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Summary. The epidermal growth factor receptor (EGFR), a growth factor receptor involved in the regulation of cellular differentiation and proliferation, is highly ...
Investigational New Drugs 17: 259–269, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Epidermal growth factor receptor inhibition in cancer therapy: biology, rationale and preliminary clinical results Shyh-Min Huang and Paul M. Harari Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin, USA

Key words: EGFR, cancer, anti-EGFR antibody, chemotherapy, radiation

Summary The epidermal growth factor receptor (EGFR), a growth factor receptor involved in the regulation of cellular differentiation and proliferation, is highly expressed by many tumor cells. In light of a relationship between overexpression of EGFR and clinically aggressive malignant disease, EGFR has emerged as a promising target for cancer therapy. In recent years, several molecular strategies have been explored to modulate either the EGFR itself, or the downstream signal beyond the cell surface receptor. One of the most promising current strategies involves the use of anti-EGFR monoclonal antibodies (mAbs), either alone or in combination with conventional cytotoxic modalities such as chemotherapy or radiotherapy. This review focuses primarily on recent progress in the development of anti-EGFR mAbs, and examines their potential in the treatment of cancer.

Introduction EGFR is a member of the receptor tyrosine kinase family and is expressed in a variety of different cell types. Ligand binding to the EGFR can activate signal transduction which, in turn, modulates cellular proliferation and neoplastic growth [1]. Epithelial tumors commonly overexpress EGFR, including many cancers of the head and neck, breast, lung, colon, prostate, kidney, cervix, ovary, and bladder [2–8]. The level of increased expression in malignant cells can reach an order of magnitude or greater than that expressed in non-malignant counterpart cells. Overexpression of EGFR often correlates with a poor clinical prognosis and therefore EGFR regulation has become an attractive target for developing new strategies in cancer therapeutics [9–12].

EGFR structure and function An appreciation of the structure and function of the EGFR is important for the development of specific inhibitors of this system, and for their application in

cancer therapy. The EGFR is an 1186-amino acid, 170-kDa transmembrane protein which is a member of the large family of Type I receptor tyrosine kinases that also includes ErB-2/neu (also known as Her-2), ErbB-3 and ErbB-4 [13,14]. The EGFR is composed of three major domains: an extracellular ligand-binding domain, a transmembrane lipophilic segment, and a cytoplasmic protein tyrosine kinase domain (Figure 1). Several ligands for EGFR have been identified. They include EGF, TGF-α, vaccinia virus growth factor, amphiregulin, and heparin binding EGF. The most widely expressed ligand for the EGFR in human tissues is TGF-α. Following ligand binding, the EGFR undergoes dimerization. Dimerization activates the intrinsic protein kinase via intermolecular autophosphorylation within the cytoplasmic domain. The tyrosine autophosphorylated region functions as a binding site for cytoplasmic messenger protein which then initiates a cascade of signals from the cytoplasm to the nucleus, eventually resulting in mitogenesis (Figure 1) [15]. Interruption in the activation of the EGFR can be accomplished through the use of antiEGFR mAbs, which block the binding of endogenous ligands, or by the use of pharmacological agents which

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Figure 1. Simplified schematic illustration of the EGFR system depicting EGFR, MAP kinase signal transduction cascade to the nucleus, and stimulation of cell cycle machinery. Potential interactions and resultant effects of radiation and selected chemotherapeutic agents on the EGFR system are discussed within text.

