clinical trials (7, 9, 10). The wtEGFR gene itself is also overexpressed in a wide variety of human epithelial tumors, including those of the breast, ovary, lung, and head and neck, as well as glioblastomas (4, 11, 12). Several anti-EGFR mAbs have been tested for radioimmunodiagnostic and radioimmunotherapeutic purposes, and the inhibition of ligand bind- ing to EGFR by certain mAbs effectively suppresses tumor growth in vitro and in vivo (5, 12, 13). Clinical trials of these antibodies in patients with squamous cell carcinoma of the head and neck or the lung (13) or with malignant gliomas (14) have yielded encouraging results. The EGFR gene is amplified in nearly 50% of human grade IV gliomas (glioblastoma multiforme; Refs. 4, 15). In the majority of these cases, amplification of the EGFR gene is associated with struc- tural rearrangements of the gene. The most frequent mutation has been observed in more than 25% of gliomas (15–17) and, also reported in carcinomas of the lung, breast, and prostate (18 –20), results from the deletion of the coding exons 2–7, which leads to an in-frame deletion of 801 bp from the mature mRNA. This corresponds to a deletion of 267 amino acids from the NH 2 terminus of the EGFR and creates a mutant EGFR with a unique extracellular domain (15). The mutant EGFR is ligand independent and constitutively active, and its ex- pression in human glioma cells enhances tumorigenicity (21, 22) and increases the invasive phenotype of the cell in vivo (23). The EGFR- selective tyrosine kinase inhibitor, AG1478, effectively suppresses the growth of EGFR-overexpressing glioblastoma cells (24), which suggests that the blockade of EGFR activation can inhibit the aggressive phenotype of glioblastomas. Correspondingly, the pres- ence of EGFR in tumors correlates with poor prognosis in glioma patients (25). The oncogenic potential mediated by EGFR together with its high level and restricted expression in tumor tissue, as well as its localization on the cell surface, make this mutant EGFR a potential target for antibody-based therapy. Here we demonstrate that systemic treatment with the novel EGFR- specific mAb, mAb 806, causes reduced phosphorylation of the constitutively active EGFR and thereby suppresses growth of intracranially implanted gliomas overexpressing this mutant receptor in nude mice and extends their survival. The inhibition of tumor growth was mediated by a decrease in proliferation and angiogenesis and increased apoptosis of the tumor cells. This suppression affected active signaling by EGFR because intracranial xenografts that were derived from cells overexpressing kinase-deficient EGFR (DK), which are recognized equally well by mAb 806, were not significantly Received 3/22/01; accepted 5/9/01. 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 Supported in part by a fellowship from the Japan Brain Foundation (to K. M.). 2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, 9500 Gilman Drive, CMM-East 3080, University of California at San Diego, La Jolla. CA 92093-0660. Phone: (858) 534-7802; Fax: (858) 534-7750; E-mail: wcavenee@ ucsd.edu. 3 The abbreviations used are: mAb, monoclonal antibody; EGFR, epidermal growth factor receptor; FACS, fluorescence-activated cell sorter/sorting; TUNEL, terminal de- oxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; NK, natural killer; MVA, microvessel area; wtEGFR, wild-type EGFR. 5349 Research. on November 17, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from