inhibit specific downstream components of the EGFR pathway (e.g. tyrosine kinase inhibitors). Characterization of anti-EGFR antibodies To date, the precise structure of the EGFR receptor ligand binding site has not been fully characterized so as to permit the synthesis of specific peptide analogues or other molecules that block ligand binding. However, a series of murine mAbs directed against the extracellular ligand binding domain of the EGFR have been produced, most notably by Mendelsohn and colleagues [16,17]. Among these, mAb 225 (IgG2a), 528 (IgG1) and 425/EMD55900 (IgG2a) have been characterized and studied in the preclinical setting to explore their potential as therapeutic agents [18,19]. These mAbs are generally similar in that they bind to the EGFR with affinity comparable to TGF-α (Kd = 2 nM) and compete with TGF-α binding [19]. However, evidence

suggests that these mAbs bind to an epitope on the EGFR that is distinct from the actual ligand binding site [20]. Therefore, it is postulated that the current mAbs do not bind to the precise TGF-α binding site, but near enough to prevent functional TGF-α from binding. Binding of mAb 225 or mAb 528 to the EGFR causes dimerization and internalization of the receptor without triggering tyrosine phosphorylation in intact cells [21–23]. This finding is significant because it implies that anti-EGFR mAb can be used to block the EGFR signaling cascade that is involved in triggering cellular proliferation in many epithelial tumors. One potential obstacle to the successful use of murine mAbs in human clinical trials is the potential for generation of human anti-mouse antibody responses. In an effort to avoid human anti-mouse antibody production, the mAb 225 was chimerized to the constant region of human IgG to produce the chimeric mAb

261 225, thereafter named C225 [24]. Comparison of these two mAbs showed that C225 had a higher affinity for the EGFR and a longer half-life in serum [25–27]. C225 was further shown to be more effective than m225 in inhibiting tumor growth of A431 xenografts in nude mice [25]. Findings from these preclinical in vitro and in vivo studies have led to the conduction of several preliminary clinical trials to investigate the safety and feasibility of delivering C225 in the treatment of human epithelial tumors [27–29]. Recently, a fully human IgG2k mAb (E7.6.3), specific to the human EGFR, has been generated [30]. Similar to C225, E7.6.3 appears to be effective in preventing human tumor formation in athymic mice and in eradicating established human tumor xenografts. Because E7.6.3 is a fully human mAb, it may prove of interest in minimizing the allergic reactions observed in some studies using chimeric mAbs [31–33].

Inhibition of tumor cell proliferation The growth inhibitory effects of anti-EGFR mAb on human tumor cells were first demonstrated in A431 epidermoid carcinoma cells which express both EGFR and TGF-α in large quantities [18]. Subsequent studies confirmed that the mAbs inhibited cellular proliferation in a variety of cultured epithelial tumor cell lines including breast, colon, lung, kidney, prostate, and head and neck [34–38]. The inhibition of cellular proliferation by anti-EGFR mAb was further characterized by modulation of cell cycle progression and apoptosis [38–40]. Treatment of tumor cells with anti-EGFR mAb generally induced accumulation of cells in G1 phase of the cell cycle associated with an increase in the level of the specific cyclindependent protein kinase (CDK) inhibitor, p27KI P . The increased level of p27KI P was accompanied by an increased association with CDK2 and resulted in a reduction in cyclin-CDK2 kinase activity. Cyclin-CDK2 serves to phosphorylate retinoblastoma protein (Rb), and therefore serves as a critical regulator for cells in their progression from G1 to S phase. Phosphorylation of Rb in turn facilitates release of the Rb-bound transcription factor E2F, which is required for the G1 to S phase transition. When cyclin-CDK2 activity is reduced, as observed in anti-EGFR mAb-treated cells, phosphorylation of Rb is similarly reduced and cells arrest in G1 phase [36,40]. The precise extent to which anti-EGFR mAb inhibits cell cycle progression appears to vary somewhat with the particular tumor cell

line tested. For example, treatment of colon carcinoma cells (DiFi) or squamous cell carcinoma of head and neck (SCC-13Y) with C225 not only inhibited cell cycle progression but also induced apoptosis [36,40]. In prostate DU-145 cells, EGFR blockade led to a steady accumulation of cells in G1 but did not induce apoptosis [38]. The human tumor xenograft model in athymic mice has been used to assess the in vivo effects of treatment with anti-EGFR mAbs. Treatment schedules of intraperitoneal injections once or twice weekly have been most commonly tested on the three day half-life of mAb in serum [41]. Injection of either m225 or m528, commencing within a few days of tumor cell implantation, induced a dose-dependent inhibition of A431 squamous tumor cell growth [41]. Successful prevention of tumor growth by anti-EGFR mAbs has now been shown in carcinoma cell lines derived from breast, colon, stomach, prostate, and head and neck (Table 1). These results demonstrate the capacity of anti-EGFR mAbs to prevent and regress existing tumor xenograft growth in vivo and suggest their promise as a cancer therapeutic agent. Findings from these initial animal studies provided additional support for the first Phase I human clinical trial, which examined the safety and feasibility of m225 as an imaging agent in patients with advanced squamous cell carcinoma of the lung [42].

Inhibition of tumor angiogenesis and metastasis In several experimental models, the capacity of antiEGFR mAb to inhibit tumor growth has proven more potent within in vivo model systems (i.e. tumor xenografts) than that identified using in vitro cell culture studies. This finding suggests that additional mechanisms may be involved in contributing to tumor growth inhibition following EGFR blockade than solely the observed antiproliferative cell cycle effects. One possible contributing mechanism for the enhanced effects observed in vivo following anti-EGFR mAb therapy appears to involve inhibition of angiogenesis. The EGFR system and angiogenesis have been independently evaluated as important cancer therapy targets, and several links between them have been recently identified [43–45]. Petit et al. showed that C225 substantially influenced the angiogenic potential of A431 cells by downregulating VEGF expression, both in cell culture and with in vivo experiments [43]. Further work by Perrott and co-workers demonstrated that se-

262 Table 1. Summary of published data regarding dose, administration schedule and antitumor effect of anti-EGFR mAbs on various human tumor xenografts in athymic mice. Cell line

Cancer derivation

Anti-EGFR mAb

Dose (single/total)a

Timing/ tumor sizeb

Growth inhibition

Reference

A431

Vulva

m225

Prostate Stomach Colon Breast Head & neck Kidney

Day 3/150 mm3 Day 5/200 mm3 Day 8/400 mm3 Day 15/100 mm3 Day 12/30 mm3 Day 7/300 mm3 Day 1 Day 7/300 mm3 Day 1 Day 10/30 mm3 Day 7/200 mm3

+++ ++ + +++ +++ +++ +++ ++ +++ +++ +++

[57]

C225/m528 m425 C225 m528 C225 m528 C225 C225

1 mg/10 mg 1 mg/9 mg 1 mg/8 mg 1 mg/4 mg 1.1 mg/2.2 mg 1 mg/10 mg 0.2 mg/0.8 mg 0.25 mg/1.5 mg 2 mg 0.2 mg/0.8 mg 1 mg/10 mg

DU145/PC-3 NMS12 GEO MDA 468 UM-SCC1/UM-SCC6 SK-RC-29

[43 [90] [25,92] [94] [79] [55] [71] [95]

a Dose of mAb delivered each injection (intraperitoneal) and total dose administered. b Day of measurement and tumor size when mAb treatment was initiated following tumor implantation.

lective downregulation of VEGF, IL-8 and bFGF by tumor cells following C225 treatment led to involution of tumor vessels [45]. These effects resulted in growth inhibition and delayed progression of human transitional cell carcinoma (TCC) of the bladder established orthotopically in nude mice [45]. Since VEGF, bFGF and IL-8 all share AP-I binding site in their promoters, it is postulated that EGFR blockade with C225 down-regulates the expression of VEGF, bFGF and IL-8 via a reduction of AP-1 activity. The level and functional activity of EGFR have also been suggested to influence the metastatic potential of human cancer cells [44,46–48]. Blockade of EGFR with anti-EGFR mAb has been shown to inhibit the development of metastasis in several tumor models [24,45,49,50]. Using a xenograft model for metastatic melanoma, the number of pulmonary metastatic foci was reduced in mice treated with m225 or with C225 [24]. In experiments with bladder TCC xenografts, control mice all developed lymph node metastases, and approximately 40% demonstrated lung metastases [45]. In contrast, none of the C225-treated mice manifested lung or lymph node metastases under the same experimental conditions. These preclinical findings further suggest that in addition to the direct antiproliferative effects on tumor cells, anti-EGFR mAb may also contribute inhibitory effects on angiogenesis and metastatic potential.

Combination with chemotherapy The EGFR signaling pathway not only influences cellular growth regulation, but can also influence the sensitivity of malignant cells to cytotoxic cell kill following conventional cancer therapy [51–54]. The strategy of enhancing cytotoxicity by combining treatment of anti-EGFR mAbs with chemotherapeutic agents has been explored. A report by Baselga et al. reported that antitumor activity was enhanced when mice with well-established A431 or MDA-468 cell xenografts were treated with doxorubicin in combination with m225 (Figure 2) [55]. Parallel studies were carried out using m225 or m528 in combination with cisplatin [55,56]. Among these latter studies utilizing A431 cell xenograft models [56,57], mAb alone or cisplatin alone were not highly effective in eradicating tumor xenografts, whereas combined therapy produced cures in tumor-bearing mice for over six months (Figure 2). Similar findings were reported in studies examining several additional tumor cell lines and chemotherapeutic drugs including taxol, cisplatinum, and topotecan [54,58,59]. Specifically, tumor eradication was much more effectively achieved with combined treatment of m225 and chemotherapeutic agent compared with single agent alone. The combined treatment strategy is now being tested in clinical trials for patients with advanced epithelial cancers. For example, C225 is being combined with doxorubicin to treat advanced prostate cancer, with cisplatin to treat advanced head and neck and lung cancers, and with

263 paclitaxel to treat breast cancer [32,33]. A recent phase Ib/IIa clinical trial of C225 plus cisplatin for recurrent head and neck cancer patients showed a complete plus partial response rate of 67% [60].

Combination with radiotherapy The well established role of radiation in cancer therapy provides a logical rationale for examining the capacity of anti-EGFR mAbs to enhance radiationinduced control of locally advanced epithelial tumors. For rapidly-proliferating tumors, the repopulation of tumor cells during radiation treatment has been identified as a factor that adversely impacts tumor response and ultimate local control [61]. This phenomenon is particularly well studied in squamous cell carcinoma of the head and neck. One experimental approach to reduce the impact of tumor cell repopulation involves the administration of antiproliferative agents which slow or inhibit tumor cell proliferation during radiotherapy [62,63]. Inhibition of the EGFR system represents one potential method to exert antiproliferative growth control on rapidly dividing tumor cells such as those that occur in the head and neck. Several studies have shown that cell survival and repopulation during a course of radiotherapy are influenced, in part, by activation of EGFR/TGF-α that is induced following radiation [64–66]. EGF-deprived cells or EGFR-inactivated cells were shown to be more radiosensitive than their EGF-stimulated, exponentially growing counterparts [67–69]. In several in vitro studies, investigators have independently demonstrated that anti-EGFR mAb increased the radiosensitivity of a spectrum of human squamous cell carcinomas [40,70,71]. Treatment of cells with anti-EGFR mAb not only enhanced reproductive cell death following radiation, but also increased the fraction of tumor cells succumbing to radiation-induced apoptosis (Figure 3) [40]. In addition, C225 was shown to augment the in vivo tumor response of squamous cell carcinoma xenografts to radiation in nude mice (Figure 4) [72–74]. Several mechanistic explanations regarding the enhanced radiosensitivity induced by anti-EGFR mAb have been proposed. These include mAb-induced alteration of cell cycle progression, reduction in the availability of reactive oxygen scavengers (glutathione), promotion of apoptosis and inhibition of DNA damage repair. Results from several studies support the involvement of these mechanisms in mediating the

radiosensitizing effect of C225. For example, treatment of tumor cells with anti-EGFR mAb was shown to induce accumulation of cells in a more radiosensitive cell cycle phase and to decrease glutathione levels, thereby facilitating cell death following exposure to radiation [75–77]. The potential involvement of DNA repair inhibition by EGFR blockade was demonstrated by Bandyopadhyay et al., who reported the observation of a specific physical interaction between internalized EGFR and cytosolic DNA-dependent protein kinase (DNA-PK) induced by C225 treatment in a variety of cell types [78]. As a result of this interaction, a reduction in the nuclear level and activity of DNAPK, with a concurrent increase in the cytosolic level of DNA-PK, were observed. DNA-PK is believed to play a major role in the repair of DNA double-strand breaks and these findings therefore suggest that DNA repair may be impaired by C225 treatment. Further research is necessary to determine the specific molecular pathways involved in the suppression of DNA repair that may be triggered by anti-EGFR mAb.

Combination with other signal transduction inhibitors A potential new strategy which targets separate signal transduction pathways by combining anti-EGFR mAb with other signal transduction inhibitors has been recently explored. Examples include the combination of anti-EGFR mAb with inhibitors of cAMP-dependent protein kinase (PKA), such as 8-chloro-cAMP, or with antisense oligonucleotide of PKA. PKA is an intracellular enzyme with serine-threonine kinase activity. It plays a key role in cellular growth and differentiation. In a series of experiments using human colon and breast cancer cell lines, the combined treatment with m528 and 8-chloro-cAMP had a more than additive antiproliferative effect [79]. Subsequently, the anticancer effect of this combined regimen was shown to be highly effective, as indicated by a strong inhibition in tumor growth and tumor-induced host angiogenesis, in an in vivo model using human GEO colon carcinoma xenografts [80]. In addition to 8-chlorocAMP, an antisense oligonucleotide of PKA was also shown to potentiate growth inhibition and induction of apoptosis in human renal cancer cells [81]. In the same report, combined treatment was also shown to induce regression of human renal tumor xenografts, whereas single agent treatment only delayed tumor growth. Future investigation of methods that simultan-

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Figure 2. Antitumor activity of C225 in combination with chemotherapeutic agents. A431 xenografts were raised subcutaneously in athymic mice. Left panel, doxorubicin (DOXO) and m225 (1 mg) were given intraperitoneally. Bars represent mean tumor size (±S.E. with n = 1O per group) at 44 days following tumor inoculation. (Adapted from Ref. #55). Right panel, cis-diamminedichloroplatinwn (cis-DDP) and m225 (1 mg) were given intraperitoneally. Bars represent mean tumor size (±S.E with n = 8–12 per group) at 37 days following tumor inoculation. (Adapted from Ref. #57).

eously target two signaling pathways may prove to be clinically applicable in anticancer therapy.

Other strategies to inhibit EGFR activity The activity and function of EGFR tyrosine kinase is necessary for effective EGFR signaling, and therefore tyrosine kinase inhibition represents another logical target for the modulation of EGFR activity. This topic is more thoroughly reviewed in a separate article within this journal issue. In brief, there are three general classes of EGFR tyrosine kinase inhibitors, namely, the tyrphostins, the dianilinophtalimides (DAPH) and the quinazolines. Many studies have investigated the effect of these tyrosine kinase inhibitors on cancer cell lines, both in vitro and in vivo. The use of a tyrphostin, i.e. RG-13022 or RG14620, was shown to inhibit the growth of human squamous cell carcinomas implanted in nude mice [82]. Oral administration of a DAPH, i.e. CGP 54211, selectively inhibited the level of EGFR phosphorylation in a mouse xenograft model [83]. In the latter study, tumor growth inhibition as well as tumor necrosis were ob-

served as a consequence of the inhibited EGFR activity. Quinazolines, such as PD153035 and AG1478, appeared to be highly selective for EGFR. Inhibition of EGFR tyrosine kinase activity was observed following drug exposure in nanomolar range (IC50: 3 nM) [84]. Treatment of human squamous cell carcinoma HN5 with PD153035 inhibited tumor growth by inducing terminal differentiation and by inducing apoptosis [85,86]. Taken together, these results suggest that tyrosine kinase inhibitors represent another promising therapeutic target against cancers that overexpress EGFR. Clinical application will require that issues of administration, dosage and selective delivery are addressed. An additional experimental method to block EGFR activation involves the prevention of translation of EGF-like growth factor mRNA by the use of antisense oligonucleotides. Studies have shown that antisense oligonucleotide directed against either amphiregulin or EGF-like growth factors CRIPTO, as well as a retroviral antisense TGF-α expression, were able to inhibit the growth of human colon carcinoma cells [87–89]. A supra-additive growth inhibitory effect

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Figure 3. C225 enhances radiation-induced apoptosis. Control or C225 treated SCC13Y cells were irradiated with either single doses of 6 Gy (left panel) or with at 2 Gy fractions for 3 consecutive days (right panel). Following radiation, cells were incubated for 3 days and processed for flow cytometric analysis using propidium iodide to stain DNA. Percentage of apoptosis shown within each panel was determined on the basis of sub-GO DNA content of the histogram.

Figure 4. Antitumor activity of C225 in combination with radiation. Squamous cell carcinoma xenografts derived from human head and neck tumor cell lines (UM-SCC1 or UM-SCC6) were raised subcutaneously in the flank of athymic mice. Injection of C225 (0.1 mg) was given intraperitoneally on days 10 and 13. Radiation (XRT) was delivered as a single fraction of 12 Gy on day 15. Bars represent mean tumor size (±S.E. with n = 6 per group) at 48 days following tumor inoculation.

266 was observed when antisense oligonucleotides directed against EGF-like growth factors were combined with anti-EGFR mAb [90]. This finding implies that the use of two different agents that target the blockade of the same receptor may be more efficient than the use of a single agent.

Conclusion and future directions Many promising new cancer therapies are now being driven by the design of agents that address specific molecular or genetic targets within the malignant cell. The concept of molecular inhibition of the EGFR signal transduction system is one such approach that has been systematically developed over the last decade. The opportunities to modulate tumor cell proliferation in this context are quite broad, ranging from agents that induce EGFR blockade to agents that facilitate inhibition of tyrosine kinase phosphorylation, MAP kinase signal transduction, cyclin-dependent kinase activity, and others. This review has focused on EGFR inhibition. The rationale and preliminary data supporting this approach in cancer therapy appears quite strong to date, particularly in combination with radiation or selected chemotherapy agents. Epithelial tumors that are rich in their expression of EGFR hold special promise for receptor blockade approaches. Squamous cell carcinomas of the head and neck are particularly rich in EGFR expression, and therefore represent a logical experimental model for EGFR inhibition. Phase I/II clinical trial evaluation combining C225 and radiation therapy in patients with advanced squamous cell carcinomas of the head and neck has produced exceptionally high major response rates [91]. The promising pre-clinical data and Phase I/II clinical trial results have led to the design and initiation of a Phase III clinical trial examining radiation therapy +/–C225 for patients with advanced head and neck tumors. This trial commenced accrual in North America in April 1999. Similar to the recent therapy successes in selected breast cancer and lymphoma patients with molecules that target specific growth receptor blockade (e.g., herceptin, rituxan), EGFR blockade plus radiation therapy in squamous cell carcinomas of the head and neck represents a promising new molecular cancer therapy approach. The broad concept of EGFR-system inhibition, either alone or in combination with radiation or selected chemotherapy agents, will be actively investigated across a spectrum of epithelial tumors in the upcoming years.

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Address for offprints: Paul M. Harari, Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, 600 Highland Ave., Madison, WI 53792, USA; Tel.: 608-263-8500; Fax: 608-263-9167